NZ240670A - Carbonyl-hydrolase mutants, dna sequences, vectors and hosts - Google Patents

Description

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DIVIDED OUT OF i-.VV:.I CATION N'' 233396 29 April 1987 PATENTS ACT, 1953 COMPLETE SPECIFICATION NON-HUMAN CARBONYL HYDROLASE MDTANTS, DNA SEQUENCES ^D.^TOftS ENCODING SAME AND HOSTS TRANSFORMED WITH SAID VECTORS Genencor" \<v\;e,r(\cvV»0OCii I'; We, GENENTECH, INC., a corporation of the State of Delaware, U.S'.'A'T, of 460 Point San Bruno Boulevard, South San Francisco, California 94QS0, United States of America hereby declare the invention for which 2 / we pray that a patent may bo granted to wve/us, ar.d the method by which it is to be performed, to bt: particularly described in and by the following statement:- - 1 - -it - i , - 5 OCT 1992 I (followed by pace la) / 240670 A4 319 8-DJB/RFT RECOMBINANT SUBTILISINS, DNA SEQUENCES AND VECTORS ENCODING SAME AND HOSTS TRANSFORMED WITH SAID VECTORS The recent development of various in vitro techniques to manipulate the DNA sequences encoding naturally-occuring polypeptides as well as recent developments in the chemical synthesis of relatively short sequences of single and double stranded DNA has resulted in the speculation that such techniques can be used to modify enzymes to improve some functional property in a predictable way. Ulmer, K.M. (1983) Science 219, 666-671. The only working example disclosed therein is the substitution of a single amino acid within the active site of tyrosyl-tRNA synthetase (Cys35-Ser) which lead to a reduction in enzymatic activity. See Winter, G., et al. (1982) Nature 29_9, 756-753; and Wilkinson, A.J., et al. ( 1983 ) Biochemistry 22, 3581-3586 (Cys35-Gly mutation also resulted in decreased activity).

When the same t-RNA synthetase was modified by substituting a different amino acid residue within the active site with two different amino acids, one of the mutants (Thr51-Ala) reportedly demonstrated a predicted moderate increase in kcat/Km whereas a second mutant (Thr51-Pro) demonstrated a massive increase in kcat/Km which could not be expla /' " o\ . y « K ls"0y I3S4 *f !, ) P "*~ certainty. Wilkinson, A.H., et al. (1984) Nature 307, 187-188.

Another reported example of a single substitution of an amino acid residue is the substitution of ''cysteine for isoleucine at the third residue of T4 lysozyme. Perry, L.J., et al. (1984) Science 226 , 555-557. The resultant mutant lysozyme was mildly oxidized to form a disulfide bond between the new cysteine residue at position 3 and the native cysteine at position 97.

This crosslinked mutant was initially described by the author as being enzymatically identical to, but more thermally stable than, the wild type enzyme. However, in a "Note Added in Proof", the author indicated that the enhanced stability observed was probably due to a chemical modification of cysteine at residue 54 since the mutant lysozyme with a free thiol at Cys54 has a thermal stability identical to the wild type lysozyme.

Similarly, a modified dihvdrofolate reductase from E.coli has been reported to be modified by similar methods to introduce a cysteine which could be crosslinked with a naturally-occurring cysteine in the reductase. Villafranca, D.E., et al. (1983) Science 222, 782-788. The author indicates that this mutant is fully reactive in the reduced state but has significantly diminished activity in the oxidized state. In addition, two other substitutions of specific amino acid residues are reported which resulted in mutants which had diminished or no activity.

New Zealand Patent Specification No. 208612 discloses the substitution of specific residues within B. aroylolique faciens subtilisin with specific amino acids. Thus, Met222 has been substituted with all 19 other amino acids, / I* ?"» p <"■ 4 w Glvl66 with 9 different amino acids anc Glyl69 with Ala and Ser.

As set forth below, several laboratories have also reported the use of site directed mutagensis to produce the mutation of more than one amino acid residue within a polypeptide.

The amino-terminal region of the signal peptide of the prolipoprotein of the E. coli outer membrane was stated to be altered by the substitution or deletion cf residues 2 and 3 to produce a charge change in that region of the polypeptide. Inovye, S., et al. (1982) ?roc. Nat. Acad. Sci. USA 7 9, 3438-3441. The same laboratory also reported the substitution and deletion of amine acid reaisues 9 and 14 to determine the effects of such substitution on the hydrophobic region cf the same signal sequence. Inouye, S., et al. (1934) J. Biol. Chem. 259, 3729-3733.

Double mutants in the active site of tyrosyl-t-RNA synthetase have also been reported. Carter, P.J., et al. (1984) Cel 1 38 , 835-840. In this report, the improved affinity of the previously described Thr51-Pro mutant for ATP was probed by producing a second mutation in the active site of the enzyme. One cf the double mutants, Gly35/Pro51, reportedly demonstrated an unexpected result in that it bound ATP in the transition state better than was expected from the two single mutants. Moreover, the author warns, at least for one double mutant, that it is not readily predictable how one substitution alters the effect caused by the other substitution and that care must be taken in interpreting such substitutions. i * 0 g 7 A mutant is disclosed in U.S. Patent No. 4,532,207, wherein a polyarginine tail was attached to the C-terminal residue of 0-urogastrone by modifying the DNA sequence encoding the polypeptide. As disclosed, the polyarginine tail changed the electrophoretic mobility of the urogastrone-polyaginine hybrid permiting selective purification. The polyarginine was subsequently removed, according to the patentee, by a polyarginine specific exopeptidase to produce the purified urogastrone. Properly construed, this reference discloses hybrid polypeptides which do not constitute mutant polypeptides containing the substitution, insertion or deletion of one or more amino acids of a naturally occurring polypeptide.

Single and double mutants of rat pancreatic trypsin have also been reported. Craik, C.S., et al. (1985) Science 2 2 8, 291-297. As reported, glycine residues at positions 216 and 226 were replaced with alanine residues to produce three trypsin mutants (two single mutants and one double mutant) . In the case of the single mutants, the authors stated expectation was to observe a differential effect on Km. They instead reported a change in specificity (kcat/Km) which was primarily the result of a decrease in kcat. In contrast, the double mutant reportedly demonstrated a differential increase in Km for lysyl and arginyl substrates as compared to wild type trypsin but had virtually no catalytic activity.

The references discussed above are provided solely for their disclosure prior to the filing date of the instant case, and nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or priority based on earlier filed applications. 2406 Based cr. the above references, however, it is apparent that the modification of the ar.ir.o acid sequence cf vild type enzymes often results in the decrease or destruction of biological activity.

Accordingly, it is an object herein to provide carbonyl hydrolase mutants in the form of recombinant subtilisins which have at least one property which is different from the same property of the carbonyl hydrolase (subtilisin) precursor from which the amino acid of said mutant is derived.

It is a further object to provide mutant DNA sequences encoding such carbonyl hydrolase mutants as well as expression vectors containing such mutant DNA sequences.

Still further, another object of the present invention is to provide host cells transformed with such vectors as well as host cells which are capable of expressing such mutants either intracellularly or extracellularly.

Sr2rv of_ t he__Invent ion The invention includes recombinant subtilisins, preferably having at least one property which is substantially different from the same property of the precursor subtilisin from which the amino acid sequence of the recombinant subtilisin is derived. These properties include oxidative stability, substrate specificity, catalytic activity, thermal stability, alkaline stability, pH activity profile and resistance to proteolytic degradation.

The amino acid sequence of the recombinant subtilisin is derived by the substitution, deletion or insertion cf one or more amino acids of the precursor subtilisin amino acid sequence.

The invention also includes mutant DNA sequences encoding such subtilisins. Further the invention includes expression vectors containing such DNA sequences as well as host cells transformed with such vectors which are capable of expressing said subti1i si ns. -rief Description of the Drawings Figure 1 shows the nucleotide sequence of the coding strand, correlated with the amino acid sequence of B. arr.viol icruefaciens subtilisin gene. Promoter (p) ribosome binding site (rbs) and termination (term) regions of the DNA sequence as well as sequences encoding the presequence (PRE) putative proseque.nce (PRO) and mature form (MAT) of the hydrolase are also shown.

Figure 2 is a schematic diagram showing the sxibW^raSe substrate, k. • . < . 'C/.-0V1994 w) binding cleft of subtilisin together with substrate. 'tA <4 ?)! ! Figure 3 is a stereo view of the S-l binding subsite of B. amviol icruefaciens subtilisin showing a lysine F-l substrate bound in the site in two different ways. Figure 3A shews Lysine P-l substrate bound to form a salt bridge with a Glu at position 156. Figure 33 shows Lysine P-l substrate bound to form a salt bridge with Glu at position 166.

Figure 4 is a schematic diagram of the active site of subtilisin Asp32, His64 and Ser221.

Figures 5A and 5B depict the amino acid sequence of subtilisin obtained from various sources. The residues directly beneath each residue of B. a:r.vlolicniefaciens subtilisin are equivalent residues which (1) can be mutated in a similar manner to that described for B. amvlolicruefaciens subtilisin, or (2) can be used as a replacement amino acid residue in B. a-vlclicruefaciens subtilisin. Figure 5C depicts conserved residues of B. aravlol icruefaciens subtilisin when compared to other subtilisin sequences.

Figures 6A and 6B depict the inactivation of the mutants Met222L and Met222Q when exposed to various organic oxidants.

Figure 7 depicts the ultraviolet spectrum of Met222F subtilisin and the difference spectrum generated after inactivation by diperdodecanoic acid (DPDA).

Figure 8 shows the pattern of cyanogen bromide digests of untreated and DPDA oxidized subtilisin Met222F on high resolution SDS-pyridine peptide gels. 2 4 0 P Figure 9 depicts a map of the cyanogen bromide fragments of Fig. 8 and their alignment with the sequence of subtilisin Met222F.

Figure 10 depicts the construction of mutations between ccdons 45 and 50 of B. amvlol icruefaciens subtilisin.

Figure 11 depicts the construction of mutations between codons 122 and 127 of B. amvlol icruefaciens subtilisin.

Figure 12 depicts the effect of DPDA on the activity of subtilisin mutants at positions 50 and 124 in subtilisin Met222F.

Figure 13 depicts the construction of mutations at codcn 166 of B. amvlol icruefaciens subtilisin.

Figure 14 depicts the effect of hydrophobicity of the P-l substrate side-chain on the kinetic parameters of wild-type B. amvlol icruefaciens subtilisin.

Figure 15 depicts the effect of position 166 side-chain substitutions on P-l substrate specificity. Figure 15A shows position 166 mutant subtilisins containing non-branched alkyl and aromatic side-chain substitutions arranged in order of increasing molecular volume. Figure 15B shows a series of mutant enzymes progressing through p- and 7-branched aliphatic side chain substitutions of increasing molecular volume.

Figure 16 depicts the effect of position 166 side-chain volumn on log kcat/Km for various P-l substrates.

Figure 17 shows the substrate specificity differences between Ilel66 and wild-type (Glyl66) B. amyloliquefaciens subtilisin against a series of alphatic and aromatic substrates. Each bar represents the difference in log kcat/Km for Ilel66 minus wild-type (Glvl66) subtilisin.

Figure 18 depicts the construction of mutations at codon 169 of B. amyloliquefaciens subtilisin.

Figure 19 depicts the construction of mutations at cocon 104 of B. amyloliquefaciens subtilisin.

Figure 20 depicts the construction of mutations at codcn 152 B. amvloliquefaciens subtilisin.

Figure 21 depicts the construction of single mutations at codon 156 and double mutations at codons 156 and 166 of B. amvloliquefaciens subtilisin.

Figure 22 depicts the construction of mutations at codon 217 for B. amvlolique faciens subtilisin.

Figure 23 depicts the kcat/Km versus pH profile for mutations at codon 156 and 166 in B. amyloliquefaciens subtilisin.

Figure 24 depicts the kcat/Km versus pH profile for mutations at codon 222 in B. amyloliquefaciens subtilisin.

I 4 0 F ~ Figure 25 depicts the constructing mutants at codcns 94, 95 and 96.

Figures 26 and 27 depict substrate specificity of various wild type and mutant subtilisins for different substrates.

Figures 28 A, B, C and D depict the effect of charge in the P-l binding sites due to substitutions at codon 156 and 166.

Figures 29 A and B are a stereoview of the P-l binding site of subtilisin BPN1 showing a lysine P-l substrate bound in the site in two ways. In 29A, Lysine P-l substrate is built to form a salt bridge with a Glu at codon 156. In 29B, Lysine P-l substrate is built to fori a salt bridge with Glu at codon 166.

Figure 30 demonstrates residual enzyme activity versus temperature curves for purified wild-type (Panel A) , C22/C87 (Panel B) and C24/C87 (Panel C).

Figure 31 depicts the strategy for producing point mutations in the subtilisin coding sequence by misin-corporation of Q-thioldeoxynucleotide triphosphates.

Figure 32 depicts the autolytic stability of purified wild type and mutant subtilisins 170E, 107V, 213R and 107V/213R at alkaline pH.

Figure 33 depicts the autolytic stability of purified wild type and mutant subtilisins V50, F50 and F50/V107/R213 at alkaline pH. 2 4 u 6 / 0 -li- Figure 34 depicts the strategy for constructing plasmids containing random cassette mutagenesis over residues 197 through 228.

Figure 35 depicts the oligodeoxynucleotides used for random cassette mutagenesis over residues 197 through 228 .

Figure 3 6 depicts the construction of mutants at codon 204 .

Figure 37 depicts the oligodeoxynucleotides used for synthesizing mutants at codon 204.

Detailed Description The inventors have discovered that various single and multiple in vitro mutations involving the substitution, deletion or insertion of one or more amino acids within a non-human carbonyl hydrolase amino acid sequence can confer advantageous properties to such mutants when compared to the non-mutated carbonyl hydrolase.

Specifically, B. amvlol icruefaciens subtilisin, an alkaline bacterial protease, has been mutated by modifying the DNA encoding the subtilisin to encode the substitution of one or more amino acids at various amino acid residues within the mature form of the subtilisin molecule. These in vitro mutant subtilisins have at least one property which is different when compared to the same property of the precursor subtilisin. These modified properties fall into several categories including: oxidative stability, substrate specificity, thermal stability, alkaline stability, catalytic activity, pH activity k-t c profile, resistance to proteolytic degradation, Km, kcat and Km/kcat ratio.

Carbonyl hydrolases are enzymes which hydrolyze 0 II compounds containing C-X bonds in which X is oxygen or nitrogen. They include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. Naturally occurring carbonyl hydrolases principally include hydrolases, e.g. lipases and peptide hydrolases, e.g. subtilisins or metalloproteases. Peptide hydrolases include o-aminoacylpeptide hydrolase, peptidylamino-acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxy-peptidase, thiol proteinase, carboxylproteinase and metalloproteinase. Serine, metailo, thiol and acid proteases are included, as well as endo and exo-proteases.

"Recombinant carbonyl hydrolase" refers to a carbonyl hydrolase in which the DNA sequence encoding the naturally occurring carbonyl hydrolase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the carbonyl hydrolase amino acid sequence. Suitable modification methods are disclosed herein and in EPO Publication No. 0130756 published January 9, 19 85.

Subtilisins are bacterial carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides. As used herein, "subtilisin" means a naturally occurring subtilisin or a recombinant subtilisin. A series of naturally occurring subtilisins is known to be produced and often secreted s .. by various bacterial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity. This class of serine proteases Ehares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. The subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine. In the subtilisin related proteases the relative order of these amino acids, reading from the amino to carboxy terminus is aspartate-histidine-serine. In the chymotrypsin related proteases the relative order, however is histidi.ne-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases.

"Recombinant subtilisin" refers to a subtilisin in which the DNA sequence encoding the subtilisin is modified to produce a mutant DNA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally occurring subtilisin amino acid sequence. Suitable methods to produce such modification include those disclosed herein and in New Zealand Patent Specification No. 208612. For example, the subtilisin multiple mutant herein containing the substitution of methionine at amino acid residues 50, 124 and 222 with phenylalanine, isoleucine and glutamine, respectively, can be considered to be derived from the recombinant subtilisin containing the substitution of glutamine at residue 222 (Q222) disclosed in EPO Publication No. 0130756. The multiple mutant thus is produced by the substitution of phenylalanine for methionine at 14 0 $ residue 50 and isoleucine for methionine at residue 124 in the Q222 recombinant subtilisin.

"Carbonyl hydrolases" and their genes may be obtained from many procaryotic and eucaryotic organisms. Suitable examples of procaryotic organisms include gram negative organisms such as E. coli or pseudomonas and gram positive bacteria such as micrococcus or bacillus. Examples of eucaryotic organisms from which carbonyl hydrolase and their genes may be obtained include yeast such as S. cerevis iae. fungi such as Aspergillus sp.; and non-human mammalian sources such as, for example, Bovine sp. from which the gene encoding the carbonyl hydrolase chymosin can be obtained. As with subtilisins, a series of carbonyl hydrolases can be obtained from various related species which have amino acid sequences which are not entirely homologous between the members of that series but which nevertheless exhibit the same or similar type of biological activity. Thus, non-human carbonyl hydrolase as used herein has a functional definition which refers to carbonyl hydrolases which are associated, directly or indirectly, with procaryotic and non-human eucaryotic sources.

A "carbonyl hydrolase mutant" has an amino acid sequence which is derived from the amino acid sequence of a non-human "precursor carbonyl hydrolase". The precursor carbonyl hydrolases include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. The amino acid sequence of the carbonyl hydrolase mutant is "derived" from the precursor hydrolase amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the "precursor DNA sequence" which encodes the amino acid sequence of the precursor carbonyl hydrolase rather than manipulation of the precursor carbonyl hydrolase per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein and in NZ 208612.

Specific residues of B. amvlol icruefaciens subtilisin are identified for substitution, insertion or deletion. These amino acid position numbers refer to those assigned to the B. amvlol icruef aciens subtilisin sequence presented in Fig. 1. The invention, however, is not limited to the mutation of this particular subtilisin but extends to precursor carbonyl hydrolases containing amino acid residues which are "equivalent" to the particular identified residues in 3. amvlol icruef aciens subtilisin.

A residue (amino acid) of a precursor carbonyl hydrolase is equivalent to a residue of B. amvlol icruefaciens subtilisin if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analagous to a specific residue or portion of that residue in B. an viol icruefaciens subtilisin (i.e., having the same or similar functional capacity to combine, react, or interact chemically).

In order to establish homology to primary structure, the amino acid sequence of a precursor carbonyl hydrolase is directly comparted to the B. amvlol icruefaciens subtilisin primary sequence and particularly to a set of residues known to be invariant in all subtilisins for which sequence is known (Figure 5C). After aligning the coase^v^i residues, allowing for necessary insertions andP*^ \ IU04 o/ deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues 5 equivalent to particular amino acids in the primary sequence of B. amvlol icruefaciens subtilisin are defined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of 2Q conserved residues is also adequate to define equivalent residues. Conservation of the catalytic triad, Asp32/His64/Ser221 should be maintained.

For example, in Figure 5A the amino acid sequence of - subtilisin from B. amvlol icruefaciens B. subtil isin var. 1168 and B. lichenformis (carlsbergensis) are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved ~ r, residues contained in each sequence. These residues are identified in Fig. 5C.

These conserved residues thus may be used to define the corresponding equivalent amino acid residues of B. amvlol icruefaciens subtilisin in other carbonyl hydrolases such as thermitase derived from Thermoactinomyces. These two particular sequences are aligned in Fig. 5B to produce the maximum homology of conserved residues. As can be seen there are a number J0 of insertions and deletions in the thermitase sequence as compared to B. amvlol icruefaciens subtilisin. Thus, in thermitase the equivalent amino acid of Tyr217 in B. amvlol icruefaciens subtilisin is the particular lysine shown beneath Tyr217.

In Fig. 5A, the equivalent amino acid at position 217 in B. amvlol icruefaciens subtilisin is Tyr. Likewise, 24 0 5 *=*7 in B. subtil is subtilisin position 217 is also occupied by Tyr but in B. licheniformis position 217 is occupied by Leu.

Thus, these particular residues in thermitase, and subtilisin from B. subtilisin and B. 1icheniformis may be substituted by a different amino acid to produce a mutant carbonyl hydrolase since they are equivalent in primary structure to Tyr217 in B. amvlol icruef aciens subtilisin. Equivalent amino acics of course are not limited to those for Tyr217 but extend to any residue which is equivalent to a residue in B. amvlol icruefaciens whether such residues are conserved or not.

Equivalent residues homologous at the level of tertiary structure for a precursor carbonyl hydrolase whose tertiary structure has been determined by x-ray crystallography, are defined as those for which the atomic coordinates of 2 or more of the main chain atoms of a particular amino acid residue of the precursor carbonyl hydrolase and B. amvlol icruefaciens subtilisin (N on N, CA on CA, C on C, and 0 on 0) are within 0.13nm and preferably O.lnm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the carbonyl hydrolase in question to the B. amylolicruefaciens subtilisin. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.

S|Fo(h) I-|FC(h) I R factor = h Z|Fo(h)| h Equivalent residues which are functionally analogous to a specific residue of B. amy lol icruef aciens subtilisin are defined as those amino acids of the precursor carbonyl hydrolases which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the B. amvlol icruef aciens subtilisin as described herein. Further, they are those residues of the precursor carbonyl hydrolase (for which a tertiary structure has been obtained by x-ray crystallography), which occupy an analogous position to the extent that although the main chain atcns of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13nm of the corresponding side chain atoms of B. amvlol icraefaciens subtilisin. The three dimensional structures would be aligned as outlined above.

Some of the residues identified for substitution, insertion or deletion are conserved residues whereas others are not. In the case of residues which are not conserved, the replacement of one or more amino acids is limited to substitutions which produce a mutant which has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such replacements should not result in a naturally occurring sequence. The carbonyl hydrolase mutants of the present invention include the mature forms of carbonyl hydrolase mutants as well as the pro- and prepro-forms of such hydrolase mutants. The prepro-forms are the preferred construction since 2 4 0 G 7 ^ t'r.is facilitates the expression, secretion and maturation of the carbonyl hydrolase mutants.

"Expression vector" refers to a DNA construct 5 containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to 10 control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once 15 transformed into a suitable host, the vector may replicate and function independently of the host cenome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid" and "vector" are sometimes used 2 0 interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions anc which are, or become, known in the art.

The "host cells" used in the present invention generally are procaryotic or eucaryotic hosts which preferably have been manipulated by the methods disclosed in New Zealand Patent Specification No. 208612 to render 0 6 them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing subtilisin is the Bacillus strain BG2036 which is 5 deficient in enzymatically active neutral protease and alkaline protease (subtilisin). The construction of strain 3G2036 is described in detail in New Zealand Parent Specification No. 208612 and further described by Yang, M.Y. et al. (19S4) J. Bacteriol. 160. 15-21. Other host cells 1 q for expressing subtilisin include Bacillus subtilis 1168 (New Zealand Patent Specification No. 208612).

Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such ■; 5 transformed host cells are capable of either replicating vectors encoding the carbonyl hydrolase mutants or expressing the desired carbonyl hydrolase mutant. In the case of vectors which encode the pre or prepro form of the carbonyl hydrolase mutant, such .. „ mutants, when expressed, are typically secreted from the host cell into the host cell medium.

"Operably linked" when describing the relationship between two DNA regions simply means that they are , _ functionally related to each other. For example, a presequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence. A q promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

The genes encoding the naturally-occurring precursor carbonyl hydrolase may be obtained in accord with the £40 6/0 General methods described herein in New Zealand Patent Specification No. 208612.

Once the carbonyl hydrolase gene has been cloned, a 5 number of modifications are undertaken to enhance the use of the gene beyond synthesis of the naturally-occurring precursor carbonyl hydrolase. Such modifications include the production of recombinant carbonyl hydrolases as disclosed in New Zealand Patent Specification i0 No. 208612 and the production of carbonyl hydrolase mutants described herein.

The carbcr.yl hydrolase mutants of the present invention may be generated by site specific 15 mutagenesis (Smith, M. (1985) Ann, Rev. Genet. 423 ; Zoeller, M.J., et al. (1982) Nucleic Acid Res. 10, 6487-6500), cassette mutagenesis (EPO Publication No. 0130756) or random mutagenesis (Shortle, D., et al. (1S85) Genetics, 110, 539; Shortle, D., et al. (1986) - 0 Proteins; Structure, Function and Genetics, _1, 81; Shcrtle, D. (1986) J. Cell. Biochem, 3 0, 281 ; Alber, T., et al. (1985) Proc. Natl. Acad, of Sci. , 8_2 , 7 4 7; Katsumura, M., et al. (1 985) J. Biochem., 26 0 , 15298; Liao, H., et al. (1986) Proc. Natl. Acad, of Sci., 83 25 576) of the cloned precursor carbonyl hydrolase. Cassette mutagenesis and the random mutagenesis method disclosed herein are preferred.

The mutant carbonyl hydrolases expressed upon 30 transformation of suitable hosts are screened for enzymes exhibiting one or more properties which are substantially different from the properties of the precursor carbonyl hydrolases, e.g., changes in substrate specificity, oxidative stability, thermal 35 stability, alkaline stability, resistance to 2 4 0 6 proteclytic degradation, pH-activity profiles and the like.

A change in substrate specificity is defined as a - difference between the kcat/Km ratio of the precursor carbonyl hydrolase and that of the hydrolase mutant. The kcat/Km ratio is a measure of catalytic efficienty. Carbonyl hydrolase mutants with increased or diminished kcat/Km ratios are described in the examples. Generally, the objective will be to secure a mutant having a greater (numerically large) kcat/Km ratio for a given substrate, thereby enabling the use of the enzyme to more efficiently act on a target substrate. A substantial change in kcat/Km ratio is preferably at least 2-fold increase or decrease. However, smaller increases or decreases in the ratio (e.g., at least 1.5-fold) are also considered substantial. An increase in kcat/Km ratio for one substrate may be accompanied by a reduction in kcat/Km ratio for another substrate. This is a shift in substrate specificity, and mutants exhibiting such shifts have utility where the precursor hydrolase is undesirable, e.g. to prevent undesired hydrolysis of a particular substrate in an admixture of substrates. Km and kcat are measured in accord with known procedures, as described in New Zealand Patent Specification No. 208612 or as described herein.

Oxidative stability is measured either by known procedures or by the methods described hereinafter. A substantial change in oxidative stability is evidenced by at least a 50% increase or decrease (preferably decrease) in the rate of loss of enzyme activity when exposed to various oxidizing conditions. Such oxidizing conditions are exposure to the organic *4067 oxidant diperdodecanoic acid (DPDA) under the conditions described in the examples.

Alkaline stability is measured either by known procedures or by the methods described herein. A substantial change in alkaline stability is evidenced by at least a 5% or greater increase or decrease (preferably increase) in the half life of the 2 0 enzymatic activity of a mutant when compared to the precursor carbonyl hydrolase. In the case of subtilisins, alkaline stability was measured as a function of autoproteolytic degradation of subtilisin at alkaline pH, e.g. for example, 0. 1M sodium 15 phosphate, pH 12 at 25* or 30'C.

Thermal stability is measured either by known procedures or by the methods described herein. A substantial change in thermal stability is evidenced 20 by at least a 5% or greater increase or decrease (preferably increase) in the half-life of the catalytic activity of a mutant when exposed to a relatively high temperature and neutral pH as compared to the precursor carbonyl hydrolase. In the case of 25 subtilisins, thermal stability is measured by the autoproteolytic degradation of subtilisin at elevated temperatures and neutral pH, e.g., for example 2mM calcium chloride, 50mM MOPS pH 7.0 at 59*C. jq The inventors have produced mutant subtilisins containing the substitution of the amino acid residues of B. amvlol icruef aciens subtilisin shown in Table I. The wild type amino acid sequence and DNA sequence of B. amvlol icruefaciens subtilisin is shown in Fig. 1. 24 0 6 Res idue -2 4- TABLE I Replacement Amino Acid Tyr21 FA Thr 22 C Ser 24 C A s p 3 2 N Q S Ser 3 3 A T Asp 3 6 A G Gly4 6 V Ala 4 8 E V R Ser49 C L Me 15 0 c F V Asn 7 7 D Ser S 7 C Ly s 9 4 C Va 19 5 C Leu 9 6 D Tyr10 4 A C D E Ile10 7 V C-lyllO c R Metl24 I L Alal52 G S Asnl55 A D H Q Glul56 Q S Glvl66 C E I L Gly169 C D E F Lysl70 E R Tyr171 F Prol72 E Q Phe189 A C D E Aspl97 R A Metl99 I Ser204 C R L P Lys213 R T Tyr217 A C D E Ser221 A C DEFGHIKLMNPQRSTVW DEGHIKLMNPQRSTVWY DEFGHIKLMNPQRSTVW • 24 0 6 7 0 ^ -25- The different amino acids substituted are represented in Table I by the following single letter designations: Amino acid 1 i u i D or residue thereof 3-letter svmbol 1-le' SVTTL Alanine Ala A Glutamate Glu E Glutamine Gin Q Aspartate Asp D Asparagine Asn N Leucine Leu L Glycine Gly G Lysine Lys K Serine Ser S Valine Val V Arginine Arg R Threonine Thr T Proline Pro P Isoleucine He I Methionine Met M Phenylalanine Phe F Tyrosine Tyr Y Cysteine Cys C Tryptophan Trp W Histidine His H Except where otherwise indicated by context, wild-type amino acids are represented by the above three-letter symbols and replaced amino acids by the above single-letter symbols. Thus, if the methionine at residue 50 in B. amvlol icruefaciens subtilisin is. i 2 4 0 6/0 1 • replaced by phenylalanine, this mutation (mutant) may be designated Met50F or F50. Similar designations are used for multiple mutants.

In addition to the amino acids used to replace the residues disclosed in Table I, other replacements of amino acids at these residues are expected to produce mutant subtilisins having useful properties. These residues and replacement amino acids are shown in Table II.

Residue Tyr-21 Thr22 Ser24 Asp3 2 Ser33 Gly46 Ala48 Ser49 Met50 Asn77 Ser87 Lys94 Val95 Tyrl04 Metl24 Alal52 Asnl55 G1U156 Glyl66 Glyl69 Tyrl71 Prol72 Phel8 9 Tyr217 Ser221 Met2 2 2 TABLE II Replacement Amino Acidfs) L K A G L K I V D N R Q L I K A C L I T M A T M L Y K R E Q D N Each of the mutant subtilisins in Table I contain the replacement of a single residue of the B. amvlol icruefaciens amino acid sequence. These particular residues were chosen to probe the influence 2i<, of such substitutions on various properties of B. amvlol icruef acien subtilisin.

Thus, the inventors have identified Metl24 and Met222 as important residues vhich if substituted with another amino acid produce a mutant subtilisin with enhanced oxidative stability. For Metl24, Leu and lie are preferred replacement amino acids. Preferred amino acids for replacement of Met222 are disclosed in New Zealand Patent Specification No. 2C86I2.

Various other specific residues have also been identified as being important with regard to substrate specificity. These residues include Tyrl04, Alal52, Glul56, Glyl66, Glyl69, Phel89 and Tyr217 for which mutants containing the various replacement amino acids presented in Table I have already been made, as well as other residues presented below for which mutants have yet to be made.

The identification of these residues, including those yet to be mutated, is based on the inventors' high resolution crystal structure of B. amvlol icruef aciens subtilisin to 1.8 A (see Table III), their experience with in vitro mutagenesis of subtilisin and the literature on subtilisin. This work and the x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J.D., et al. (1972) Biochemistry 11. 2439-2449), product complexes (Robertus, J.D., et al. (1972) Biochemistry 11, 4293-4303), and transition state analogs (Matthews, D.A., et al (1975) J. Biol. Chem. 250. 7120-7126; Poulos, T.L., et al. (1976) J. Biol. Chem. 251. 1097-1103) , has helped in identifying an extended peptide binding cleft in subtilisin. This substrate binding cleft together with substrate is schematically 2 4 Q Q 7 diagramemed in Fig. 2, according to the nomenclature of Schechter, I., et al. (1967) Biochera Bio. Res. Commun. 27./ 157. The scissile bond in the substrate is identified by an arrow. The P and P1 designations refer to the amino acids which are positioned respectively toward the amino or carboxy terminus relative to the scissle bond. The S and S' designations refer to subsites in the substrate binding cleft of subtilisin which interact with the corresponding substrate amino acid residues.

D 2 4 6* 7 Atomic Coordinates for the Apoenzyme Form of B, Amvloliquefaciens Subtilisin to 1.8AResolution (following on pages 31-45) 1 A1A N 19 .4 34 53 . .195 -21 .756 2 a~ a c 18 .731 50 , .885 -21 .324 x ALA C3 21 , .099 51 . .518 -21 , .183 2 gln CA 17 , .219 49 . .008 -21 , .434 2 GLN O IS .7 65 47 .165 -21 .691 2 GLN CG . 02S 47 .805 -21, .927 2 GLN OKI 13 . 023 48 . 612 -22 .867 SEP. 17 .477 47 .205 -19 .852 SER c 1 6 , .735 44 . . 918 -19, .490 3 SEP.

C3 IS .588 45 . .838 -18 .069 VAL *,T 1 6 . 931 43 . 646 -19 .725 4 Vnij C IS . 129 41 . 934 -18 , .290 4 VAL CB 1 6 .008 41 . 622 -20 .822 4 VAL CG2 16 . 037 42 . .266 -22 .186 J ?F.O CA . .384 41 . .415 -16, . 027 PRO O 14 .885 39 .263 -17 . 146 PP.0 CG 13 . 841 43 . .215 -15, . 921 0 TYR N 16, . 3 63 39 . .240 -15, .487 6 TYR C .359 36 . .975 -15, .528 6 TYR CB 17 . . 824 37 . .323 -14 . .834 TYR CD 1 18 .437 . . 452 -16, .34 6 6 T'VS CE1 18 .535 34 . .070 -16 . 653 6 TYR.

CZ 18 . 222 33 . . 154 -15. . 628 7 GLY N 14 , .464 37 . .362 -14 . , 630 *7 GLY C 12 . .400 36. . 535 -15. . 670 8 \ ^vL N 12 . .441 37. .529 -16, .541 8 VAL C 12 . .363 36. .433 -18 , .735 g VAL CB 11 .765 38 . . 900 -18 , .567 0 VAL CG2 . . 991 39. . 919 -17 . .733 9 SER CA 14 . .419 . . 342 -19. .562 9 SER O 14 . .112 33. . 014 -19, .801 9 SER CG 16. .162 36. .747 -20, .358 1 0 GLN CA 13 . . 964 32. . 636 -16, .876 GLN C 12. .785 . . 642 -17 , .413 GLN CG 14 . .295 31. . 617 -14 . .588 GLN OE1 14. .554 33. .068 -12, .744 11 ILE N 11 . . 625 32 . .575 -17, . 670 11 ILE C . .209 31. ,792 -19. .605 11 ILE CB 9. . 132 32 . , 669 -17, .475 11 ILE CG2 9. , 162 32. , 655 -15. . 941 12 LYS N 11. .272 32. .185 -20. .277 12 LYS C . . 456 33. . 006 -22, .522 1 2 LYS CB 11 . . 257 . . 646 -22 . .216 12 LYS CD 12 . .543 28. .517 -22, . 159 12 LYS NZ 14 . , 476 27. , 680 -20 , .935 13 ALA CA 9. , 325 . .198 -22 , . 631 1 3 ALA O 9. . 338 . . 804 -24 , . 901 14 PRO N 11. . 332 . . 950 -23, .893 14 PRO C 11. . 786 . .557 -26, .317 14 PRO CB 13 . .462 36. 580 -24 , . 692 14 PRO CD 12 . .281 . . 936 -22 .758 ALA CA 11 . . 379 33. , 450 -27, .367 ALA O . . 008 33. .710 -29, .278 16 LEU N 9 . . 085 34 . , 138 -27, .240 16 LEU C 7 . 912 . 925 -28 . .521 16 LEU C3 6. .746 34 . . 623 -26 . 698 16 LEU CD1 . .001 33. .234 -27, .309 17 KIS N 8. , 6 65 36. 828 -27 , . 922 17 HIS C 9. .510 37. , 981 -29, .890 17 HIS CB 9. , 708 39. , 100 -27 , .652 17 HIS ND1 9. , 930 39. .887 -25, .272 17 HIS CE1 9. .226 39. . 914 -24 , .144 18 SER N . , 443 37. .033 -30, .022 18 SER C . . 159 36. .123 -32, .353 18 SER CB 12. .311 . ,799 -31, .172 19 GLN N 9. .080 . .485 -31 . 943 *4# .< !irj * 2,^y w ALA CA 19 .811 51 . 774 -21 . 965 ALA O 18 .376 51 .197 -20 . 175 GLN N 18 .268 49 .886 -22 . 041 GLN C 17 . 875 47 .706 -20 . 992 GLN C3 16 .125 48 .7 60 -22 . 449 GLN CD 13 . 912 47 .762 -22 . 930 GLN NE2 14 . 115 46 . 917 -23 . 926 SER CA 17 . 950 45 .8 68 -19 .437 SER O .590 45 .352 -19 .229 SER OG 17 . 682 46 .210 -17 .049 VAL CA . 946 42 . 619 -19 . 639 VAL O 17 . 123 41 .178 -18 .086 VAL CGI 14 . 874 40 .572 -20 .741 PRO N .239 42 . 106 -17 .331 PRO c .501 39 . 905 -16 .249 PRO C3 14 . 150 41 .880 -15 .263 PRO CD 14 . 044 42, . 986 -17. .417 TYR CA 16 . 628 37, . 803 -15, .715 TYR O .224 , . 943 -16, .235 TYR CG 18 . 021 , . 847 -15. .055 T Y?.

CD2 17 . 696 34 . 908 -14, .071 TYR CE2 17 . 815 33 .539 -14, .379 TYR OH 18 . 312 31, . 838 -15. .996 GLY CA 13 .211 36, . 640 -14. .376 GLY O 11 . 747 , .478 -15. .883 VAL CA 11 .777 37, .523 -17, . 836 VAL O 11 . 639 , .716 -19, . 470 VAL CGI 11 . 106 38, . 893 -19, .943 SER N 13 . 661 36, ,318 -18. .775 SER C 14 . 188 33. . 920 -18, .965 SER C3 . 92 6 , . 632 -19. .505 GLN N 14 .115 33. . 887 -17. .662 GLN C 12 . 687 31. . 887 -17. .277 GLN C3 14 . 125 32, . 885 -15, .410 GLN CD 14 .486 31. , 911 -13. ,147 GLN NE2 14 .552 . . 960 -12. .251 ILE CA .373 31, . 904 -18. .102 ILE O 9 .173 31. ,333 -20. .180 ILE CGI 9 .066 34. , 117 -18. .049 ILE CD1 7, .588 34. . 648 -17. 923 LYS CA 11 .388 32. .119 -21. ,722 LYS O . 173 32. .703 -23. .686 LYS CG 12, .283 29. .830 -21. .423 LYS CE 13, .023 27. ,467 -21. .166 ALA N . .109 34. .138 -21. .991 ALA C , .026 . .716 -23, ,863 ALA CB 8 .885 36, .195 -21. ,565 PRO CA 11, . 985 36, .430 -25, .120 PRO O 11, .778 36. ,047 -27. ,445 PRO CG 13. .328 36. ,978 -23. ,221 ALA N 11, .560 34, .236 -26. .129 ALA C , .082 33, .795 -28. 032 ALA C3 11, .552 31. .969 -27. ,062 LEU CA 7 , .791 34 , .558 -27. 828 LEO- O 7. .342 36. ,126 -29. 588 LEU CG , .790 33, .465 -26. ,522 LEU CD2 6, . 694 32, .287 -26. ,283 HIS CA 8. .890 38. ,151 -28 . 530 KIS O 9. .107 38. .622 -30. 856 KIS CG 9, .185 39. ,288 -26. 262 HIS CD 2 8. .008 38. .924 -25. 694 HIS NE2 8. .079 39. .328 -24 . 381 SER CA 11, ,109 36. .739 -31. ,322 SER O . ,547 36. ,112 -33. 534 SER OG 13. ,321 36, .450 -30. 399 GLN CA 8. .082 34 , ,962 -32. ,878 f N.Z. PATr-«-> f MAY f<?93 ^ i i 2 2 2 2 2 3 3 3 4 4 4 6 6 6 6 6 6 7 7 8 8 8 9 9 9 11 11 11 11 12 12 12 12 13 13 13 14 14 14 16 16 16 16 17 17 17 17 17 18 18 18 19 1 G2N C 7 142 36 .111 -33 303 1 ? GLN CB 7 221 33 .849 -32 280 i 9 GX-U CD 6 923 31 .707 -31 181 J. 9 GLM MS 2 7 362 .852 -30 256 2 j GLY CA 6 369 38 .387 -32 859 2 3 GLY 0 4 263 39 .276 -32 215 2 1 Tip.

CA 4 118 37 .831 -29 763 ^ 1 T - P. 0 422 38 .074 -27 756 2 1 T CG 2 973 .784 -30 708 2 1 TiP.

C02 3 650 34 .794 -31 397 2 1 ^ Y P.

CE2 3 193 34 .261 -32 588 2 1 T J'P.

OH 1 501 34 .241 -34 250 2 2 THP.

CA 4 262 40 .527 -27 129 2 2 THP. 0 3 287 41 .725 -25 325 2 2 THR 0G1 4 319 42 457 -28 597 G1A* N 1 939 40 .285 -26 453 2 3 GX.Y C -0 157 41 .631 -26 118 2 -j SSP, N -0 023 41 967 -27 371 «_ SEP.

C _ n 383 42 626 -27 864 SEP.

C3 -0 734 43 120 -29 520 2 5 A S N N -3 059 43 692 -27 515 2 r A. N C -5 015 42 675 -26 205 2 5 ASN C3 -5 165 43 227 -28 700 2 ~ ASN GDI -4 965 43 .767 -31 083 ^ ,* VAL N - 4 177 42 449 -25 292 VAL C -4 792 42 652 -22 987 CB -3 714 40 .503 -23 821 VAL CG2 -3 598 39 .576 -25 018 LYS CA -6 133 43 524 -21 175 2 LYS 0 -6 405 41 873 -19 413 _ " L Y 3 CG -8 0 4 6 44 .575 -22 490 ; 7 L Y S CE -10 304 45 497 -23 137 2 ?.

VAL N -4 813 43 .462 -19 200 2 a VAL C -4 758 43 959 -16 828 o « V A.L CB -2 926 42 666 -17 932 2 3 VAL CG2 -2 667 41 805 -19 173 2 9 ALA CA -5 747 44 330 -14 639 2 J .ALA 0 -4 666 42 845 -13 104 VAL N -4 057 45 . 033 -13 072 3 0 VAL C -3 958 45 409 -10 681 3 0 VAL CB -1 886 45 810 -12 149 3 0 VAL CG2 -1 053 45 236 -13 307 i ILE CA -5 328 44 846 -8 679 3 L ILE 0 -3 825 43 915 -6 997 3 1 ILE CGI -7 298 43 707 -9 798 31 ILE CD 1 -8 617 42 856 -9 717 ^ 2 ASP CA _2 94 4 46 .467 -6 255 3 2 AS? 0 -4 197 48 418 -5 502 -i "» ASP CG -0 483 45 702 -6 273 ASP OD2 -0 081 46 . 429 -5 330 3 3 SEP.

CA -1 895 49 . 857 -4 801 33 SER 0 -1 706 52 .136 -5 363 3 3 S EP OG 0 535 50 025 -4 774 3-; GLY CA _ 2 255 51 728 -8 165 34 GLY 0 -0 144 50 831 -8 761 ILE CA 0 208 52 . 438 -10 995 ILE 0 -0 327 54 638 -11 744 ILE CGI -0 530 50 210 -12 097 ILE CD1 -0 962 49 485 -13 424 36 ASP CA 2 359 55 618 -11 232 36 ASP 0 3 004 55 . 471 -13 579 36 ASP CG 4 339 57 . 099 -10 804 36 ASP OD2 448 57 .277 -10 263 37 SER CA 1 183 57 .221 -14 512 37 SER 0 2 545 58 .303 -16 151 37 SER OG -0 090 59 133 -13 879 V \ ■f 1 GLN O 6 .297 . 972 -34 .219 GLN CG 7 . 975 32 . 602 -31 .823 GLN OE1 .719 31 . 833 -31 .444 GLY N 7 .205 37 .223 -32 .587 GLY C . 181 38 .4 92 -31 .880 TYR N .202 37 . 801 -30 .761 TYR C 4 .579 38 .552 -28 .525 TYR C3 3 . 498 36 .431 -29 .443 TYR CD1 1 .795 36 . 332 -31 .258 TYR CE1 1 .306 .797 -32 .446 TYR CZ 2 .003 34 .755 -33 .067 THR N 3 . 902 39 . 680 -28 .288 THR C 3. .091 40 . 922 -26 .244 THR C3 . 133 41 .759 -27 . 611 THR CG2 6 .476 41 .323 -28 .229 GLY CA 0 . 809 40 . 600 -25 .542 GLY O -1 .013 42 . 095 -25 .330 SER CA -0, .897 42 . 957 -28 .012 SER O -2 . 813 41 .508 -28 . 160 SER OG 0 .563 43 . 652 -29 .728 ASN CA -4 , .519 43 . 687 -27, .393 ASN O -6. .233 42 . 668 -26 . 190 ASN CG -4 . .960 44 . 170 -29, .885 ASN ND2 -4 . .747 45 .461 -29, .594 VAL CA -4 . . 674 41 . 679 -24, .143 VAL O -3. .858 43 . 419 -22, . 689 VAL CGI -4 . .160 39 .802 -22, .548 LYS N -5. .910 42 . 613 -22 .301 LYS C -5. .815 42 . 872 -19, .841 LYS C3 -7. .590 43 . 981 -21, .149 LYS CD -9. .321 45 .302 -22. .020 LYS N2 -9. .686 46 .253 -24. .264 VAL CA -4 , .457 42 . 950 -17. .897 VAL O -4 , .209 45 . 095 -16. .817 VAL CGI -2 . .466 42 . 105 -16. .589 ALA N -5. .484 43 .527 -15. .813 ALA C -4 . .750 44 . 010 -13. .553 ALA CB -7 . .172 44 . 107 -14. , 101 VAL CA -3, .146 44 . 962 -11. .910 VAL O -4 , .155 46 . 648 -10. .578 VAL CGI -0. .996 45 . 901 -10. .900 ILE N -4 . .514 44 .515 -9. .877 ILE C -4 . ,346 44 . 933 -7. .54 6 ILE C3 -6. .457 43 . 776 -8. ,501 ILE CG2 -7 , .278 44 . 038 -7. ,225 ASP N -4 . .044 46 . 193 -7. ,227 ASP C -3. .071 47 . 889 -5. .705 ASP C3 -1. .695 46 . 129 -7. ,092 ASP OD1 0 . .034 44 , .592 -6. 576 SER N -1 . .931 48 .512 -5. .394 SER C -1 . .952 50 . 976 -5. ,808 SER cs -0 . , 621 49 . 922 -3. 939 GLY N -2 . .173 50 . 740 -7, ,084 GLY c -1. ,035 51, . 648 -9. ,057 ILE N -0 . ,965 52 , . 431 -10. 102 ILE C 0 . .568 53 , . 919 -11. 263 ILE CB -0 . ,042 51 . . 694 -12. 367 ILE CG2 1. 149 51 . .741 -13. 362 ASP N 1. .816 54 . .253 -10. 971 ASP C 2 . 281 55 . . 956 -12. 702 ASP CB 3 . .712 55, .720 -10. ,514 ASP OD1 3. ,755 57, . 974 -11. 429 SER N 1. ,304 56. .822 -13. 111 SER C 2. .377 58 . .095 -14 . 949 SER CB -0. ,093 58. .049 -14 . 788 SER N 3. 163 58 . . 614 -14. 001 N.Z. PA: -h MAY 1993 19 19 19 21 21 21 21 21 21 22 22 22 22 23 23 24 24 24 26 26 26 27 27 27 27 27 28 28 28 29 29 29 31 31 31 31 32 32 32 32 33 33 33 34 34 36 36 36 36 37 37 37 33 33 SER CA 4 .261 59 . 505 -14 .487 36 SER O 6, .543 59 . 251 -15 .285 36 SER CG .376 59 . 8 65 -12 .234 39 KIS CA 6 . 637 56. 574 -15 .291 39 KIS O .738 55 . 873 -17 .419 3 9 KIS CG 8 .014 54 . 609 -14 .456 39 KIS CD2 8 .769 54 . 345 -13 .389 3 9 KIS NE2 9 . 986 53 . 910 -13 .808 40 FRO CA 7 . 988 56 . 697 -18 .831 ^ 1 ^ PRO O 8 .032 55 . 097 -20 .578 * r* *i ^ ??.o CG . .053 57 . 405 -17 .902 41 AS? N 8 .481 54 . 328 -18 .485 41 AS? ODl .325 51 . 395 -20 .429 41 AS? C3 9. .799 52 . 239 -18 .224 41 AS? C 7 , .311 52 . 163 -18 .839 4 Z LEU N 6 .185 52 . 803 -18 .558 4 -< *1 k LEU C 3 . 924 52 . 907 -19 .376 LEU CB A . . 421 52 . 153 -17 .008 •1 — LEU CD1 4 . .535 51 . 546 -14 .581 4 3 LYS N 3 .018 52 . 135 -19 .946 4 3 LYS C 0 . 637 52 . 156 -20 .018 4 3 LYS CB 2 . 021 52 . 389 -22 .169 4 3 LYS CD 0 . .998 52 . 8 62 -24 .339 4 j- LYS NZ 0 , .337 51 . 757 -26 .418 1 « VAL CA _ 1 .407 52 . 639 -18 .765 VAL 0 . 623 53 . 906 -20 .434 VAIJ CGI -2 . . 724 52 . 941 -16 .582 4 5 ALA N -3, .494 51. 951 -19 . 871 4: ALA r -5 .841 52 . 507 -20 .053 H J ALA CB -4 .831 50 . 580 -21 .389 H t GLY CA -7 , .082 52 . 837 -18 .001 4 6 GLY O -5 , . 938 52 . 006 -16 .035 4 ' GLY CA -8 , .014 52 . 246 -14 .388 .4 GLY O -9. .988 53 . 481 -14 .185 4 8 ALA CA -10 . .255 52 . 870 -11 .382 4 "J ALA O -9 , .066 51 . 720 -9 .725 49 SEP.

N -10. .149 53 . 547 -9 .037 *1 .* SER C -10 . . 947 52 . 986 -6 .783 49 SER C3 -9. .092 54 . 588 -7 .029 0 MET N -10 , .835 52 . 007 -5 .932 50 MET C -11. .463 51 . 962 -3 .561 50 CB -12 . .012 50 . 018 -4 . 996 c, T SD -13, .460 49 . 889 -7 .256 51 VAL N -10 , .427 52 . 760 -3 .422 51 VAL C -10. . 630 54 . 562 -1 . 907 -1 VAL CB -8 , .443 53 . 155 -2 .000 51 VAL CG2 -7 , .764 51 . 815 -2 .302 52 PRO CA -12 . .372 55 . 933 -0 . 821 52 PRO O -11 . .771 58 . 220 -0 .925 52 PRO CG -13 , .583 54 . 103 0 .085 53 SEP.

N -10, .442 56 . 906 0 .299 53 SER C -8 , .420 58 . 245 -0 .326 53 SER CB -9, .004 57 . 707 2 .069 54 GLU U -8 . .254 57 . 523 -1 .393 54 GLU c -7 , .767 57. 303 -3 .785 54 GLU CB -6. .134 56 . 599 -2 .154 54 GLU CD -4 . .066 56 . 042 -0 . 920 54 GLU OE2 -3. . 900 55 . 777 0 .271 55 TKR CA -9, .433 58 . 121 -5 .441 55 THR 0 -9. .433 57 . 919 -7 . 810 55 THR 0G1 -9, .885 60 . 510 -5 .418 56 ASN N -7. .482 58 . 403 -6 .877 56 ASN ODl -5. .075 58 . 967 -10 .337 56 ASN CB -5. .898 59. 694 -8 .208 56 ASN C -6. .012 57. 094 -8 .305 57 PRO N -6. .362 56. 261 -9 .258 . .. ;• 'v b ; t SER C .466 58 .705 -14 . 992 SER CB 4 .742 60 .435 -13 . 398 HIS N .454 57 .390 -14 . 892 HIS C 6 . 681 56 .401 -16 .778 HIS CB 6 . 637 55 .203 -14 .515 HIS ND1 8 .795 54 .356 -15 .561 KIS CE1 9 .970 53 .930 -15 .130 PRO N 7 .807 56 .836 -17 .387 PRO C 8 . 156 55 .280 -19 .357 PRO CB 9 .247 57 .533 -19 . 161 PRO CD 8 . 988 57 .452 -16 .776 AS? OD2 11 . 148 50 .399 -18 . 668 ASP CG .473 51 .307 -19 .211 ASP CA 8 . 645 52 .959 -18, . 966 ASP O 7 .396 50 .947 -18, . 977 LEU CA 4 . 892 52 .147 -18, .466 LEU O 3 . 993 54 .163 -19, . 490 LEU CG . 182 51 .363 -15. . 946 LEU CD 2 . 273 49 .877 -16. ,350 LYS CA 1 .893 52 .685 -20, .721 LYS O 0 . 504 50 . 920 -19, . 820 LYS CG 0 . 685 52 .436 -22, . 910 LYS CE -0 . 180 52 .584 -25. ,260 VAL N -0 . 191 53 .035 -19. .490 VAL C -2 .571 52 .887 -19. .731 VAL CB -1 . 480 53 .351 -17, .383 VAL CG2 -0 . 197 53 .194 -16. .553 ALA CA -4 . 619 51 . 977 -20. .810 ALA O -6 .703 53 .085 -20. .703 GLY N -5 . 910 52 .356 -18. .768 GLY C -6 . 987 52, .443 -16. .538 GLY N -8 . 092 52 .658 -15. ,793 GLY C -9 . 179 52, .757 -13. ,572 ALA N -9 .221 52 .446 -12. ,330 ALA C -9 .790 52, .675 -9. .968 ALA CB -11, .558 52. .100 -11. .617 SER CA -9, .752 53. .355 -7. , 652 SER O -11 . 972 53 .677 -6. .908 SER OG -8 . 879 54 , .255 -5. .650 MET CA -11. . 852 51. .549 -4 . . 974 MET O -11, . 997 51. .398 -2. ,575 MET CG -11 . 912 49. .463 -6. .389 MET CE -12, . 808 50, .111 -8. .903 VAL CA -9, . 968 53. . 170 -2. .067 VAL O -10, .237 55. .437 -2. 682 VAL CGI -7, .892 53. .579 -0. ,631 PRO N -11, . 621 54 . . 693 -1. 056 PRO C -11, .490 57. .123 -0. ,440 PRO CB -13, .400 55. .594 0. 244 PRO CD -12 .164 53. . 620 -0. ,175 SER CA -9 .538 57. . 982 0. , 682 SER O -7, . 679 59. ,224 -0. ,038 SER OG -8 .256 56. ,521 2 . 127 GLU CA -7, .204 57. . 648 -2 . 421 GLU O -7, .533 56, .243 -4 . ,379 GLU CG -5, .289 56. . 959 -0. ,927 GLU OE1 -3, .545 55. , 694 -1. 968 THR N -8, .571 58. .251 -4 . 249 THR C -8, .764 58. .139 -6. ,779 THR CB -10, ,586 59. .200 -5. 303 THR CG2 -11. .432 59. , 143 -4 . 017 ASN ND2 -4 . . 930 61. ,179 -9. 881 ASN CG -5. ,273 59. 925 -9. 555 ASN CA -6. .762 58. ,425 -8. 200 ASN O -5. . 104 56. ,866 -7 . 470 PRO CG -7. ,123 55. 257 -11. 177 N.Z. PAT^f'< 'c -A MAY 1993 HEoLi /fID 38 38 39 39 39 39 39 40 40 40 40 41 41 41 41 42 42 42 42 43 43 43 43 44 44 44 44 45 45 46 46 47 47 48 48 48 49 49 49 50 50 50 50 51 51 51 52 52 52 52 53 53 53 54 54 54 54 55 55 55 55 56 56 56 56 57 c ~ Fr.o CD _ " 384 56 433 -10 . c, 7 FP.O CA -5 679 54 . 961 -9. c *"• FRO 0 - 3 509 54 . 128 -9. c c F HE CA -2 747 56 577 -11. 58 pke C -0 635 57 .497 -10. r.

F HE CG - 3 983 56 . 968 -13 . e c F HE CD2 -5 211 57 . 630 -13. 3 FHE CE2 -6 194 57 .095 -14 . 9 GLN >; -2 04 4 57 . 119 -8 . 9 ' v C -0 807 56 .403 -7 . 9 gln CB -1 862 58 .668 -7 . c z gln CD -1 790 60 . 157 -5. 9 gln NE2 _ o 959 59 . 685 -4 . € 0 a3 p CA 0 851 54 .792 -6 . 6 0 ASF C 2 827 55 .550 -5. 6 0 ASP CG 2 077 52 .538 -6. €c CD 2 2 915 51 .841 -7 . €1 ASM ND2 - x 364 57 .747 -2 . 6 z ASN CG -0 040 57 . 670 -2 . 6 1 ajm CA 1 557 55 .734 -2. fcl ASH 0 2 933 54 .862 -0 . l asm CA 2 877 52 .348 -1. 62 asn 0 4 951 51 .313 -1. 62 asm CG 2 371 50 .103 -0 . 62 ASM ND2 2 622 50 .208 0. 6 3 SEP.

CA 189 51 . 696 -4 . 63 SER 0 593 49 .790 -6. 63 SEP.

CG 6 871 50 . 698 -3. 6 •; H13 CA 3 994 48 . 059 -4 .

HIS 0 3 861 4 6 974 -7 . 6 ♦« his CG 3 144 46 .021 -3.

•» HIS CD 2 4 054 45 .194 -3 .

HIS MS 2 3 556 43 .920 -3. 6 5 GL: CA 1 552 48 .264 / . 65 GLY 0 2 230 48 .078 -10 . 6 6 THP.

CA 4 064 50 .117 -9. 6 6 THP. 0 333 48 .789 -11. 6-5 THR 0G1 3 637 52 . 425 -9. a HIS n 685 48 .443 -9 . 6 7 HIS C 6 091 46 .141 -10 . 67 HIS CB 7 300 47 .071 -8 . 6 7 HIS mi) 1 8 590 44 . 907 -8 .

HIS CS1 9 857 44 .491 -8 . 68 V AL m 4 892 45 .749 -9. 68 VAL C 3 856 44 .860 -11. 6C VAL CB 2 939 44 .252 -9. t: q VAL CG2 3 319 43 .705 -8 . 6 9 ALA CA 3 037 46 .468 -13.

C ALA 0 4 028 45 . 913 -15 . 7 0 gly m 340 46 .782 -13. 7 0 gly C 7 046 45 .370 -15 . thp. m 6 820 44 .431 -14 . 7; 7 hp.

C 6 224 42 .506 -15. 7 i THP. cb 7 119 42 . 070 -13 .

THR cg2 7 274 40 .583 -13.

VAI< CA 3 976 42 .491 -16 . 7 VA". 0 4 341 42 .380 -18 . 7 3 VAI, cgi 1 512 42 .480 -17 . 7 3 a;.a t; 4 504 44 417 -17 . 7 3 ALA c 433 46 333 -19. 7 3 ALA cb 3 107 45 441 -19. 7 *» ALA CA 7 470 47 .591 -18. 7 m ALA 0 7 959 46 . 640 -21. 7 5 LEU N 7 650 48 .784 -21. 7 ^ LEU C 9 192 48 .568 -22. 7 u LEU cb 7 548 50 471 -22 . <? M' • J*:- 'ii j, V 57 PRO CB -6 . 644 54 . 178 -10 . 235 57 PRO C -4 . 301 55 . 082 -9 . 966 58 PKE N -3 . 998 56 .262 -10 . 491 58 PKE C -1 .712 57 . 129 -10 .253 58 PKE C3 -2 . 943 57 .502 -12 . 423 58 PKE CD1 -3 .756 55 .780 -14 . 059 58 PKE CE1 -4 . 722 55 .255 -14 . 928 58 FKE CZ -5 . 949 55 . 939 -15 . 051 59 GLN CA -1 . 172 57 .583 -7 . 934 59 GLN 0 -1 . 639 56 .083 -6 . 115 59 GLN CG -0 . 942 59 .261 -6 .034 59 GLN 0E1 -1 .404 61 .288 -4 .836 60 ASP N 0, .410 55 .895 -7 .211 60 ASP C 1 . 631 55 .267 -5 .090 60 AS? CB 1 .596 53 .744 -7 .188 60 ASP ODl 1 . 746 52 .337 -5 .190 61 ASN N 0 . 959 55 .265 -3 . 950 61 ASN ODl 0 . 666 58 .566 -2 .875 61 ASN C3 0 .531 56 . 401 -1 .784 61 ASN C 2, .291 54 . 632 -1 . 940 62 ASN N 2, .210 53 .434 -2 .468 62 ASN C 4 .124 51 .893 -2 .479 62 ASN CB 1 , .783 51 .319 -1 .421 62 ASN ODl 2 .633 49 . 077 -1 .343 63 SER N 4 .152 52 .104 -3 .761 63 SER C .071 50 .256 -5 .209 63 SER CB 6 .523 51 . 958 -4 .012 64 HIS N 4 .202 49 .475 -4 .639 64 HIS C 3 . .366 47 .759 -6 .261 64 HIS CB 3, .184 47 .501 -3 .747 64 HIS ND1 2 .107 45 .247 -4 .241 64 HIS CE1 2 .416 43 . 966 -4 .054 65 GLY N 2 .287 48 .428 -6 .587 65 GLY C 2 .392 48 . 636 -9 .037 66 THR N 3 .233 49 .659 -8 .832 66 THR C . .089 49 .009 -10 .291 66 THR CB 4 . .744 51 .511 -9 .667 66 THR CG2 , .536 52 .078 -10 .849 67 HIS CA 6 . .703 47 , 361 -9 .458 67 HIS 0 6, .649 45 . 638 -11 .150 67 HIS CG 8 . .595 46 .275 -8 .148 67 HIS CD2 9 .904 46 . 678 -8 .076 67 HIS NE2 . 678 45 .514 -8 .186 68 VAL CA 4 .142 44 . 607 -10 .266 68 VAL O 4 .114 43 . 942 -12 .535 68 VAL CGI 1 , , 960 43 .260 -10 .020 69 ALA N 3. ,373 46 .049 -12 .113 69 ALA C 4 , ,193 46 .390 -14 .411 69 ALA CB 2 , ,332 47 . 851 -13 .386 70 GLY CA 6. ,595 46 .805 -14 .670 70 GLY O 7 , , 604 45 .154 -16 .119 71 THR.

CA 7 . .177 43 .019 -14 .446 71 THR O 6 , , 602 41 . 828 -16 .495 71 THP.

OG1 8 , ,191 42 .592 -12 .390 72 VAL N 4 , , 930 42 .887 -15 .427 72 VAL C 4 . .312 43 . 084 -17 .831 72 VAL CB 2. ,516 42 . 867 -16 .085 72 VAL CG2 2. .142 42 .327 -14 .723 73 ALA CA 4 . ,587 45 .091 -19 .167 73 ALA 0 . .062 47 . 188 -20 .216 74 ALA N 6. ,544 46, . 429 -18 .635 74 ALA C 7 . .740 47 .648 -20 .342 74 ALA CB 8 , ,653 47 . 446 -17 .925 75 LEU CA 7 . .812 48, . 968 -22 .456 75 LEU O . ,162 48, .750 -22 ,253 75 LEU CG 6. ,123 50, . 913 -22 .379 4 MAY 1993 272 332 945 222 680 357 459 276 990 000 089 150 742 304 231 380 030 347 399 700 902 709 770 697 601 709 269 418 935 108 726 135 368 830 134 954 461 406 274 143 064 276 299 731 740 386 000 429 565 914 021 138 543 191 596 484 860 170 880 355 433 859 054 039 966 809 / c 7 c 7 c 7 3 73 79 79 79 73 eo so s: e i 82 82 82 6 2 6 3 S3 a-; 8-4 64 85 8 5 65 8 6 8 6 8 5 8 ~i 87 87 88 88 e« 89 89 8 9 90 90 90 90 91 91 91 91 91 91 92 92 93 93 93 93 94 9 4 9 4 94 let; cd 1 6. 075 52 . 4 3 6 -22 . 300 75 leu cd2 . 096 50 .442 -23. 405 asn* 9. 147 48 . 103 -24 . 169 76 asn nd2 12 . 335 46 .432 -26. 304 asn odl . 550 45 . 840 -27 . 928 76 ash cg 11 . 195 46 .274 -26. 802 asn cs . 010 4 6 . 651 -25. 908 76 asn ca . 359 47 .738 -24 . 938 asn c . 783 49. 048 -25. 643 76 asn o . 157 49 .479 -26. 619 asn n 11 . 804 49 . 654 -25 . 071 77 asn ca 12 . 220 50 . 957 -25. 681 asn r~ 13 . 707 51 . ,029 -25 . 348 77 asn o 14 . 364 49 .979 -25. 313 * tv nC •« c3 11 . 335 52 . , 076 -25. 117 77 asn cg 11. 250 52 .027 -23. 616 a^n ODl 12 . 032 51 . ,346 -22 . 917 77 asn nd2 . 294 52 .741 -23. 025 SER N 14 . 125 52 . ,267 -25. 164 78 sep. ca . 513 52 . 614 -24. 906 SER c . 810 52 . ,742 -23. 4 3 6 78 ser o 16. 982 53 .071 -23. 164 SER c3 . 905 53 . , 941 -25. 587 73 ser og . 926 53 .870 -26. 999 ILE N 14 . 85 8 52 . ,565 -22 . 529 79 ile ca . 155 52 .784 -21. 120 ILE c 14 . 617 51 . . 633 -20 . 230 79 ile o 13. 843 50 .841 -20. 67 9 ILE cb 14 . 471 54 . , 174 -20. 697 79 ile cgi 12. 945 54 .032 -20. 814 ILE cg2 14 . 997 55 . ,320 -21. . 612 79 ile cd1 12. 135 55 .176 -20. 155 CjL "i N 14 . 995 51 . ,768 -18 . 981 80 gly ca 14 . 476 50 . 940 -17. 913 GLY C 14 . 612 49 . ,443 -18 . 219 80 gly o . 719 48 . 994 -18. 544 V ■ n 13. 513 48 . ,766 -17 . 980 81 val ca 13. 411 47 .286 -18. 061 val C 12 . 511 46 . , 919 -19. 217 81 val o 12 . 260 47 .739 -20. 117 VAL CE 13 . 001 46 . ,755 -16 . 677 81 val cgi 14 . 030 47 .084 -15. 573 VAL cg2 11 . 638 47 . .261 -16 . 231 82 leu n 12. 126 45 .645 -19. 216 LEU CA 11 . 312 45 . . 020 -20 . .256 82 leu c . 390 44 .028 -19. 510 LEU 0 . 858 43 . .356 -18 . . 600 82 leu cb 12 . 206 44 .219 -21. 229 LEU CG 11 . 430 43 . .563 -22. .366 82 leu cd1 . 796 44 .657 -23. 223 LEU CD 2 12 . 359 42 , . 675 -23. . 192 83 gly n 9. 131 44 .180 -19. 816 GLY ca 8 . .133 43 . .321 -19. , 114 83 gly c 8 . 027 42 .011 -19. 925 GLY 0 8 . . 546 41 . . 822 -21. , 026 84 val n 7. 272 41 .112 -19. 283 val ca 6 . . 973 39 . .807 -19. , 888 84 val c 6. 164 40 .030 -21. 140 v al 0 6 . , 424 39 , . 472 -22 . ,194 84 val cb 6. 256 38 .920 -18. 841 val CGI . . 680 37 , . 677 -19. .557 84 val cg2 7 . 190 38 .507 -17. 705 n . . 156 40 , . 926 -21. . 024 85 ala ca 4 . 217 41 .194 -22. 158 ala c 4 . ,213 42 , . 683 -22 . , 396 85 ala o 3. 260 43 .401 -22. 030 ala cb 2 . , 846 40 , . 663 -21. .748 86 pro n . 240 43 .186 -23. 059 PF.O ca . ,413 44 , . 635 -23. .205 86 pro c 4 . 321 45 .371 -23. 947 PRO 0 4 . .291 46 , . 605 -23. .849 86 pro cb 6. 822 44 .784 -23. 813 PRO CG 7 . , 030 43 , .468 -24 , .546 86 pro cd 6. 377 42 .440 -23. 636 SF.R N 3. , 548 4 4 , . 676 -24 , .769 87 ser ca 2. 489 45 .324 -25. 529 SER c 1 . . 103 45, . 132 -24 , .897 87 ser o 0. 162 45 .513 -25. 619 SER cb 2 . . 401 44 .777 -26. . 927 87 ser og 3. 591 45 .143 -27. 583 ala t; 1 , .017 44 .564 -23, .742 88 ala cb -0. 163 43 .510 -21. 828 KZJi ca -0 . , 273 44 .353 -23, . 084 88 ala c -0. 898 45 .717 -22. 690 ,\LA 0 -0 , .174 46 .717 -22 , .435 89 ser n -2. 219 45 .691 -22. 678 SER OG -4 . . 146 47 . 102 -24 , .280 89 ser cb -4. 343 46 .903 -22. 898 SER ca -3. . 001 46 . 867 -22, .227 89 ser c -3. 136 46 .780 -20. 727 SER. 0 - 3 , .793 45 .864 -20 .209 90 leu n -2. 446 47 .656 -20. 037 LEU ca -2 , . 378 47 . 667 -18 .593 90 leu c -3. 483 48 .430 -17. 864 LEU 0 -3, . 582 49 . 604 -18, .215 90 leu cb -0. 951 48 .273 -18. 426 LEU CG -0 , . 233 47 . 851 -17 . 174 90 leu cd1 -0. 028 46 .341 -17. 219 LEU cd2 1 . .160 49 . 524 -17 , . 047 91 tyr n -4 . 264 47 . 944 -16. 938 TYR CA -5. . 258 48 . 678 -16 . 137 91 tyr c -4 . 873 48 .750 -14 . 685 TYR 0 ~ *i . .496 47 . 749 -14 .023 91 tyr. cb -6. 686 48 .093 -16. 314 TYR CG -7 . .094 43 .237 -17 . 741 91 tyr cd1 -6. 595 47 .415 -18. 755 TYR cd 2 -7 . 971 49 .275 -18 . 149 91 tyr ce1 -6. 905 47 .572 -20. 090 TYR CE2 -8 . 315 49 .421 -19 .492 91 tyr cz -7. 794 48 .582 -20. 463 T Y R CH -8 .182 43 .752 -21 .764 92 ala n -4 . 895 49 . 958 -14. 104 ala CA - 4 . 549 50 . 199 -12 .707 92 ala c -5. 823 50 .033 -11. 903 ALA 0 -6 . 723 50 . 898 -12 .050 92 ala cb -3. 997 51 .621 -12. 488 V .* vi j N -5 . 959 48 . 993 -11 . 129 93 val ca -7 . 183 48 .854 -10. 325 val C -6 . 708 49 . 014 -8 .899 93 val o -6. 181 47 .993 -8. 372 val cb -7 . 957 47 .555 -10 . 611 93 val cgi -9. 213 47 .488 -9. 725 val cg2 -8 .195 47 .370 -12 .072 94 lys n -6. 907 50 .217 -8. 327 LYS ca -6 . 378 50 .464 -6 . 999 94 lys c -7. 331 49 .985 -5. 894 LYS 0 -8 . 458 50 .480 -5 .783 94 lys cb -6. 051 51 .976 -6. 818 LYS CG -5 .394 52 .320 -5 .467 94 lys cd -4 . 868 53 .785 -5. 582 LYS ce -4 .399 54 .208 -4 .199 94 lys nz -3. 735 55 .544 -4 . 387 5 95 55 5 6 c 5 95 9 5 97 " 95 95 9 9 9 3 9 j 95 oc cc 01 0 1 01 02 02 03 03 03 03 0 3 0-4 04 o-; 04 0 4 0 4 05 05 05 06 06 06 06 06 0 6 0 6 07 07 07 07 oe 09 08 08 0 9 09 09 09 11 11 11 11 12 12 12 12 VAL N - 5 , . 909 49 . 071 -5, .026 VAL C -6. . 919 48 .499 -2 . .568 VAL C3 -8 . .104 47 .030 -4 . .319 VAL.

CG2 -6. . 900 4 6 . 100 -4 , .332 LEU C A -t , .782 49 . 103 -1 , .486 LEU 0 -3 , . 942 51 . 121 -2, .336 LEU CG -3 , .593 46 .799 -2 , .072 LEU CD2 _ A 1 . . 489 46 .082 -1 .045 GLY CA -3. .890 52 . 307 0 .287 GLY 0 -1 . 619 51 .463 0, .165 ALA C3 -0 . 428 55 . 478 1 .510 ALA C 0 .188 53 . 118 1 .917 ASP N -0 . .504 52 .573 2 .912 AS? ODl -2 . .730 50 . 902 4 .003 AS P CB -0 . . 648 51 . 603 .175 AS ? C 0 , . 146 50 .165 3 .320 GLY r; -0. .424 49 . 883 2, .163 GLY c -1. .520 47 . 651 2 , .002 SEP.

N -2 . . 342 48 . 128 2 , .908 SER C -4 . ,759 47 .894 2 , .532 SER CB -3 . ,716 47 .447 4 , .817 GLY N -5 . , 821 47 .092 2. .577 GLY C -8 . ,166 46 .536 2 , .528 GLN N -9. , 377 47 . 058 2 , .498 GLN C -10 . , 963 45 .232 2 , .022 GLN C3 -11 . , 671 47 .307 3, .274 GLN CD -12 . .360 49 . 104 4 , .915 GLN NE2 -13. .419 49 .197 4 , .112 TYR.

CA -12 . .068 43 . 126 1, .508 TYR 0 -12 . . 939 43 .276 -0, .687 TYR.

CG -11 , . 629 40 . 829 2 .472 TYR.

CD2 -10 . .379 40 . 959 1, .860 TYR CE2 -9. .352 40 .057 2 . 171 TYR OH -8 . .481 38 .191 3, .324 SER CA -14 . . 877 45 .166 -0 .034 SER O -14 . .759 45 .935 -2 .258 SER OG -15 , .209 47 .039 1 .450 TRP CA -12 . . 421 47 .391 -1 . 948 TRF O -12 . . 021 46 . 648 -4 .245 TRP CG -11 . . 645 49 .111 -0 .206 TRP CD2 -10 , . 658 49 .812 0 .581 TRP CE2 -11 , . 359 50 .573 1 .561 TRP' CZ2 -10 . . 671 51 .318 2 .500 TRP CH2 -9. .293 51 .291 2 .455 ILE CA -10 .765 44 .250 -3 .325 ILE O -11 . 695 43 .474 -5 .398 ILE CGI -8 . 634 43 .784 -1 .936 ILE CD1 -8 . .283 42 .998 -0 . 627 ILE CA -14 , .116 42 .722 -4 .321 ILE O -14 . ,894 43 .329 -6 .552 ILE CGI -14 . ,726 41 . 077 _o .482 ILE CD1 -15 . . 452 40 .845 -1 . 131 ASN CA -15 . .204 46 .018 -5 . 916 ASN O -14 . . 660 46 .272 -8 .235 ASN CG -16 , .528 47 .486 -4 .353 ASN ND2 -16 . . 633 48 . 447 -3 .442 GLY CA -11 , . 952 45 . 917 -7 .865 GLY O -11 , . 929 44 .929 -10 .034 ILE CA -12 . . 603 42 .334 -9 .099 ILE 0 -13. . 921 42 .384 -11 .148 ILE CGI -11. .421 40 .501 -7 . 655 ILE CD1 -11, .588 39 .706 -6 .336 GLU CA -16, . 118 43 .376 -10 .046 GLU O -16, .467 44 .130 -12 .246 GLU CG -17 . 847 42 .917 -8 .135 GLU OE1 -19, . 041 40 .866 -8 .016 ^ 7 ft VAL CA -7 . 646 48 . 457 -3 . 920 VAL O -7 . 425 48 . 156 -1 .501 VAL CGI -8 .868 46. 852 -5 . 619 LEU N -5 . 676 48 . 974 -2 . 604 LEU C -4 .331 50. 559 -1 . 321 LEU CB -3 .509 48 . 241 -1 .573 LEU CD1 -2 .207 46. 184 -2 . 163 GLY N -4 .326 50. 975 -0 .086 GLY C -2 . 363 52. 437 0 .385 ALA N -1 . 954 53. 648 0 .758 ALA CA -0 .563 54 . 068 0 . 965 ALA O 1 .393 52. 921 1 . 663 ASP OD2 -2 . 631 51. 042 6 . 151 ASP CG -2 . 083 51. 131 . 040 AS? CA 0 . 101 51. 610 3 . 855 AS? O 0 .735 49. 313 4 .029 GLY CA -0, .343 48. 521 1 . 615 GLY O -1, . 649 46. 512 1 .479 SER CA -3, .542 47 . 388 3 .315 SER O -4 . . 758 48. 972 1 . 907 SER OG -4 . . 411 48 . 634 .209 GLY CA -7 . . 077 47. 422 1 . 896 GLY O -7 . . 888 45. 431 3 .030 GLN CA -10 . .535 46. 297 3 . 020 GLN O -10 . .779 45. 482 0 . 817 GLN CG -11. .368 48 . 005 4 .586 GLN OE1 -12 . . 159 49. 816 . 902 TYR N -11. . 611 44 . 141 2 .451 TYR C -13. .031 43. 690 0 . 473 TYR CB -12. . 697 41. 866 2 . 143 TYR CD1 -11. .819 39. 789 3 .377 TYR CE1 -10 . .805 38. 885 3 .707 TYR CZ -9. .564 39. 022 3 . 081 SER N -13. . 909 44 . 572 0 . 903 SER C -14 . .172 45. 920 -1 .159 SER CB -15. . 880 46. 121 0 . 601 TRP N -13. .079 46. 625 -0 . 834 TRP C -11. . 895 46. 436 -3 . 012 TRP CB -11. .321 48. 254 -1 .355 TRP CD1 -12 . .862 49. 524 0 .264 TRP NE1 -12. . 691 50. 358 1 .360 TRP CE3 -9. .275 49. 852 0 .576 TRP CZ3 -8. .568 50. 563 1 .525 ILE N -11. . 339 45. 330 -2 .481 ILE C -11. . 855 43. 594 -4 .190 ILE CB -9. . 944 43. 183 -2 .523 ILE CG2 -9. . 632 41. 930 -3 .381 ILE N -12. . 994 43. 292 -3 .577 ILE C -14 . . 639 43. 694 -5 .386 ILE CB -15 . ,246 42 . 265 -3, . 320 ILE CG2 -16 . ,568 42. 024 -4 , .095 ASN N -14 . ,751 44 . 958 -4 , . 981 ASN C -14 . ,232 46. 067 -7 . 084 ASN CB -15. ,280 47 . 359 -5, .207 ASN ODl -17 . ,455 46. 695 -4 , . 646 GLY N -12 . , 951 45. 908 -6, .774 GLY C -12 . , 108 44 . 712 -8, .812 ILE N -12 . , 379 43. 539 -8, .246 ILE C -13. ,859 42. 560 -9, . 942 ILE CB -12 . 734 40. 948 -8, .364 ILE CG2 -13. , 122 39. 791 -9, .347 GLU N -14 . ,893 43. 075 -9, .280 GLU C -15. 872 44 . 347 -11, .171 GLU CB -17 . 229 43. 899 -9, .141 GLU CD -18. ,724 41. 824 -8, . 685 GLU OE2 -19. ,123 41. 928 -9, .866 N.Z. PATF' '-" - — -4 MAY 1993 t • 6- 95 95 95 96 96 96 96 97 97 98 98 98 99 99 99 99 100 100 101 101 101 102 102 103 103 103 103 104 104 104 104 104 104 105 105 105 106 106 106 106 106 106 106 107 107 107 107 108 108 108 108 109 109 109 109 110 110 111 111 111 111 112 112 112 112 112 113 TF.P N -15 .094 45 .403 -10 . 971 113 TP.? C -14 .076 45 . 663 -13 . 140 113 T?J? C3 -13 .882 47 .553 -11 .434 113 TP.? CDl -14 . 143 49 .736 -12 . 681 113 TCP NE1 -13 .597 50 .443 -13 .723 113 TP.? CE3 -11. .451 47 . 645 -13 .809 11 j TPP CZ3 -10 . 610 47 .899 -14 . 879 114 ALA N -13 , .089 44 . 801 -12 .832 114 ALA C -13 .199 43 . 179 -14 .752 114 ALA CE -11 .299 43 .192 -13 .140 115 ILE CA -15 . 070 41 . 640 -14 .897 115 ILE 0 -16 .077 42 .225 -17 .070 115 ILE CGI -15 .218 39 . 836 -13 .043 115 ILE CDl -16, .004 39 .411 -11 .743 116 ALA CA -17 .390 44 .440 -16 .050 116 AiiA 0 -17 , .323 45 .255 -18 .343 117 ASM V" -15, . 423 45 . 390 -17 .122 117 ASM C -13 . 827 44 . 974 -19 .034 1 ' T ASN CB -13 . 615 4 6 . 958 -17 .426 117 ASN ODl -14 .565 49 . 082 -17 .773 113 ASN N -14 , .223 43 .725 -18 .967 113 ASN C -12 .240 42 .444 -19 .843 113 ASN C3 -14 , .247 42 .863 -21 .279 IIS ASM ODl -16 , .510 42 . 321 -20 .759 119 MET ;; -11 .636 42 .500 -IS .675 119 MET c -10 , .025 40 . 734 -18 . 928 119 MET CB -9 .810 42 .461 -17 .055 119 MET SD -8 .788 44 . 943 -17 .526 120 AS? N -S . 904 40 . 437 -19 .584 120 ASP C -7 , .822 38 .390 -18 .856 120 AS? C5 -7 , .555 39 . 156 -21 .236 120 ASP ODl -7 , .801 40 .706 -23 .084 121 VAL N -7 .021 39 . 117 -16 .115 121 VAL C -6 .296 39 .534 -15 .786 121 VAL CB -4 .755 38 .587 -17 .496 121 VAL CG2 -4 .707 37 . 916 -18 .846 122 ILE CA -6, .248 39 .799 -13 .397 122 ILE O -4 , .829 38 . 012 -12 .469 122 ILE CGI -8 , . 686 40 .392 -13 .063 122 ILE CDl -9 , .976 39 .788 -12 .383 123 ASN CA -3 , .145 39 .854 -11 .232 123 ASN O -3 .708 41 . 631 -9 .833 123 ASN CG -0 . 692 40 .048 -10 .777 123 ASN ND2 -0 , .346 40 . 747 -9 .720 124 MET CA -3, .650 39 . 973 -7 .438 124 MET O -2 , .306 38 .508 -6 .090 124 MET CG -6 , .158 40 . 082 -7 .473 124 MET CE -7 , .940 38 .095 -7 .542 125 SER CA -0 , .193 40 .287 -5 .769 125 SER O 0 .235 41 . 617 -3 .805 125 SER OG 1, .444 40 .496 -7 .575 126 LEU CA -1 , .842 40 . 347 -2 .386 126 LEU O -2 . 844 38 . 136 -2 .529 126 LEU CG -3 . 988 41 .447 -3 .333 126 LEU CD2 -4 , .179 42 .760 -4 .073 127 GLY CA -3 , .035 37 . 871 0 .193 127 GLY O -2 , .446 39 . 030 2 .220 123 GLY CA -4 .475 37 .496 3 . 642 128 GLY O -4 .903 . 158 3 .276 129 PRO CA -4 , . 671 34 . 525 .998 129 PRO O -6 .338 32 . 887 6 .305 129 PRO CG -4 , .419 36 . 116 7 .727 130 SER N -7, .051 .015 .912 130 SER C -9, .218 34 .884 4 .726 130 SER CB -9, .069 .351 7 .216 131 GLY N -10 .083 33 .967 4 .349 / ■>' i'i i U TRP CA -14 .756 46 . 400 -12 .000 TRP O -14 .319 45 . 932 -14 .332 TRP CG -13 .486 48 .556 -12 .481 TRP CD 2 -12 .441 48 .552 -13 .463 TRP CS2 -12 .545 49 .761 -14 .215 TRP CZ2 -11 .696 50 .045 -15 .274 TRP CK2 -10 .752 49 . 074 -15 . 603 ALA CA -12 .333 44 .065 -13 .874 ALA O -12 .963 43 .074 -15 .978 ILE N -14 .174 42 .540 -14 .119 ILE C -15 .928 42 . 485 -15 .856 ILE C3 -16 .000 40 . 840 -13 .922 ILE CG2 -17 .151 40 .163 -14 .755 ALA N -16 .534 43 .527 -15 .267 ALA C -16 .706 45 .069 -17 .278 ALA C3 -18 .011 45, .510 -15 .151 ASN CA -14 .553 45 . 967 -18 .139 ASN O -12 .997 45 .436 -19 .820 ASN CG -14 .400 48, .177 -16 .939 ASN ND2 -14 .931 48, .249 -15 .736 ASN CA -13 .760 42, . 642 -19 .832 ASN O -11 .617 42, .309 -20 .932 ASN CG -15, .737 43, . 060 -21 .395 ASN ND2 -16, .136 44 . .096 -22 .133 MET CA -10 .232 42, .222 -18 .478 MET O -10, .888 39, .838 -18 .759 MET CG -9 .880 43, .883 -16 .502 MET CE -9, .982 46. .061 -18 .263 ASP CA -8 , .480 39, .118 -20 .030 ASP O -8 , .038 37. .189 -18 . 690 ASP CG -8 , .237 39. .730 -22 .454 ASP OD2 -9. .327 39. .135 -22 .739 VAL CA -6, .226 38. . 601 -16 .974 VAL O -6, .284 40. .788 -15 .909 VAL CGI -3, .758 38. .176 -16 .427 ILE N -6 , .318 38. .978 -14 .590 ILE C -5, .020 39. ,262 -12 . 627 ILE C3 -7 . .476 39. , 604 -12 .466 ILE CG2 -7 . .221 39. ,883 -10 .954 ASN N -4 . .263 40. 222 -12 .110 ASN C -3 . .502 40. .404 -9 .861 ASN CB -1, .828 40. .478 -11 .697 ASN ODl -0 , .0 63 38. , 990 -11 .018 MET N -3. .458 39. ,604 -8 .832 MET C -2 . .423 39. , 603 -6 . 614 MET CB -4 . .943 39. ,387 -6 .890 MET SD -7 . .585 39. ,472 -6 .450 SER N -1. .454 40. ,496 -6 .502 SER C -0 . .422 40. ,712 -4 .326 SER CB 1 , .021 41 . ,027 -6 . 328 LEU N -1, .433 40. ,075 -3 .775 LEU C -2 . .438 39. ,056 -1 .807 LEU CB -2 , .791 41. ,568 -2 . 410 LEU CDl -5 . .278 41. ,131 -2 .578 GLY N -2 . .522 39. 082 -0 .481 GLY C -3 . ,176 38. ,180 1 . 682 GLY N -4 . ,121 37. 443 2 .222 GLY C -4 . .644 36. ,038 4 . 104 PRO N -4 . .519 . ,857 . 402 PRO C -6. .116 34 . 086 6 .082 PRO CB -4 . ,060 34. 684 7 .384 PRO CD -4 . ,239 36. 870 6 .418 SER CA -8. ,470 34. 611 6 .023 SER O -8 . ,949 . 881 4 . 029 SER OG -8 . 723 34 . 626 8 . 403 GLY CA -10 . ,824 34. ,229 3 . 074 N.Z PATF\'T ^cr ■ "c J -4 MAY 1993 j 7- 113 113 113 113 113 113 113 114 114 115 115 115 115 116 116 116 117 117 117 117 118 118 118 118 119 119 119 119 120 120 120 120 121 121 121 122 122 122 122 123 123 123 123 124 124 124 124 125 125 125 126 126 126 126 127 127 128 128 129 129 129 129 130 130 130 131 131 GLY r -12 .205 34 . 713 3 . 542 131 GLY 0 -12 .4 95 34 .722 4 .751 1 32 SEP.

N -13 .040 .058 2 . 594 132 SEP.

CA -14 .407 .433 3 .011 1" 2 SER C -15 .289 34 . 805 1. 936 132 SER 0 -14 .799 34 .586 0 .824 132 SEP.

C3 -14 .590 36 . 927 3. 145 132 SER OG -14 .693 37 .539 1 .875 133 7 A N -16 .547 34 .588 2. 294 133 ALA CA -17 .507 34 .057 1 .324 13 2 AI-A C -17 .650 34 . 965 0 . 097 133 ALA 0 -17 .743 34 .437 -1 .014 *> ~ 3 A "* ~ CB -18 .866 33 .828 1. 996 134 ALA N -17 . 683 36 .288 0 .294 134 A 1.

CA -17 .872 37 .259 -0 . 792 134 ALA C -16 .635 37 .369 -1 .674 i .'•! A " r. -16 .781 37 .585 -2 . 869 134 ALA C3 -18 .263 38 600 -0 .187 13 5 LEU ;; -15 .473 37 .229 -1. 046 135 LEU CA -14 .197 37 .244 -1 .804 135 LEU C -14 .156 36 . 005 -2 . 705 135 LEU 0 -13 .794 36 020 -3 .890 135 LEU CB -13 .038 37 .328 -0 . 798 135 LEU CG -11 .693 37 .130 -1 .508 1 35 LEU CDl -11 .460 38 .415 -2. 292 135 LEU CD2 -10 .582 36 .807 -0 .519 13-5 LYS i; -14 .509 34 . 825 -2. 173 136 LYS CA -14 .543 33 597 -3 .013 136 LYS C -15 .544 33 .739 — 4 . 150 136 LYS 0 -15 .279 33 431 -5 .305 13 6 LYS CB -14 . 903 32 .341 -2 . 186 136 LYS CG -14 .743 31 067 -3 043 1 3c LYS CO -15 .033 29 892 -2. 134 136 LYS CE -15 743 28 707 -2 778 13 6 L Y 3 ri" -15 .308 28 . 411 -4 . 160 137 ALA N -16 744 34 260 -3 847 137 ALA CA -17 .795 34 .416 -4 . 883 137 ALA C -17 .338 303 -6 045 137 rt.1.1 A c -17 .705 . 049 -7 . 208 137 ALA CB -19 .094 34 941 -4 263 1 3? ALA ?! -16 .529 36 . 301 -5 . 729 138 ALA CA -16 001 37 311 -6 685 1 3 $ A.j.-\ C _ i * . 903 36 696 -7 . 557 138 ALA 0 -14 985 36 843 -8 762 133 A' .A CB -15 .522 38 .567 -5 . 934 139 VAL N -13 950 959 -7 027 139 V.AL C A -12 . 946 .291 -7 . 837 139 VAL C -13 623 34 228 -8 720 130 VAL 0 -13 .208 34 .070 -9 . 877 139 VAL CB -11 .830 34 671 -6 968 I ? VAL CGI -10 .919 33 . 856 -7. 866 139 VAL CG2 -11 .078 780 -6 253 i o ASP N -14 .593 33 .536 -8 . 122 140 ASP CA -15 .274 32 496 -8 929 ! AS P r -16 .023 33 . 131 -10. 084 140 ASP 0 -16 .080 32 579 -11 190 1 -» A L P CB -16 .149 31 .549 -8 . 138 140 ASP CG -15 .388 640 -7 186 1-5 0 ASP ODl -14 .178 403 -7. 202 140 ASP 002 -16 .139 132 -6 329 i. 1 -I LYS N -16 . 658 34 .263 -9. 820 141 LYS CA -17 .373 006 -10 868 1-51 LYS C -16 .373 415 -11. 946 141 LYS O -16 .700 248 -13 111 1-51 LYS C3 -13 .039 36 .275 -10. 325 141 LYS CG -18 .884 37 056 -11 306 1-51 LYS CD -19 .586 38 . 187 -10. 536 141 LYS CE -20 .572 39 051 -11 250 1-51 LYS NZ -21 .138 40 .037 -10. 275 142 ALA N -15 .167 848 -11 566 1 -12 ALA CA -14 .173 36 . 192 -12. 614 142 ALA C -13 .818 010 -13 521 1-5 2 ALA 0 -13 .770 . 169 -14 . 755 142 ALA CB -12 .870 36 697 -11 948 1-13 VAL t; -13 .532 33 . 886 -12. 832 143 VAL CA -13 .168 32 705 -13 650 143 VAL C -14 . 346 32 .233 -14 . 496 143 VAL O -14 .140 31 886 -15 639 1-53 VAL CB -12 .551 31 . 673 -12. 714 143 VAL CGI -12 .300 370 -13 461 143 VAL CG2 -11 .305 32 .195 -12. 014 144 ALA N -15 .531 32 238 -13 875 1-5-1 ALA CA -16 .744 31 . 834 -14 . 641 144 ALA C -16 928 32 681 -15 861 i •; •; ALA 0 -17 .380 32 .263 -16. 958 144 ALA CB -17 942 31 968 -13 700 1 -5 5 SEP.

N -16 .507 33 . 948 -15. 706 145 SER CA -16 682 34 917 -16 786 145 SER C -15 .609 34 .773 -17 . 829 145 SER 0 -15 .910 321 -18 893 1-55 SSR C3 -17 .016 36 . 376 -16 . 414 145 SER OG -15 882 36 955 -15 849 146 GLY II -14 .577 33 . 986 -17. 565 146 GLY CA -13 619 33 799 -18 675 1 4 6 GLY C -12 .273 34 .491 -18 . 385 146 GLY O -11 420 34 386 -19 266 147 VAL N -12 .150 .162 -17. 254 147 VAL CA -10 874 856 -16 912 147 VAL C -9 .850 34 .836 -16. 323 147 VAL O -10 171 33 991 -15 486 147 VAL CB -11 .152 36 . 977 -15. 889 147 VAL CGI -9 896 37 803 -15 570 147 VAL CC-2 -12 .340 37 . 915 -16. 230 148 VAL N -8 583 018 -16 603 148 VAL CA -7 .432 34 . 230 -16 . 003 148 VAL C -7 157 34 907 -14 701 148 VAL 0 -6 .840 36 . 133 -14 . 750 148 VAL CB -6 273 34 126 -16 950 148 VAL CGI -5 .079 33 .403 -16 . 281 148 VAL CG2 -6 590 33 432 -18 262 149 VAL N -7 .258 34 . 355 -13. 531 149 VAL CA -6 987 34 965 -12 249 149 VAL C -5 .700 34 . 385 -11. 613 149 VAL 0 -5 624 33 173 -11 439 149 VAL CB -8 .224 34 .890 -11. 315 149 VAL CGI -7 893 619 -10 009 149 VAL CG2 -9 .456 . 386 -12. 096 150 VAL N -4 732 301 -11 404 150 VAL CA -3 .393 34 . 987 -10. 901 150 VAL C -3 157 625 -9 559 150 VAL 0 -3 .592 36 . 778 -9. 400 150 VAL CB -2 274 . 305 -11 951 150 VAL CGI -0 .973 34 . 633 -11. 461 150 VAL CG2 -2 675 34. 843 -13 301 151 ALA N -2 .568 34 .946 -8. 595 151 ALA CA -2 361 . 582 -7 287 151 ALA C -1 .080 .036 -6. 657 151 ALA 0 -0 618 33. 889 -6 904 151 ALA C3 -3 .557 .390 -6. 307 152 ALA N -0 490 . 907 -5 822 152 ALA CA 0 .714 .438 -5. 112 152 ALA C 0 304 34. 320 -4 158 N.Z. PATENT Pr. -4 MAY 1993 0 -0. 728 34 . ,4 66 -3 . 457 153 ALA N 1. 125 33 . ,302 -3 . 912 3 ALA.

C 0 . 931 32 . ,725 -1 . 511 153 ALA CB 1. 750 31. ,038 -3 . 195 4 GLY CA 2. 043 34 . .211 0 . .125 154 GLY 0 A 1 , 189 33 . .267 -0 . ,118 155 ASN CA . 344 34 . .787 2 . ,037 155 ASN 0 6. 101 34 . .829 4 . ,295 155 ASN CG . 890 3 6 . .702 0 . ,500 155 ASN ND2 . . 454 37 . . 9 65 0 . ,352 6 GLU CA 4 . 633 32 . .537 4 . ,970 156 GLU 0 . . 374 . . 637 6. ,222 6 GLU CG 2 . .491 32 . .442 6 . .368 6 GLU OE1 1. . 744 34 , .322 . .312 157 GLY N 6 . . 389 31, .057 4 . ,227 157 GLY C 6. .503 28 , .622 4 . .553 8 THR N 7 . 147 27 . .793 . ,382 158 TKR OG1 8. 707 . .487 6 . ,217 152 THR CA 6. 552 26 . ,487 . 702 158 TKR.

O 6. 479 27 . ,335 7 . 977 159 SER OG 3 . 141 . ,904 . 525 ? SER CA 4 . 835 . ,210 8 . 855 159 SER O 3 . 3 3 9 23 . ,281 9 . 030 160 GLY CA . 434 21 . ,504 8 . 895 160 GLY 0 4 . 808 21 . .326 6. 555 161 SER CA 2 . 654 19. .777 7 . 054 161 SER 0 0. 696 . .347 . ,869 161 SER OG 1. 854 18 . .028 8 . .585 162 SER CA 0. .167 22 , .725 7 . ,113 162 SER O 1. . 533 23 , .840 . ,394 162 SER OG 0. , 184 23 , .091 9. ,480 163 SER CA -0. . 611 24 . .750 3. ,990 16 3 SER O -1. , 078 26. .548 . ,504 163 SER OG -1. , 992 . .718 2. .331 164 THR CA 0. , 609 28 . .340 4 . .312 164 TKR O 0. . 485 .502 3. .278 164 THR.

OG1 2. , 984 28 .282 3. . 692 165 VAL N -0 . , 513 28 .742 2 . .190 165 VAL C -2. . 028 .545 1. .497 165 VAL CB -1. . 339 28 . 624 -0 , .161 165 VAL CG2 -0. . 210 27 .716 -0 , . 699 166 GLY CA -2 . 945 32 .778 1, . 626 1 6 o GLY O -4 . . 124 32 .106 -0 , .396 167 TYR CA -6. . 223 34 .046 0 , .113 167 TYR O -5 , . 474 36 .283 0 .084 167 TYR CG -7 . . 791 32 .984 1 .709 1 67 TYR CD2 -8. .710 32 .116 1 .133 16 7 T YP.

CE2 -9. .068 . 955 1 .809 167 TYR OH -8. . 880 29 .481 3 . 658 168 PRO CG -6. . 943 36 .376 -3, . 938 168 PRO CB -7 . . 964 .344 -3 , .505 168 PRO C -6, .398 33 .336 -3, .270 169 GLY N -5. .086 33 .193 -3, .189 169 GLY C _ A 1 , . 937 .702 -3, .470 170 LYS N -5, .402 .579 -2 .255 170 LYS C -7. . 055 28 .773 -2 .516 170 LYS CB -6. .246 29 .294 -0 .286 170 LYS CD -6, . 250 28 .289 2 .031 170 LYS NZ -4 . . 259 27 .463 3 .215 171 TYR CA -9. . 012 29 .043 -3 .859 171 TYR O -7. .760 28 .714 -5 . 928 171 TYR CG -10. .497 .984 -3 .047 171 TYR CD2 -10 . 456 32 .374 -3 .026 171 TYR CE2 -10. . 941 33 .088 -1 .936 171 TYR OH -12 . 008 33 .119 0 . 170 172 PRO CA -9 .093 26 .417 -6 .596 f (ft ^ n / i; ALA C3 1, .266 36 . 607 -4, .294 ALA CA 0, . 840 32 .250 -2, .943 ALA O 0 , .317 32 .192 -0, .599 GLY N 1, .827 33 . 693 -1, .244 GLY C 3, .519 34 .069 0, .550 ASN N 3, . 958 .34 .788 1, .568 ASN C , .399 34 .258 3, .462 ASN CB 6, .008 36 .158 1, .904 ASN ODl 6 . 123 36 .0 65 -0, .534 GLU N 4 .711 33 . 168 3, . 675 GLU C .522 31 . 328 , .183 GLU C3 3 .205 31 . 980 , .100 GLU CD 2 .394 33 . 951 6, .270 GLU OS2 3 .106 34 .456 7, . 146 GLY CA 7 .306 29 . 917 4, .387 GLY O .416 28 .346 4, .009 THR CG2 8 , . 079 .396 3. . 850 TKR CB 7 , .564 .346 , .296 THP.

C 6. .100 26 .480 7. ,157 SER N . .338 .441 7. ,497 SER CB 3. .673 26 .105 9. ,212 SER C 4 . .494 23 .720 8. . 944 GLY N . .574 22 . 967 8. .835 GLY C 4 , .576 21 .045 7. .738 SER N 3. .525 .310 8. .116 SER C 1. . 477 .708 6. .786 SER C3 2 . . 344 18 .293 7, .271 SER N 1. .303 21 .841 7. ,459 SER C 0. .430 23 .552 . ,848 SER CB -0, .213 23 .666 8. .242 SER N -0. . 679 23 . 921 . .197 SER C -0. . 441 26 .177 4 . ,513 SER CB -1. .890 24 . 642 3. .211 THR N 0 . .387 26 . 952 3. .852 THR C 0. .185 29 .286 3. .194 THR CB 2. .095 28 .518 4. .818 THR CG2 2, .397 27 . 610 6. .001 VAL CA -0 , . 959 29 .542 1, .010 VAL O -2, . 929 .132 2. ,280 VAL CGI -1, . 947 29 .357 -1, .374 GLY N -1, . 910 31 .821 1. ,129 GLY C -4 , .098 32 .859 0. , 617 TYR N -5, .054 33 .730 0, ,970 TYR C -5, . 993 .389 -0, ,606 TYR CB -7 , .464 34 .252 0. , 964 TYR CDl -7 , .208 32 .703 2, , 947 TYR CE1 -7 , .567 31 .528 3, . 615 TYR CZ -8, .486 . 671 3, .046 PRO N -6, .380 .499 -1, .850 PRO CD -6 , .273 36 . 752 -2. , 624 PRO CA -7 . .134 34 .457 -2. ,560 PRO O -7 . .097 32 .520 -3. , 912 GLY CA -4 . .446 32 . 077 -3. .927 GLY O -4 . .880 29 .733 -4. ,249 LYS CA -5, .856 29 .265 -1. ,745 LYS O -7. .308 27 .554 -2. ,524 LYS CG -5, .795 28 .106 0. ,585 LYS CE -5, .731 27 .271 3, ,029 TYR N -7, .838 29 . 616 -3. .148 TYR C -8. . 603 28 .309 -5. , 113 TYR CB -9. .962 .224 -4. ,242 TYR CDl -11. .060 .303 -1, .982 TYR CE1 -11, .520 31 .003 -0. .867 TYR CZ -11, .528 32 .398 -0, ,886 PRO N -9, .297 27 .204 -5. ,374 PRO C -9, .233 27 .156 -7. ,909 N.Z PATC-M-r rxr - -c "A MAY 1993 152 153 153 154 154 155 155 155 155 156 156 156 156 156 157 157 153 158 158 159 159 159 160 160 161 161 161 162 162 162 163 163 163 164 164 164 164 165 165 165 166 166 167 167 167 167 167 167 168 168 168 168 169 169 170 170 170 170 171 171 171 171 171 171 172 172 7 3 73 « 11 7 4 74 7 4 7 5 75 7 5 7 5 7 6 7 6 77 7 7 7 7 7 3 7 c 79 7 9 90 90 9 0 •j -J c - 91 C 1 82 93 83 B 3 3 4 3-3 34 8 4 8 5 9 5 85 85 8 6 36 36 9 6 36 96 8 7 87 38 8 3 8 3 39 8 9 89 39 89 89 90 90 90 .91 PP.0 0 -8 , .525 26 . .784 -8 , .881 172 PRO C3 -10 .167 . 329 -6 .513 P?.0 CG -10 . . 600 , .271 -5 . .096 172 PRO CD -10 . 364 26 . 669 -4 .514 SEP.

N -10 . .097 28 . .167 -8 . .019 173 SEP.

CA -10 . 220 28 . 818 -9, .330 SEP.

C -9 . . 025 29 , .773 -9 , .595 173 SEP.

O -8 .966 .233 -10 .742 SEP.

C3 -11 . .528 29, . 623 - 9 , .481 173 SEP.

OG -11 . 595 .546 -8 .406 VAL N -8 , .162 29 , . 944 -8 , .614 174 VAL CA -7 . 053 . 891 -8 .855 VAL C -5 , .754 , .131 -9 , .068 174 VAL O -5 . 612 29 .152 -8 .344 VAL C5 -6 . .899 31. .775 -7 . .596 174 VAL CGI -5 .796 32 . 837 -7 . 617 VAL CG2 -8 . .220 32, .503 -7 , .323 175 ILE N -4 . 911 .729 -9 .885 ILE CA -3 . .569 , .15 6 -10 . .024 175 ILE C -2 .714 .736 -8 .894 ILE 0 _2 .450 31. .958 -8 . .955 175 ILE C3 -2 . 953 .524 -11 .419 ILE CGI -3 . .857 29, . 978 -12 , .524 175 ILE CG2 -1 . 451 .089 -11 .512 ILE CDl -3 . . 692 , .529 -13, .946 176 ALA N -2 .220 .028 -7 .925 ALA C A _ T_ . 335 , .517 -6 .870 176 ALA C 0 .120 .301 -7 .310 ALA 0 0 . .453 29, .215 -7 .838 176 ALA CB - x . 639 29 . 838 -5 .541 VAL N 0 , .864 31, .410 -7 , .180 177 VAL CA 2 .261 31 .534 -7 . 656 VAL C 3 , .225 31. . 693 -6, .473 177 VAL O 3 .178 32 . 657 -5 .721 VAL C3 2 . .439 32. , 607 -8 , .755 177 VAL CGI 3 . 842 32 . 667 -9, .392 VAL CG2 1 , .374 32. ,552 -9. .845 178 GLY N 4 .077 . 654 -6 , .358 GLY CA , .168 . .703 -5, .339 178 GLY C 6 .446 31 .233 -6 .074 GLY 0 6 , .499 31. , 435 -7 , .286 179 ALA N 7 .512 31 .447 -5, .287 ALA CA 8 .715 32. .037 -5 .859 179 ALA c 9 . 939 31 .099 -5 .779 ALA 0 . .198 . . 481 -4 , .719 179 ALA CB 9 . 025 33 .251 -4 .973 VAL N , . 659 31. , 162 -6, .885 180 VAL CA 11 . 970 .482 -6 .981 VAL C 13 .048 31, .585 -7, .171 180 VAL O 12 .712 32 .691 -7 .627 VAL C3 12 . .075 29. .514 -8, .166 180 VAL CGI 11 .271 28 .251 -7 .855 \ V ^ , CG2 11 , . 675 . , 129 -9, .500 181 AS? N 14 .267 31 .203 -6, .800 AS? CA .451 32. . 108 -7, .039 181 ASP C . 942 31 .804 -8 .462 ASP 0 , .339 31. . 090 -9, .292 181 ASP CB 16 .446 31 .921 -5 .914 ASP CG 17 . . 120 . .534 -5, .971 181 ASP ODl 17 .105 29 .785 -6 .972 AS? 0D2 17 . . 680 , .256 -4 .887 182 SER N 17 .087 32 .386 -8 .847 SER CA 17 . . 622 32. .214 -10 , .191 182 SER C 18 .153 . 817 -10, .494 SSR 0 13 , .365 . . 452 -11 .670 182 SER CB 18 . 678 33 .313 -10 .464 SER OG 18 , . 016 34 . .561 -10, .475 183 SER N 18 .258 .042 -9, .423 SER CA 18 , .716 28. . 645 -9, .444 183 SER C 17 .581 27 . 614 -9, .547 SER 0 17 . . 859 26. . 415 -9 .397 183 SER CB 19 .256 28 .323 -8 .007 SER OG . .589 28. . 615 -8, .251 184 ASN N 16 .373 28 .094 -9, .602 ASN CA . . 144 27. . 317 -9, .580 184 ASN C 14 . 931 26 .720 -8 , .197 ASM 0 14 , . 13B . .759 -8 .097 184 ASN CB .014 26 .341 -10 .722 ASM CG 14 , . 990 26. . 998 -12, .076 184 ASN ODl 14 .700 28 .184 -12, .277 ASN ND2 , .352 26, .210 -13 .076 185 GLN N .542 27 .247 -7 .159 GLN CA , .276 26 , . 646 -5 .835 185 GLN C 14 .200 27 .494 -5, .203 GLN 0 14 , . 159 28 . .726 -5 .386 185 GLN CB 16 .599 26 .568 -5, .101 GLN CG 16, .539 26. . 242 -3 . 614 185 GLN CD 18 .011 26 .102 -3, .206 GLN OE1 18 , .864 , .799 -4 .061 185 GLN NE2 18 .266 26 .386 -1, .934 ARG M 13, .278 26. . 958 -4 .448 186 ARG CA 12 .185 27 .774 -3. .841 ARG C 12 , .780 28 . .782 -2 .866 186 ARG O 13 . 698 28 .384 -2, .093 ARG CB 11, .215 26 , . 843 -3 .116 186 ARG CG .214 27 .471 -2, .161 ARG CD 9 .467 26 . 337 -1 .468 186 ARG NE 9 .866 26 .333 -0 .117 ARG CZ 9, . 961 26, . 879 1 .059 186 ARG NH1 9 .367 27 . 880 1 .658 ARC- NH2 , . 966 26. . 321 1 .783 187 ALA N 12 .294 .009 -2 , .853 ALA CA 12 .728 31 . 064 -1 .895 187 ALA C 12 .262 . 604 -0, .517 A-..-v 0 11, . 150 , .043 -0 . 387 187 ALA CB 12 . 144 32 .402 -2 , . 344 SEP.

N 13, .051 , .770 0 .549 188 SER CA 12 . 671 .286 1 , .868 SER C 11 . 356 . 847 2 .412 188 SER O .740 . Ill 3, .212 SEP.

CB 13 .767 , . 456 2 .938 188 SER OG 14 .137 31 .826 2, .841 PHE N , .943 32 , .010 1 . 974 189 PHE CA 9 . 697 32 . 688 2. .418 PHE C 8 .499 32 , .198 1 .609 189 PKE 0 7 .389 32 .556 2, .011 PHE CB 9, .787 34 .217 2 .243 189 PHE CG .117 34 . 696 0. .867 PHE CDl 9 .147 34 .830 -0 .121 189 PHE CD 2 11 .415 .116 0, .567 PHE CEl 9 . 483 . 187 -1 .411 189 PKE CE2 11 .769 .545 -0. .701 PHE CZ , .786 .586 -1 .725 190 SER N 8 .703 31 .526 0. .499 SER CA 7 . 626 31 .096 -0 .391 190 SER C 6 .663 .162 0, .328 SER 0 7 , .034 29 .083 0 .866 190 SER CB 8 .181 .590 -1. ,708 SER OG 7 , .136 .337 -2 .618 191 SER N .388 .551 0. .326 SEP.

CA 4 . 341 29 . 686 0 .987 191 SER C 4 .261 28 .330 0. ,223 1 S1 SER 0 4 .543 28 . 268 -0 . 995 131 SE?.

OG 2 . 729 31. 285 1. 954 192 VAL CA 3 . 629 . , 932 0 . 391 192 VH. " .

O 1 . 559 . , 698 1. 598 192 VAL CGI 6 . 144 . ,727 0 . 722 193 GLY N 1 . 938 24 . , 172 0 . 047 193 GLY C 0 . 031 23. , 029 -0 . 901 1 94 PRO N _ T_ . 023 22. 289 -0 . 722 194 PRO C -2 .237 22. , 605 -2 . 914 194 FRO C3 -2 .769 . ,783 -1. 210 134 pf-c CD _ T_ . 633 21. , 954 0 . 578 1 95 GLU CA -3 . 145 24 . , 850 -3 . 252 1 35 GLU O _2 . 516 26 . .398 -4 . 936 195 GLU CG -4 . 942 . .134 -1 . 435 1 95 GLU oe1 -3 . 110 24 . . 960 0 . 165 136 leu n -0 . 829 . .264 -3 . 870 196 LEU C 0 . 228 , .376 -6 . 059 196 LEU C3 1 . 540 . ,739 -3 . 854 196 LEU CDl 2 . 739 27 . .716 -4 . 639 1 97 A3? N 0 . 140 26 . .208 -7 . 093 1 37 A. 3 ? C 1 . 307 . ,738 -9 . 293 1 97 AS? C3 -1 . 067 26. ,598 -9 . 191 1 97 A3? CDl _ o . 804 . . 155 -8 . 354 1 90 VAL n 2 . 013 26. . 889 -9 . 344 193 VAL C 4 . 157 27 . , 950 -9 . 514 1 93 VAL CB 2 . 894 27 . , 476 -11 . 637 193 VAL CG2 2 . 337 28 . , 919 -11 . 484 1 99 met CA 6 . 439 28 . . 802 -9 . 498 193 met o 6 . 696 29 . . 518 -11. 793 193 met CG 7 .365 26 , .849 -8 . 139 1 99 met CE 8 . 227 27 , .755 -5 . 587 200 ALA CA 7 . 991 31 , . 929 -11 . 055 2 GO ALA o 0 . 127 32. . 524 -9 . 060 2 01 PRO N 9 . 927 33 , .455 -10 . 951 201 PRO C .450 . .127 -9 . 238 2 01 PRO cb 11 . 817 34 . .723 -11. 400 2 01 PRO CD 9 . 941 33. . 616 -12 . 405 2 02 GLY CA . 473 36. .204 -7 . 044 2 02 GLY O 11 . 352 37, .124 -4 . 979 2 03 VAL CA 13 . 948 36, . 929 -5 . 716 203 VAL O .133 37 , .731 -7 . 593 2 0 3 VAL CGI 16 .096 36 , . 106 -4 . 612 2 0 4 SEP.

N 14 .865 39 , .182 -5 . 859 2 0 4 SER C .067 40 , . 619 -7 . 872 2 04 SER CB 17 . 087 39 .976 -6 . ,326 2 05 ILE N 13 . 771 40 , .865 -8 . ,008 2 0 5 ILE C 13 .207 42 .749 -9 . , 478 205 ils CB 11 . 532 40 . 833 -9 . . 144 205 ile CG2 .899 41 .281 -10 . ,467 206 GLN N 13 . 956 43. . 095 -10 . 489 206 gl:j C 13 . 002 44 , . 978 -11 . 630 206 GLN CB . 455 44 . .708 -11 . 740 206 GLN CD 17 .285 45, . 145 -10 . 007 206 GLN me 2 16 . 556 46 . 260 -9 . 857 207 ser CA 11 .217 46 .571 -11 . ,987 207 ser o 11 . 919 48 . 657 -11 . ,004 207 ser og 8 . 993 46 .056 -12 . ,613 208 thr cg2 9 .171 50 .339 -14 . ,754 208 thp. cb 8 .620 50 .415 -13. ,357 208 thr c 9 .197 50 .488 -10 . ,803 209 leu n 9 .656 51 . 613 -10 . ,228 209 leu c 8 . 673 53 .610 -9. ,262 209 leu CB .335 52 .192 -7 . .958 209 leu CDl 11 .968 51 .114 -6. ,472 210 pro N 7 .790 54 .139 -8 . ,444 210 pro C 8 .383 56 .573 -8 . ,639 SER CB 3, .015 .411 0 .911 VAL N 3, , 756 27 .310 0 . 928 VAL C 2, .254 .291 0 .686 VAL CB 4 , ,781 , .127 1, .088 VAL CG2 4 , . 617 , .104 2 .592 GLY CA 0 , . 629 23 .564 0 .410 GLY O 0. .530 23 .244 -2 .015 PRO CA -1, . 662 21, . 651 -1, .873 PRO O -2, .403 22, .244 -4 , .085 PRO CG -2 , ,311 , .622 0, .213 GLU N -2, ,522 23 .793 -2 .439 GLU C -2, .095 .631 -4 .058 GLU CB -4 , .043 .786 -2 .470 GLU CD -4 , .315 24 .860 -0 .100 GLU OE2 -5, .138 24 .520 0 .785 LEU CA 0, .241 .929 -4 . 664 LEU O 0. .305 24 . .121 -6, .153 LEU CG 2. ,770 26, .178 -4, , 643 LEU CD 2 4 . . 027 , .721 -3. , 911 ASP CA 0. ,032 . .774 -8 . ,480 ASP O 1. , 655 24 . .734 -9. , 914 ASP CG -2. ,406 26. .351 -8. .549 ASP OD2 -3. ,035 27 , .327 -8 . .088 VAL CA 3. .206 26, .970 -10, .209 VAL O 3. , 752 28. .699 -8. .587 VAL CGI 1. , 930 26, .726 -12. .537 MET N . .374 27 , .916 -10. .016 MET C 6, , 845 29, .810 -10, .578 MET CB 7 , . 660 27 , .970 -9. .077 MET SD 6, ,755 27, .449 -6, .568 ALA N 7 . . 42 6 , .942 -10. .103 ALA C 9. .088 32, .666 -10, .272 ALA CB 6, . 932 32, .870 -11, . 638 PRO CA 11, .013 34 , .130 -10, ,238 PRO O 9. ,579 , ,907 -9, ,682 PRO CG 11, ,392 34. ,040 -12. .678 GLY N , ,925 . .204 -8. .021 GLY C 11. .580 3 6. .658 -6. .115 VAL N 12, ,815 36. .503 -6, ,613 VAL C 14 , .706 38, .017 -6, .469 VAL CB 14 , . 814 , .688 -5, .351 VAL CG2 14 . , 079 34 . .741 -4 . .378 SER CA , ,572 40, .281 -6. .487 SER O . .786 40. .685 -8. .889 SER OG 17, ,752 41. .186 -6. .672 ILE CA 13. .069 41, .234 -9. .225 ILE O 12, . 675 43, .498 -8. , 648 ILE CGI 11, . 436 39, .336 -8. ,810 ILE CDl 12 . .257 38, .412 -9. ,771 GLN CA 14 . 204 44 . ,517 -10. 834 GLN O 12 . , 669 44 , ,318 -12 . 621 GLN CG 16 . 684 44 . .163 -10. ,980 GLN OE1 18 . 328 44 . .936 -9. ,353 SER N 12 . , 359 46. .064 -11. ,214 SER C 11. , 089 48. .093 -11. ,749 SER CB 9. , 918 45. .853 -11. ,569 THR N . , 054 48. ,664 -12 , .326 THR OGl 7. ,570 49. ,414 -13, .144 THR CA 9. , 675 50. ,092 -12. ,173 THR O 8. ,423 49. ,807 -10. ,049 LEU CA 9. ,192 52. ,158 -8. ,959 LEU 0 9. , 140 54, .227 -10. ,222 LEU CG . , 804 50. .816 -7 , ,416 LEU CD2 9. , 607 50. ,282 -6, ,649 PRO CA 7. .273 55, .517 -8, .649 PRO O 9. .491 56, .445 -8. .104 PA ~ - •. T 4 MAY 1993 i- 191 192 192 192 192 193 193 194 194 194 195 195 195 195 195 196 196 196 196 197 197 197 197 198 198 198 199 199 199 199 200 200 200 201 201 201 202 202 203 203 203 203 204 204 204 205 205 205 205 206 206 206 206 207 207 207 208 208 208 208 209 209 209 209 210 210 2 1Z PRO 6 302 55. 733 - 7 517 210 PP.0 CO 7 193 53 491 -7 271 211 Gil' ca Q 069 58 765 -9 410 211 GL- 0 11 176 59 005 -10 259 212 a5n ca 803 57 422 -12 643 212 AJ.I C 13 188 57 181 -12 420 212 asn CG 11 S 0 3 58 185 -14 814 212 as:; no 2 12 273 59 159 -15 576 2 * ~ 1.1* s CA. 12 810 54 946 -10 537 213 lys 0 11 775 53 039 -11 613 2 13 lys C3 13 206 56 694 -3 767 213 ly 3 CE 14 105 58 218 -6 670 -i. *1 t I?. n 13 6 31 52 703 -10 444 214 typ. r 14 383 50 600 -9 489 «_ . -i T Y ?.

CB 14 641 50 981 -11 984 214 typ.

CDl 14 689 52 847 -13 678 214 TVS CEl 14 230 53. 475 -14 814 4- ~ 1 11?. cz 13 4 52 895 -15 550 2 • c, GLY n 14 058 49 347 -9 158 2 15 gly C 14 130 47 325 -7 749 2 1 ala n 14 810 46 638 -6 831 2 1 t ala r 13 682 4 4 922 -5 512 216 ala C3 715 44 354 -6 887 2 17 t y ?.

CA. 11 9 b 4 43 488 -4 440 t i-?. 0 12 202 41 442 -5 656 " • - rv:- tg 117 45 291 -4 214 ?:" t v p. cnr. 9 016 45 933 -4 785 v r- C7-.2 0 654 47 219 -4 381 t i p.

OH 8 953 49 160 -2 988 2 - c asn C.a 11 64 0 39 942 -3 227 2 1" as:; 0 Q 763 40 347 -1 817 ^ * 0 asn CG 14 031 39 566 -2 343 r. , £ asn no 2 14 660 39 644 -1 165 /■» gly ca 8 382 38 130 -2 649 2 19 gly 0 7 873 37 500 -4 876 220 thr ca 697 936 -4 179 220 thp. 0 4 417 36 742 -5 958 O o thp.

OGl 4 136 543 -2 451 221 SEP. n 4 738 38 238 -4 303 22 1 SER C 4 760 39. 641 -6 388 2 2 1 ser cb 3 323 40 383 -4 546 2 2 2 met n 6 060 39. 389 -6 485 o o met sd 7 768 41 533 -4 993 O O 0 met C3 8 351 40. 015 -7 218 222 met c 6 877 38. 435 -8 567 2 2 3 ala n 6 554 37. 246 -8 041 223 ala C 200 36. 068 -9 707 22 3 ala CB 6 509 34 807 -7 923 2 2 4 sep. ca 2 758 36. 488 -9 700 2 2 4 ser 0 2 145 36. 593 -12 057 2 2 4 SEP.

OG 0 492 36. 899 -9 157 225 FRO ca 3 095 39 130 -12 439 22 5 ?p.o 0 3 406 38 650 -14 804 225 PRO cg 4 411 40 402 -10 764 226 k13 n 4 769 37 626 -13 299 226 his c 4 418 . 947 -15 061 226 his cb 6 608 36. 046 -13 765 226 his nd1 8 048 37 488 -12 170 226 kis CEl 9 270 38. 052 -12 236 227 val n 3 593 . 366 -14 199 221 val C 1 479 . 197 -15 421 227 val CB 2 103 33. 444 -13 619 227 val CG2 3 204 32. 665 -12 891 228 ala CA 0 011 37. 109 -15 517 228 ALA 0 -0 253 37. 435 -17 828 229 GLY N 1 791 38. 028 -16 941 "Tf 7 i PRO CG 6 . 004 54 .379 -6 .944 GLY N 8 .077 57 . 6 65 -9 .355 GLY C .094 53 . 454 -10 .490 ASN N 9 . 851 57 .770 -11 .537 ASN C 12 .059 56 .753 -12 . 056 ASN CB 11 .224 58 .595 -13 . 499 ASN ODl 11 .853 57 . 054 -15 .323 LYS N 11 .803 55 .749 -11 .247 LYS C 12 . 668 53 . 459 -10 .866 LYS CB 12 .769 55 .241 -9 .059 LYS CD 13 .246 57 .030 -7 . 312 LYS NZ . 048 58 . 705 -7 . 921 TYR.

CA 13 .800 51 .246 -10 .722 TYR 0 .211 51 .253 -8 .817 TYR CG 14 .130 51 . 621 -13 .246 TYR CD2 13 . 129 51 . 065 -14 .014 TYR CE2 12 . 654 51 .669 -15. . 178 TYR OH 12 .756 53 . 458 -16. .696 GLY CA 14 . 622 48 .772 -7, .905 GLY O 13 .249 46 . 917 -8, .521 ALA CA 14 . 454 45 . 203 -6, .781 ALA 0 13 . 948 45 . 527 -4, .475 TYR N 12 .758 43 . 982 -5, .575 TYR C 12 .033 41 . 928 -4, .547 TYR CB .473 43 . 862 -4. .570 TYR CDl .846 45 . 991 -3. ,236 TYR CEl .459 47 .267 -2, .790 TYR CZ 9 .358 47 . 882 -3, .391 ASN N 11 . 750 41 . 386 -3. ,391 ASN C .204 39 . 636 -2. ,749 ASN CB 12 .553 39 .340 -2, .154 ASN ODl 14 . 612 39 . 709 -3, ,422 GLY N 9 . 670 38 . 554 -3. ,239 GLY C 7 .570 37 .384 -3, .681 THR N 6 .561 36 . 638 -3, .205 THR C 4 .879 37 . 044 -4, .864 THR CB 4 .825 34 . 819 -3. .526 THR CG2 .704 33 . 696 -2. .900 SER CA 3 .984 39 .201 -5, .169 SER 0 4 . 117 40 .208 -7. .277 SER OG 3 .435 40 .282 -3. ,149 MET CE 6 . 471 42 .771 -5. ,173 MET CG 8 .506 41. .399 -6. ,602 MET CA 6 . 916 39 . 670 -7. ,638 MET 0 7 . 084 38, .567 -9. ,775 ALA CA 6 .469 36, . 020 -8. 885 ALA 0 . .133 , . 948 -10. 929 SER N 4 .076 36, .360 -9. ,033 SER C 2. . 661 37, .161 -11. ,039 SEP.

C3 1 . 801 36, .995 -8 . ,603 PRO N 3. . 156 38, .411 -11. ,159 PRO C 3 .764 38, .469 -13. .626 PRO C3 3 . 653 40, .511 -12. ,054 PRO CD 3. .735 39, .224 -10. ,054 HIS CA , .446 36, .879 -14 . 362 KIS 0 4 .425 .809 -16. ,293 HIS CG 7. . 814 36, .859 -13. ,358 HIS CD 2 8. . 883 37 , .118 -14 . 167 HIS NE2 9. .771 37. , 866 -13. 443 VAL CA 2, .583 34 , ,388 -14 . ,727 VAL 0 1. .018 34 , .773 -16. 4 90 VAL CGI 1. .076 32. , 476 -14. 246 ALA N 1. .003 36. ,242 -14 . 814 ALA C 0. .543 37. ,538 -16. 863 ALA CB -0. . 307 38 , .353 -14 . ,668 GLY CA 2. .352 38. .408 -18 . 239 4 MAY 1993 o _ 210 211 211 212 212 212 212 213 213 213 213 213 214 214 214 214 214 214 215 215 215 215 217 217 217 217 217 217 218 218 218 218 219 219 220 220 220 220 221 221 221 222 222 222 222 223 223 224 224 224 225 225 225 225 226 226 226 226 226 227 227 227 228 228 228 229 2 2 9 GLY C 2 420 37 197 -19 . 2 3 3 ALA N 2 711 988 -18 . 230 ALA c 1 424 34 500 -20 . 2 3 0 m ~ C3 3 298 33 624 -18 . 231 ALA CA — 1 010 34 416 -19. 2 31 ALA 0 -1 909 056 -21. 232 ALA N -0 778 36 657 -20 . 2 32 ALA C -0 281 37 284 -23 . 232 ALA C3 -0 742 39 121 -21. 232 LEU CA. 1 617 3 6 293 -24 . 2 33 LEU C 0 696 231 -26. 2 33 L EL- CG 3 995 36 994 -23 . ^ - q LEU CD2 *t 241 37 853 -24 . o ■; « ILE CDl 0 306 664 -21. » *1 I LE C3 -0 811 32 014 -23 . z j11 ILE CA -0 405 33 076 -24 . 2 j 4 ILE C _1 883 33 144 -26 . 2 35 LEU CA -3 596 028 -25. 2 35 LEU O 4 109 914 -27 . 2 3 5 LEU CG -5 140 34 899 -23 . 2 35 LEU CD2 -6 252 34 138 -24 . ^ jn SER CA -1 7 64 37 237 -27 . 2 3 *5 SER O _ t_ 746 3 6 634 -30 . 2 3*: SER C'G 0 599 37 571 -27 .

LYS CA -0 84 6 34 085 -29 . r 3 *" LYS 0 — 2 378 32 951 -31. _ j LY 3 CG 0 677 32 240 -30 .

L Y 3 CE 2 345 762 -31 . .. ~2 :~::3 jj _2 951 32 989 -29. 238 HIS C -5 334 32 899 -28. 2 H I 3 CB -3 948 8 62 -28 . 2 38 HIS ND1 -1 707 29 679 -28 . 2 3 S HIS CEl -1 086 28 851 -29 . 2 3 9 PRO N -5 848 33 917 -29. 239 PRO C -8 204 34 052 -28 . 239 PRO C3 -7 018 977 -29. 239 PRO CD -5 436 34 439 -30. 2 •; 0 ASN CA -9 529 32 041 -29. 2 -SO ASN 0 -10 540 610 -27 .

ASN CG -7 971 827 -30 . 2-10 ASN ND2 -7 670 29 509 -30 . 2-11 TRP CA -8 304 124 -26 . 2-11 TPP O -9 043 31 833 -24 . 2 41 TPP CG -6 094 28 903 -26 . 2 -11 TRP CD2 — 4 839 28 324 -26. 2 -11 TRP CE2 -4 .114 27 -4 76 -27 . o . t TPP CZ2 -3 195 26 786 -27 . 2 -11 TRP CH2 _2 470 26 873 -26 . 2 42 THR CA -10 458 119 -22 . 2 -12 THP.

O -s 335 29 674 -21 . 2-12 THR CGI -10 837 27 786 -22 . 2-13 ASN N -9 946 659 -20 . 2-13 ASN ODl -11 698 341 -18 . 2 4 3 ASN CB -9 708 31 530 -18 . 2-13 ASN C -8 657 29 303 -19. 244 THR N -9 564 28 362 -19 . 2-14 THR C -8 133 26 393 -19. 2-14 THR CB -10 665 26 088 -19. 2 44 THR CG2 -10 503 24 595 -19. 245 GLN CA -6 964 26 362 -21. 245 GLN 0 -4 573 26 393 -21. 245 GLN CG -8 265 526 -23. 2 45 GLN OE1 -9 306 26 769 -25. 246 VAL N -5 697 28 304 -21. 246 VAL C -3 936 28 462 -19. 246 VAL CB -4 779 555 -20. 229 GLY 0 2 . .189 37 . . 375 -20 .384 230 ALA CA 2 . .794 34 . . 801 -19 .54 6 230 ALA 0 1. , 380 34 . .205 -21 .343 231 ALA N 0 . 385 34 . . 623 -19 .328 231 ALA C -1. .256 . . 423 -20 .864 231 ALA CB -1. . 932 34 . . 664 -18 .549 232 ALA CA -1 . 013 37. . 663 -21 .792 232 ALA O -0 . 841 37. .501 -24 .187 233 LEU N 0 . 935 36. .726 -22 .967 233 LEU C 0 . 821 . . 169 -24 .880 233 LEU CB 3. 063 . . 877 -23 .907 233 LEU CDl . 259 36. . 342 -22 .921 234 ILE N 0 . 357 34 . . 199 -24 .047 234 ILE CGI 0 . 454 31. , 223 -23 .105 234 ILE CG2 -1. 803 . . 900 -24 .091 234 ILE C -1. 621 33. .597 -25 .434 235 LEU N -2 . 390 34 . . 465 -24 .779 235 LEU C -3. 258 . , 843 -26 .672 235 LEU C3 -4 . 432 . .765 -24 .378 235 LEU CDl -5. 652 . , 683 -22 .145 236 SER N -2 . 094 36. 438 -26 .798 236 SER C -1. 491 36. 292 -29. . 144 236 SER CB -0 . 633 38 . 234 -27. .733 237 LYS N -1. 046 . 067 -28, .882 237 LYS C -2 . 113 33. ,277 -30, .268 237 LYS CB 0. 272 33. 112 -29, .551 237 LYS CD 2 . 020 31. 535 -30, . 442 237 LYS NZ 3. 525 29. 848 -31, .596 238 KIS CA -4 . 168 32 . 163 -29. .379 238 KIS 0 -5. 713 32. 584 -27. .562 238 HIS CG -3. 009 29. 921 -29. .237 238 KIS CD2 -3. 137 29. 258 -30. .394 238 KIS NS2 -1. 948 28 . 600 -30. .599 239 PRO CA -6. 908 34 . 779 -28. .773 239 PRO O -8 . 949 34 . 519 -27. . 662 239 PRO CG -6. 666 . 294 -31, .027 240 ASN N -8 . 386 32. 969 -29. .227 240 ASN C -9. 508 31. 180 -27. . 980 240 ASN CB -9. 403 31. 249 -30. .535 240 ASN ODl -7 . 008 31. 590 -31. .147 241 TRP N -8 . 354 31. 006 -27. .304 241 TRP C -9. 106 . 638 -24. .936 241 TRP CB -6. 879 29. 830 -25. . 679 241 TRP CDl -6. 338 28 . 433 -27. .818 241 TRP NE1 -5. 362 27 . 547 -28. .211 241 TRP CE3 -4 . 097 28 . 406 -24. , 981 241 TRP CZ3 -2 . 912 27 . 667 -24. , 943 242 TKR N -9. 727 29 . 781 -24 . ,142 242 THR C -9 . 469 . 176 -21. ,747 242 THP.

CB -11 . 579 29 . 032 -22. , 675 242 THR.

CG2 -12 . 494 28 . 907 -23. ,895 243 ASN ND2 -11. 545 31. 654 -16. .792 243 ASN CG -11 . 093 31. 131 -17. .905 243 ASN CA -9 . 053 . 731 -19. ,444 243 ASN 0 -7 . 593 29. 136 -18. ,440 244 THR CA -9 . 381 26. 934 -19. ,059 244 THR 0 -7 . 324 . 757 -19. ,111 244 THR OGl -11. 735 26. 675 -18. , 684 245 GLN N -8 . 082 26. 716 -21. ,073 245 GLN C -5. 647 27. 020 -21. 520 245 GLN CB -7 . 330 26. 599 -23. ,397 245 GLN CD -8. 493 . 873 -25. 428 245 GLN NE2 -7. 745 . 312 -26. 370 246 VAL CA -4 . 477 29. 040 -20. 770 246 VAL 0 -2. 705 28. 227 -19. 361 246 VAL CGI -3. 544 31. 272 -20. 027 r TP" rt • i f * ^ . r ! : '■ r • i j f — — - - - J ■4 MAY 1 993 1 i 187 646 153 709 744 852 721 078 377 209 111 453 680 657 570 644 544 423 589 342 120 986 290 582 952 444 716 729 310 697 511 835 642 365 532 713 668 216 576 889 986 120 686 557 155 216 174 005 911 937 476 611 645 332 010 283 802 494 158 962 447 989 727 218 467 621 1 ~~ 1 / •-I I t I 4 0 C J* 4 9 49 0 50 0 o \j ~ j - 7 '? *i V ■ i 55 55 55 6 6 6 6 6 57 57 7 57 58 58 59 59 59 59 50 60 60 61 61 61 61 61 62 62 62 62 62 52 2 4 r 6 ? 0 val cg2 -5 169 31 138 -21 959 a_P.G ca — *1 380 27 714 -17 168 a.p.g 0 -2 705 985 -16 764 ap.g cg - 4 987 27 095 -14 852 a_?.g ne -5 440 2 6 757 -12 546 arg nh1 -7 064 27 484 -11 210 sep. — -1 4 = 0 505 -18 131 SEP. c _ 2 657 24 086 -19 073 sep. cb -5 034 23 408 -19 372 sep. >; _ 2 500 24 853 -20 136 sep. c -0 071 302 -19 940 sep. cb -1 369 758 -22 068 leu -0 2s9 26 333 -19 160 leu cdl -0 373 453 -17 268 leu cb 0 178 28 063 -17 505 leu c 1 092 694 -17 265 gln7 n 0 068 007 -16 714 gen CEl _2 795 460 -12 347 gen CG _ i 213 24 814 -13 994 gin ca 0 381 23 941 -15 745 gen C 1 743 22 014 -15 616 ca 1 082 21 206 -18 282 0 2 809 442 -19 768 a..~n CG -1 036 19 926 -18 573 a^'.« nd2 _ o 234 19 834 -19 161 T :■•:?.

CA. 4 256 22 717 -19 713 THP. 0 6 343 23 733 -19 427 thp CGI 3 595 24 957 -20 423 THP. t; 218 23 177 -17 551 THP.

C 7 466 22 700 -16 612 THR CB 6 6 4 23 558 -15 132 thp. cg2 4 530 24 549 -14 802 thp. ca 9 771 22 594 -15 817 thr 0 9 439 22 786 -13 474 thp. cgi 11 082 23 709 -17 321 lys n 9 606 702 -14 314 lys c 522 333 -12 063 lys c5 9 024 18 590 -13 249 lys cd 286 16 948 -11 777 lys ne 9 24 3 14 869 -11 054 leu ca 11 272 21 038 -9 893 LEU 0 12 096 565 -7 732 leu CG 11 357 23 620 -10 568 leu cd2 12 678 23 468 -11 325 gly ca 602 18 793 -6 879 gly 0 8 283 18 956 -7 202 as? ca 7 757 17 896 -4 516 asp 0 6 859 039 -4 214 as? cg 6 781 17 128 -2 241 asp 0D2 7 098 16 299 -1 321 ser ca 4 481 19 587 -5 529 ser 0 3 500 21 503 -4 446 sep. og 2 745 17 937 -5 448 PHE ca 3 831 468 -1 885 phe 0 3 944 22 848 -1 432 phe cg 3 549 337 0 715 phe cd2 4 401 21 060 1 558 phe ce2 3 945 21 602 2 748 tyr n 778 21 758 -2 305 tyr c 6 820 23 689 -3 545 tyr c3 8 123 22 455 -1 851 tyr CDl 8 084 484 -0 364 tyr CEl 8 062 19 .873 0 882 tyr CZ 8 069 .672 2 018 tyr n 6 626 23 104 -4 693 tyr C 626 23 680 -6 956 AP.G N -4 .767 23 . .240 -18 .462 ARG C -3 . 770 26 . ,292 -17 .340 ARG C3 -5 . 533 27 . , 667 -16 . 149 ARG CD -6 .056 27 . , 179 -13 .793 ARG CZ -5 .893 26. ,866 -11 .315 ARG NH2 -5 . 177 26. ,428 -10 .270 SSR CA -4 . 039 24 . ,131 -18 .426 SER 0 -1 . 848 23. ,253 -18 .583 SSR OG -6 . 146 23 . ,090 -18 .532 SER CA -1 .223 24 . ,874 -20 . 851 SER O 1 . 026 24 . ,705 -20 .049 SSR OG -0 .300 . ,419 -22 . 956 LEU CD2 1 . 824 29 . ,814 -18 .222 LSU CG 0 . 352 29. ,438 -18 .151 LSU CA 0 .718 26 . 837 -18 .216 LEU O 2 .283 . 421 -17 . 032 GLN NE2 -2 . 905 23 . 353 -13 .010 GLN CD -2 , . 345 24 . 550 -13, .034 GLN CB -0. . 857 23 . 621 -14 , . 877 GLN C 0 . . 959 22 . 664 -16, .361 ASN N 0. . 633 22 . 394 -17, .590 ASN C 2 . .394 21. 359 -18, . 991 ASN CB 0 . . 004 . 780 -19, .292 ASN ODl -0 . . 836 19 . 355 -17. .502 THR N 3. .018 22 . 505 -18. . 923 THR C . .381 23 . 247 -18. .818 THR CB 4 . .086 23. 672 -20. . 952 THR CG2 3. . 147 23 . 130 -22. .032 THR CA 6. .216 23 . 612 -16. .588 THR O 7 , .402 21. 580 -17 , .095 THR OGl . . 129 22. 178 -15. . 040 THR N 8. .499 23. 296 -16, .076 THR C 9. . 621 22 . 031 -14 , .414 THR CB 11. .080 23. 455 -15, .897 THR CG2 12 . .286 22. 628 -15. .406 LYS CA 9. .364 . 063 -13. ,010 LYS O 11. . 662 . 274 -12. ,592 LYS CG 9. .018 17 . 805 -11, . 921 LYS CE . .212 . 940 -10, .623 LEU N . .212 . 674 -10. .824 LEU C 11. .250 . 232 -8 , , 614 LEU CB 11. , 187 22 . 547 -9. .522 LEU CDl 11. ,245 . 003 -9. , 921 GLY N . .431 19. 282 -8 . ,298 GLY C 9. .168 18 . 703 -6. ,373 ASP N 9. .024 18 . 282 -5, ,150 ASP C 6. , 659 18 . 941 -4 , .709 ASP CB 7 . . 996 17 . 540 -3. .053 ASP ODl . . 611 17. 527 -2, ,354 SER N . 560 18 . 610 -5 . ,312 SER C 4 . 046 . 362 -4 . 289 SER CB 3. 345 18 . 919 -6. 289 PHE N 4 . 241 19 . 778 -3. 112 PHE C 4 . 544 21. 846 -1. 863 PHE CB 4 . 053 19 . 749 -0. 563 PHE CDl 2. 206 . 163 1. 125 PHE CEl 1. 737 . 717 2 . 315 PHE CZ 2. 605 21. 465 3. 114 TYR CA 6. 688 22 . 914 -2. 251 TYR 0 7. 201 24 . 853 -3. 393 TYR CG 8 . 146 21 . 892 -0 . 454 TYR CD2 8. 149 22 . 669 0. 698 TYR CE2 8. 114 22 . 069 1. ,962 TYR OH t . 965 . 029 3. ,205 TYR CA 6. 812 23. 655 -6. ,022 TYR 0 . 781 24 . 117 -8. 111 N Z. PATffJT orr-'CF -4 MAY 1993 247 247 247 247 247 247 243 243 243 249 249 249 250 250 250 250 251 251 251 251 252 252 252 252 253 253 253 253 254 254 254 255 255 255 255 256 256 256 256 257 257 257 257 258 258 259 259 259 259 260 260 260 261 261 261 261 261 261 262 262 262 262 262 262 263 263 iV; 1 i ' ' ' ' I 6 3 T'.'p cb 7 . . 928 22 . .768 -6. . 681 263 TYp.

CG 9 .279 23 . 035 -6 .068 2 63 t ye. cel . .0 64 24 . . 046 -6. .657 263 TYR CD2 9 .800 22 . 342 -4 .995 2 62 typ. cel 11 . . 335 24 . .323 -6. .168 263 TYR CE2 11 .062 22 . 640 -4 .491 2 63 typ. cz 11 . . 838 23 . 613 -5. .106 263 TYR OH 13 .065 23. ,949 -4 .597 2 — 4 gly n 4 . . 471 23 , .161 -6, .516 264 GLY CA 3 .301 23 . .064 -7 .412 2 i «t gly c 3 . . 847 22 . .196 -8 , .556 264 GLY 0 4 . 647 21. 274 -8 .365 2 65 lys 3 . .436 22 . .477 -9. .754 265 LYS CA 3 .834 21. 798 -10 . 971 o 65 lys c , .188 22 . .232 -11, .464 265 LYS O .684 21. 563 -12 .386 2 6 5 lys c3 2 . . 755 22 . .071 -12, . 044 265 LYS CG 1 .521 21 . 416 -11 .373 65 ly s cd 0 . . 624 . . 700 -12, .333 265 LYS CE -0 .813 . 839 -11 .736 2 65 lys NZ -0 . . 724 . .252 -10, .360 266 GLY N .787 23 . 22 6 -10 . 817 2 6 gly ca 7 , . 120 23 . . 612 -11, .325 266 GLY C 7 .155 . 052 -11 .818 2 c 6 gly c 6 , . 177 , .793 -11, .648 267 LEU N 8 .2 62 . 336 -12 .480 2 6 " le u ca 6 . .490 26. . 660 -13, .097 267 LEU C 7 .804 26. 771 -14 .437 2 6 7 LE j 0 7 . . 953 . . 909 -15, .298 267 LEU C3 .010 26. 855 -13 .214 o 67 lsu cg . . 432 28 . .060 -14 , .058 2 67 LEU CDl .096 29 . 331 -13 .250 2 6 7 lf.:j cd 2 11 . . 924 27 . . 921 -14 , .327 263 ILE N 7 .064 27 . 863 -14 . 632 2 '5 : i le ca 6 . . 406 28 . . 035 -15, . 944 268 ILE C 7 .426 28 . 246 -17 . 065 o 6 c ile 0 8 . .539 28. .793 -16. .912 268 ILE CB .369 29 . 210 -15 . 899 2 6 ~ ile cgi 6 . . 099 . . 541 -15. .592 268 ILE CG2 4 .243 28 . 925 -14 . 867 2 6 6 i le cd i . . 399 31 . .769 -16, .262 269 ASN N 7 .007 27 . 843 -18 .237 2 6 9 asn" ca 7 . . 802 27 . . 975 -19. .457 269 ASN C 6 .839 28 . 554 -20 .485 2 6 9 asn 0 . . 965 27 . .760 -20. . 943 269 ASN CB 8 .432 26. 653 -19 . 895 2 6 9 A 5N cg 9 . . 161 26. . 806 -21. .210 269 ASN ODl 8 . 993 27 . 626 -22 . 122 ■"* nd2 .

. Oil . .796 -21, .472 270 VAL N 6 . 908 29. 868 -20 .724 *.* *>_ t f ca . .8 63 . .418 -21, . 614 270 VAL C 6 .059 . 007 -23 . 054 2 ' 'j VAL 0 . .097 29 . . 969 -23, .872 270 VAL CB . 656 31. 950 -21. . 422 CGI 6 . . 849 32 . . 797 -21, .879 270 VAL CG2 4 .420 32 . 362 -22, .232 2!,t N 7 . , 325 29 . .701 -23, .352 271 GLN CA 7 .603 29 . 270 -24 .744 2 r.-.:; C 6 . , 869 27 , . 934 -25, .031 271 GLN O 6 .213 27 . 806 -26, .091 ' 1 cr- g . . 104 29 . .220 -24 , .964 271 GLN CG 9 .406 28 . 618 -26, .338 2 CLN cd . . 901 28 . .585 -26, .582 271 GLN OE1 11. .369 28 . 579 -27, .718 2 7 1 gln NE2 11 . 702 28 . .553 -25, .510 272 ALA N 6, . 977 26. 999 -24, .092 2 " z. al/i ca 6. , 224 . .712 -24 , .240 272 ALA C 4 .701 . 958 -24, .164 2 7 L ali-—. 0 3 . . 898 . . 505 -25, .001 272 ALA CB 6 .743 24 . 742 -23, .172 7 3 a. la N 4 . , 247 26. . 661 -23, .135 273 ALA CA 2 .840 26. 921 -22. .859 o 7 3 ALA c 2. . 081 27. .528 -24, .020 273 ALA O 0 , .899 27 . 219 -24. .255 2 7 3 Aix/\ cb 2 . . 736 27. .773 -21, .585 274 ALA N 2 .755 28. 404 -24, .762 2 / H ala cb 2 . . 952 . . 391 -26 .210 274 ALA CA 2 .109 29. 144 -25. .847 2 / M a:, a c 1 . , 730 28 . . 367 -27 .090 274 ALA O 0, . 980 28. 949 -27. .921 2 7 :j GLN N 2 . , 350 27 . .194 -27, .314 275 GLN CA 2 .048 26 . 389 -28. .527 2 7 S gln c 2 . . 147 27 , .261 -29 .777 275 GLN O 3 .260 27 . 807 -29, .916 ~ 7 5 GLN ot 1 . , 193 27 , . 361 -30 .590 275 GLN CB 0 .666 . 734 -28, .520 2 7 S GLN cg 0. , 501 24 . . 664 -27 .447 275 GLN CD -0 .823 23. 936 -27. .631 2 7 5 gln 0e1 -1. . 376 23. .895 -28, .729 275 GLN NE2 -1, .373 23. 411 -26. .538 The above structural studies together with the kinetic data presented herein and elsewhere (Philipp, M. , et al. (1983) Mol. Cell. Biochem. 51, 5-32; Svendsen, I. B. (197 6) Carl sberq Res. Comm. 41, 237-251; Markland, S.F. Id; Stauffe, D.C., et al. (1965) J. Biol. Chem. 244. 5333-5338) indicate that the subsites in the binding cleft of subtilisin are capable of interacting with substrate amino acid residues from P-4 to P-21.

The most extensively studied of the above residues are Glyl66, Glyl69 and Alal52. These amino acids were identified as residues within the S-l subsite. As seen in Fig. 3, which is a stereoview of the S-l : 5 subsite, Glyl66 and Glyl69 occupy positions at the bottom of the S-l subsite, whereas Alal52 occupies a position near the top of S-l, close to the catalytic Ser2 21. 2 0 All 19 amino acid substitutions of Glyl66 and Glyl69 have been made. As will be indicated in the examples which follow, the preferred replacement amino acids for Glyl66 and/or Glyl69 will depend on the specific amino acid occupying the P-l position of a given 2 5 substrate.

The only substitutions of Alal52 presently made and analyzed comprise the replacement of Alal52 with Gly and Ser. The results of these substitutions on P-l specificity will be presented in the examples.

In addition to those residues specifically associated with specificity for the P-l substrate amino acid, Tyrl04 has been identified as being involved with P-4 specificity. Substitutions at Phel89 and Tyr217, however, are expected to respectively effect P-2' and P-l' specificity.

The catalytic activity of subtilisin has also been modified by single amino acid substitutions at Asnl55. The catalytic triad of subtilisin is shown in Fig. 4. As can be seen, Ser221, His64 and Asp32 are positioned to facilitate nucleophilic attach by the serine hydoxylate on the carbonyl of the scissile peptide bond. Crystallographic studies of subtilisin (Robertus, et al. (1972) Biochem. 11, 4293-4303; Matthews, et al. (1975) J. Biol. Chem. 250. 7120-7126; Poulos, et al. (1976) J. Biol. Chem. 250, 1097-1103) show that two hydrogen bonds are formed with the oxyanion of the substrate transition state. One hydrogen bond donor is from the catalytic serine-221 main-chain amide while the other is from one of the NE2 protons of the asparagine-155 side chain. See Fig. 4.

Asnl55 was substituted with Ala, Asp, His, Glu and Thr. These substitutions were made to investigate the the stabilization of the charged tetrahedral intermediate of the transition state complex by the potential hydrogen bond between the side chain of Asnl55 and the oxyanion of the intermediate. These particular substitutions caused large decreases in substrate turnover, kcat (200 to 4,000 fold), marginal decreases in substrate binding Km (up to 7 fold), and a loss in transition state stabilization energy of 2.2 to 4.7 kcal/mol. The retention of Km and the drop in kcat will make these mutant enzymes useful as binding proteins for specific peptide sequences, the nature of which will be determined by the specificity of the precursor protease. «>? a • i h 0 6 / Various other amino acid residues have been identified which affect alkaline stability. In some cases, mutants having altered alkaline stability also have 5 altered thermal stability.

In B amvlol icruefaciens subtilisin residues Asp3 6, Ilel07, Lysl70, Ser204 and Lys213 have been identified as residues which upon substitution with a different 2; amino acid alter the alkaline stability of the mutated enzyme as compared to the precursor enzyme. The substitution of Asp3 6 with Ala and the substitution of Lysl7 0 with Glu each resulted in a mutant enzyme having a lower alkaline stability as compared to the wild type subtilisin. When Ilel07 was substituted with Val, Ser204 substituted with Cys, Arg or Leu or Lys213 substituted with Arg, the mutant subtilisin had a greater alkaline stability as compared to the wild type subtilisin. However, the mutant Ser204P demonstrated a decrease in alkaline stability.

In addition, other residues, identified as being associated with the modification of other properties of subtilisin, also affect alkaline stability. These residues include Ser24, Met50, Glul56, Glyl66, Glyl69 and Tyr217. Specifically the following particular substitutions result in an increased alkaline stability: Ser24C, Met50F, Glyl56Q or S, Glyl66A, H, K, N or Q, Glvl69S or A, and Tyr217F, K, R or L. The q mutant Met50V, on the other hand, results in a decrease in the alkaline stability of the mutant subtilisin as compared to wild type subtilisin.

Other residues involved in alkaline stability based on 5 the alkaline stability screen include Aspl97 and Met222. Particular mutants include Aspl97(R or A) and Het 222 (all other amino acids).

Various other residues have been identified as being involved in thermal stability as determined by the thermal stability screen herein. These residues include the above identified residues which effect alkaline stability and Metl99 and Tyr21. These latter two residues are also believed to be important for alkaline stability. Mutants at these residues include 1199 and F21. 1 ^ The amino acid sequence of B. amvlol icrue faciens substilisin has also been modified by substituting two cr more amino acids of the wild-type sequence. Six categories of multiply substituted mutant subtilisin , _ have been identified. The first two categories comprise thermally and oxidatively stable mutants. The next three other categories comprise mutants which combine the useful properties of any of several single mutations of B. amvlol icrue faciens subtilisin. The last category comprises mutants which have modified ... ! J alkaline and/or thermal stability.

The first category comprises double mutants in which two cysteine residues have been substituted at various amino acid residue positions within the subtilisin -j molecule. Formation of disulfide bridges between the two substituted cysteine residues results in mutant subtilisins with altered thermal stability and catalytic activity. These mutants include A21/C22/C87 and C24/C87 which will be described in more detail in su ' Example 11.

The second category of multiple subtilisin mutants comprises mutants which are stable in the presence of various oxidizing agents such as hydrogen peroxide or peracids. Examples 1 and 2 describe these mutants which include F50/I124/Q222 , F50/I124, F50/Q222, F50/L124/Q222, I124/Q222 and L124/Q222.

The third category of multiple subtilisin mutants comprises mutants vith substitutions at position 222 coriined with various substitutions at positions 166 or 169. These mutants, for example, combine the property of oxidative stability of the A222 mutation with the altered substrate specificity of the various 166 or 169 substitutions. Such multiple mutants include A166/A222, A166/C222, F166/C222, K166/A222, K166/C222, V166/A222 and V166/C222. The K166/A222 mutant subtilisin, for example, has a kcat/Km ratio which is approximately two times greater than that of the single A222 mutant subtilisin when compared using a substrate with phenylalanine as the P-l amino acid. This category of multiple mutant is described in more detail in Example 12.

The fourth category of multiple mutants combines substitutions at position 156 (Glu to Q or S) with the substitution of Lys at position 166. Either of these single mutations improve enzyme performance upon substrates with glutamate as the P-l amino acid. When these single mutations are combined, the resulting multiple enzyme mutants perform better than either precursor. See Example 9.

The fifth category of multiple mutants contain the substitution of up to four amino acids of the B. amvloliquefaciens subtilisin sequence. These mutants have specific properties which are virtually identicle to the properties of the subtilisin from B. licheniformis. The subtilisin from B. licheniformis differs from B. arovlolicruefaciens subtilisin at 87 out of 275 amino acids. The multiple mutant t *t 0 6 F50/S156/A169/L217 was found to have similar substrate specificity and kinetics to the licheniformis enzyme. (See Example 13.) However, this is probably due to only three of the mutations (S156, A169 and L217) which are present in the substrate binding region of the enzyme. It is quite surprising that, by making only three changes out of the 87 different amino acids between the sequence of the two enzymes, the B. amvlolicruifaciens enzyme was converted into an enzyme with properties similar to B. lichen!formis enzyme. Other enzymes in this series include F50/Q156/N166/L217 and F50/S156/L217.

The sixth category of multiple mutants includes the combination of substitutions at position 107 (lie to V) with the substitution of Lys at position 213 with Arg, and the combination of substitutions of position 204 (preferably Ser to C or L but also to all other amino acids) with the substituion of Lys at position 213 with R. Other multiple mutants which have altered alkaline stability include Q156/K166, Q156/N166, S156/K166, S156/N166 (previously identified as having altered substrate specificity), and F50/S156/A169/L217 (previously identified as a mutant of B. a my1o1i gu i f a c i e n s subtilisin having properties similar to subtilisin from B. licheniformis). The mutant F50/V107/R213 was constructed based on the observed increase in alkaline stability for the single mutants F50, V107 and R213. It was determined that the V107/R213 mutant had an increased alkaline stability as compared to the wild type subtilisin. In this particular mutant, the increased alkaline stability was the result of the cumulative stability of each of the individual mutations. Similarly, the mutant F50/V107/R213 had an even greater alkaline stability as compared to the V107/R213 mutant indicating that 2*0? the increase in the alkaline stability due to the F50 mutation was also cumulative.

Table IV summarizes the multiple mutants which have been made including those not mentioned above.

In addition, based in part on the above results, substitution at the following residues in subtilisin is expected to produce a multiple mutant having increased thermal and alkaline stability: Ser24, Met50, Ilel07, Glul56, Glyl66, Glyl69, Ser204, Lys213, Gly215, and Tyr217.

TA3LE IV - Li Double Mutants C22/C87 C24/C87 V4 5/V4 8 C49/C94 C49/C95 C50/C95 C50/C110 F50/I124 F50/Q2 2 2 I124/Q222 Q156/D166 Q156/K166 Q156/N166 S156/D166 S15 6/K16 6 S15 6/N16 6 S156/A169 A166/A222 A166/C222 F166/A222 F166/C222 K166/A2 2 2 K166/C22 2 V166/A22 2 V166/C222 A169/A222 A169/A222 A169/C222 A21/C2 2 Triple, Quadruple or Other Multiple F50/I124/Q222 F50/L124/Q222 F50/L124/A2 2 2 A21/C22/C87 F5 0/S156/N166/L217 F50/Q15 6/N166/L217 F50/S156/A169/L217 F50/S156/L217 F50/Q156/K166/L217 F50/S156/K166/L217 F50/Q15 6/K166/K217 F50/S156/K166/K217 F50/V107/R213 [S15 3/S156/A158/G159/S16 0/A161-164/I165/S166/A169/R17 0] L204/R213 R213/204A, E, Q, D, N, G, K, V, R, T, P, I, M, F, Y, W or H VI07/R213 In addition to the above identified amino acid residues, other amino acid residues of subtilisin are Z 4 o r * also considered to be important with regard to substrate specificity. Mutation of each of these residues is expected to produce changes in the substrate specificity of subtilisin. Moreover, multiple mutations among these residues and among the previously identified residues are also expected to produce subtilisin mutants having novel substrate specificity.

I C Particularly important residues are His67, Ilei07, Leul2 6 and Leul35. Mutation of His67 should alter the S-l1 subsite, thereby altering the specificity of the mutant for the P-l1 substrate residue. Changes at this position could also affect the pH activity prcfile of the mutant. This residue was identified based on the inventor's substrate modeling from product inhibitor complexes.

Ilel07 is involved in P-4 binding. Mutation at this position thus should alter specificity for the P-4 substrate residue in addition to the observed effect on alkaline stability. 21el07 was also identified by molecular modeling from product inhibitor complexes.

The S-2 binding site includes the Leul26 residue. Modification at this position should therefore affect P-2 specificity. Moreover, this residue is believed to be important to convert subtilisin to an amino peptidase. The pH activity profile should also be modified by appropriate substitution. These residues were identified from inspection of the refined model, the three dimensional structure from modeling studies. A longer side chain is expected to preclude binding of any side chain at the S-2 subsite. Therefore, binding would be restricted to subsites S-l, S-l', S-2', S-31 24 0 6 and cleavage would be forced to occur after the amino terminal peptide.

Leul3 5 is in the S-4 subsite and if mutated should -> alter substrate specificity for P-4 if mutated. This residue was identified by inspection of the three-dimensional structure and modeling based on the product inhibitor complex of F222.

In addition to these sites, specific amino acid residues within the segments 97-103, 126-129 and 213-215 are also believed to be important to substrate binding.

— Segments 97-103 and 126-129 form an antiparallel beta sheet with the main chain of substrate residues P-4 through P-2. Mutating residues in those regions should affect the substrate orientation through main chain (enzyme) - main chain (substrate) interactions, since the main chain of these substrate residues do not interact with these particular residues within the S-4 through S-2 subsites.

Within the segment 97-103, Gly97 and Asp99 may be mutated to alter the position of residues 101-103 within the segment. Changes at these sites must be compatible, however. In B. amvlol icrui faciens subtilisin Asp99 stabilizes a turn in the main chain tertiary folding that affects the direction of 30 residues 101-103. B. 1icheniformis subtilisin Asp97, functions in an analogous manner.

In addition to Gly97 and Asp99, SerlOl interacts with Asp99 in B. amvlicuefaciens subtilisin to stabilize 35 the same main chain turn. Alterations at this residue should alter the 101-103 main chain direction. u e Kutations at Glul03 are also expected to affect the 101-103 main chain direction.

The side chain of Glyl02 interacts with the substrate P-3 amino acid. Side chains of substituted amino acids thus are expected to significantly affect specificity for the P-3 substrate amino acids.

All the amino acids within the 127-129 segment are considered important to substrate specificity. Gly 127 is positioned such that its side chain interacts with the S-l and S-3 subsites. Altering this residue thus should alter the specificity for P-l and P-3 residues of the substrate.

The side chain of Glyl28 comprises a part of both the S-2 and S-4 subsites. Altered specificity for P-2 and P-4 therefore would be expected upon mutation. Moreover, such mutation may convert subtilisin into an amino peptidase for the same reasons substitutions of Leul2 5 would be expected to produce that result.

The Prol29 residue is likely to restrict the conformational freedom of the sequence 126-133, residues which may play a major role in determining P-l specificity. Replacing Pro may introduce more flexibility thereby broadening the range of binding capabilities of such mutants.

The side chain of Lys213 is located within the S-3 subsite. All of the amino acids within the 213-215 segment are also considered to be important to substrate specificity. Accordingly, altered P-3 substrate specificity is expected upon mutation of this residue. 2 0 The Tyr214 residue does not interact with substrate but is positioned such that it could affect the conformation of the hair pin loop 204-217.

Finally, mutation of the Gly215 residue should affect the S-3' subsite, and thereby alter P-31 specificity.

In addition to the above substitutions of amino acids, the insertion or deletion of one or more amino acids within the external loop comprising residues 152-172 may also affect specificity. This is because these residues may play a role in the "secondary contact region" described in the model of streptomyces subtilisin inhibitor complexed with subtilisin. Hirono, et al. (1984) J. Mol. Biol. 178 , 389-413. Thermitase K has a deletion in this region, which eliminates several of these "secondary contact" residues. In particular, deletion of residues 151 through 164 is expected to produce a mutant subtilisin having modified substrate specificity. In addition, a rearrangement in this area induced by the deletion should alter the position of many residues involved in substrate binding, predominantly at P-l. This, in turn, should affect overall activity against proteinaceous substrates.

The effect of deletion of residues 161 through 164 has been shown by comparing the activity of the wild type (WT) enzyme with a mutant enzyme containing this deletion as well as multiple substitutions (i.e., S153/S156/A158/G159/S160/A 161-164/I165/S166/A169/ R170) . This produced the following results: 2406 TA3LE V kcat Kit, kcat/Km WT 50 1.4xlO~4 3.6xl05 Deletion mutant 8 5.0xl0~^ 1.6x10^ The WT has a kcat 6 times greater than the deletion mutant but substrate binding is 28 fold tighter by the in deletion mutant. The overall efficiency cf the deletion mutant is thus 4.4 times higher than the WT enzyme.

All of these above identified residues which have yet 15 tc be substituted, deleted or inserted into are presented in Table VI.

TABLE VI Substitution/Insertion/Deletion Res idues His 6 7 Ala 152 Leul26 Alal53 Leul35 Glyl54 Gly9 7 Asnl55 Asp99 Glyl56 SerlOl Glyl57 Glyl02 Gly160 Glul03 Thrl58 Leul26 Ser159 Glyl27 Serl61 Gly128 Serl62 Prol2 9 Ser163 Tyr214 Thrl64 Gly215 Vail 65 Glyl66 Gly169 Tyr167 Lys170 Prol68 Tyr171 Pro172 <toe: j The following disclosure is intended to serve as a representation of embodiments herein, and should not be construed as limiting the scope of this application. These specific examples disclose the - construction of certain of the above identified mutants. The construction of the other mutants, however, is apparent from the disclosure herein and that presented ir. New Zealand Patent Specification No. 208612.

All literature citations are expressly incorporated by reference.

EXAMPLE 1 ^■5 Identification of Peracid Oxicizable Residues of Subtilisin Q2 2 2 and L222 As shown in Figures 6A and 6B, organic peracid cxicar.ts inactivate the mutant subtilisins Met222L and Met222Q (L222 and Q222) . This example describes the —' identification of peracid oxidizable sites in these mutant subtilisins.

First, the type of amino acid involved in peracid oxidation was determined. Except under drastic " conditions (Means, G.E., et al. (1971) Chemical Modifications of Proteins, Holden-Day, S.F., CA, pp. 160-162), organic peracids modify only methionine and tryptophan in subtilisin. Difference spectra of the enzyme over the 2 50nm to 3 50nm range were determined during an inactivation titration employing the reagent, diperdodecanoic acid (DPDA) as oxidant. Despite quantitative inactivation of the enzyme, no change in absorbance over this wavelength range was noted as shown in Figures 7A and 7B indicating that 5 tryptophan was not oxidized. Fontana, A., et al: (1980) Methods in Peptide and Protein Secuence Ar. a 1 v sis (C. Eirr ed.) Elsevier, New York, p. 305 . The absence of tryptophan modification implied oxidation cf one or more of the remaining methionines cf E. amyloliquefaciens subtilisin. See Figure 1.

To confirm this result the recombinant subtilisin MetI22F was cleaved with cyanogen bromide (CNBr) both before ar.c after oxidation by DPDA. The peptides produced by CNBr cleavage were analyzed on high resolution SDS-pvricine peptide gels (SPG).

Subtilisin Met22 2F (F22 2) was oxidized in the following manner. Purified F222 was resusper.ded in C.l M socium borate pH 9.5 at 10 mg/ml and was added to a final concentration of 26 diperdodecanoic acid (DPDA) at 26 mg/ir.l was added to produce an effective active oxygen concentration of 30 ppm. The sample was incubated for at least 30 minutes at room temperature ar.c then quenched with 0.1 volume of 1 M Tris pH 8.6 buffer to produce a final concentration of 0.1 M Tris r:! £.6). 3mM phenylmethylsulfonyl fluoride (PMSF) was added ar.c 2.5 ml of the sample was applied to a Pharmacia PD10 column equilibrated in 10 mM sodium phosphate pH 6. 2, 1 mM PMSF. 3.5 ml of 10 irM sodium phcsphate pH6.2, lmM PMSF was applied and the eluant collected.

F2 22 and DPDA oxidized F222 were precipitated with 9 volumes of acetone at -20°C. The samples were resuspended at 10 mg/ml in 8M urea in 88% formic acid and allowed to sit for 5 minutes. An equal volume of 200 mg/ml CNBr in 88% formic acid was added (5 mg/ml protein) and the samples incubated for 2 hours at room temperature in the dark. Prior to gel electrophoresis, the samples were lyophilized and resuspended at 2-5 mg/ml in sample buffer (1% & H (J ^ cvrid.ne, 5* N'aDccSO , 5» glycerol and brorr.oDhenol •3 blue) and disassociated at 95°C for 3 minutes.

The samples were electrophoresed on discontinuous ? pclyacrylamice gels (Kyte, J. , et al. (1983) Anal. Bioch. 133 , 515-522). The gels were stained using the Pharmacia silver staining technique (Sarrjr.ons , D.W., et al. (1981) Elect rophores is 2 13 5-141).

The results of this experiment are shown in Figure 8. As can be seen, F222 treated with CNBr only cives nine resolved bar.ds on SPG. However, when F222 is alsc treated with DPDA prior to cleavage, bands X, 7 and 9 -disappear whereas bands 5 and 6 are greatly increased in. intensity.

Ir. order to determine which of the methionines were 0 effected, each of the CNBr peptides was isolated by reversed phase HPLC and further characterized. The buffer system in both Solvent A (aqueous) and Solvent E (organic) for all HPLC separations was 0.05% triethvlamime/trifloroacetic acid (TEA-TFA). In all •'5 cases unless noted, solvent A consisted of 0.05% TEA-TFA in H^O, solvent B was 0.0 5% TEA-TFA in 1-propanol, and the flow rate was 0.5 ml/minute.

For HPLC analysis, two injections of 1 mg enzyme 30 digest were used. Three samples were acetone precipitated, washed and dried. The dried 1 mg samples were resuspended at 10 mg/ml in 8M urea, 88% formic acid; an equal volume of 200 mg/ml CNBr in 88% formic acid was added (5 mg/ml protein). After 35 incubation for 2 hours in the dark at room temperature, the samples were desalted on a 0.8 cm X 7 Ik Or cr. cclurr.r. cf Tris Acryl GF05 coarse resir. (IBF, Paris, France) equilibrated with 40% solvent B, 60% solvent A. 200 ul samples were applied at a flow rate of 1 ml a minute and 1.0-1.2 ml collected by monitoring the absorbance at 2 80r.rr.. Prior to injection on the HPLC, each desalted sample was diluted with 3 volumes of solvent A. The samples were injected at 1.0 ml/min (2 minutes) and the flow then adjusted to 0.5 ml/min (1001 A). After 2 minutes, a linear gradient to 60% B at 1.0s 5/ir.ir. was initiated. From each 1 mg run, the pooled peaks were sampled (50ul) and analyzed by gel electrophoresis as described above.

Each polypeptide isolated by reversed phase HPLC was further analyzed for homogeneity by SPG. The position of each peptide on the known gene sequence (Wells, J.A., et al. (1983) Nucleic Acids Res. 11 7911-7924) was obtained through a combination of amino acid compositional analysis and, where needed, amino terminal sequencing.

Prior to such analysis the following peptides were to rechromatographed. 1. CNBr peptides from F222 not treated with DPDA: Peptide 5 was subjected to two additional reversed phase separations. The 10 cm C4 column was equilibrated to 80%A/ 20%B and the pooled sample applied and washed for 2 minutes. Next an 0.5% ml B/min gradient was initiated. Fractions from this separation were again rerun, this time on the 25 cm C4 column, and employing 0.05% TEA-TFA in acetonitrile/l-propanol (1:1) for solvent B. The gradient was identical to the one just described. 3 0 2 4 u 6 / o Peptide "X" was subjected to one additional separation after the initial chromatography. The sample was applied and washed for 2 minutes at 0.5ml/min {10 0 % A) , ana a 0.5% ml B/min gradient was initiated.

Peptides 7 and 9 were rechromatographed in a similar .manner to the first rerun of peptide 5.

Peptide 8 was purified to homogeneity after the initial separation. 2. CNBr Peptides from DPDA Oxidized F222: Peptides 5 and 6 from a CNBr digest of the oxidized F 222 were purified in the same manner as peptide 5 from the untreated enzyme.

Ami no acid compositional analysis was obtained as follows. Samples (-InM each amino acid) were dried, hycrolyzec in vacuo with 100 ul 6N HC1 at 106°C for 24 hours ar.c then dried in a Speed Vac. The samples were analyzed on a Beckmann 6300 AA analyzer employing r.inhycrin detection.

Amino terminal sequence data was obtained as previously described (Rodriguez, H., et al. (1984) Anal. Biochem. 134, 538-547).

The results are shown in Table VII and Figure 9. & ♦ V « TABLE VII Amino and COOH terminii of CNBr fragments Terminus and Method Fragment amino, method COOH. method X 1, sequence 50, composition 9 51, sequence 119, composition 7 125, sequence 199, composition 8 200, sequence 275, composition 5ox 1, sequence 119, composition 6ox 120, composition 199, composition Peptides 5ox and 6ox refer to peptides 5 and 6 isolated from CN3r digests of the oxidized protein where their respective levels are enhanced.

From the data in Table VII and the comparison of SPG tracks for the oxidized and native protein digests in Figure 8, it is apparent that (1) Met50 is oxidized leading to the loss of peptides X and 9 and the appearance of 5; and (2) Metl24 is also oxidized Jleading to the loss of peptide 7 and the accumulation of peptide 6. Thus oxidation of B. amvloliguifaciens subtilisin with the peracid, diperdocecanoic acid leads to the specific oxidation of methionine at residues 50 and 124. :o EXAMPLE 2 Substitution at MetSO and Metl24 in Subtilisin Met222Q ^r' The choice of amino acid for substitution at Met50 was based on the available sequence data for subtilisins froa B. 1 icheni f ormis (Smith, E.C., et al. (1968) J. Biol. Chem. 243. 2184-2191), B. DY (Nedkov, P., et al. (1983) Hoppe Savler's Z. Physiol. Chem. 364 1537-1540), B. amylosacchariticus (Markland, F.S., et al. (1967) J. Biol. Chem. 242 5198-5211) and B. subtilis (Stahl, M.L., et al. (1984) J. Bacteriol. 155, 411-418). In all cases, position 50 is a phenylalanine. See Figure 5. Therefore, Phe50 was chosen for construction.

At position 124, all known subtilisins possess a methionine. See Figure 5. Molecular modelling of the x-ray derived protein structure was therefore required to determine the most probable candidates for substitution. From all 19 candidates, isoleucine and leucine were chosen as the best residues to employ. In order to test whether or not modification at one site but not both was sufficient to increase oxidative stability, all possible combinations were built on the Q222 backbone (F50/Q222, I124/Q222, F50/I124/Q222).

A. Construction of Mutations Between Codons 45 and 50 All manipulations for cassette mutagenesis were carried out on pS4.5 using methods disclosed in EPO Publication No. 0130756 and Wells, J.A., et al, (1985) Gene 34., 315-323 . The p£50 in Fig. 10, line 4, mutations was produced using the mutagenesis primer shown in Fig. 10, line 6, and employed an approach designated as restriction-purification which is described below. Briefly, a M13 template containing the subtilisin gene, M13mpll-SUBT was used for heteroduplex synthesis (Adelman, et aj. (1983) , DNA 2, 183-193). Following transfection of JM101 (ATCC 33876), the 1.5 kb EcoRI-BamHI fragment containing the ^40670 subtilisin gene was subcloned from M13mpll SU3T rf into a recipient vector fragment of pBS4 2 the construction of which is described in EPO Publication r No. 0130756. To enrich for the mutant sequence (p.i50, line 4), the resulting plasmid pool was digested with KmI. and linear molecules were purified by polyacrylamide gel electrophoresis. Linear molecules were ligated back to a circular form, and transformed into E. coli MM2S4 cells (ATCC 31446). Isolated ^ plasnids were screened by restriction analysis for the Km I site. Kml"1" plasmids were sequenced and confirmed the p^50 sequence. Asterisks in Figure 11 indicate the bases that are mutated from the wid type sequence (line 4). pi 50 (line 4) was cut with StuI ar.c EcoRI and the 0.5 Kb fragment containing the 5' half of the subtilisin gene was purified (fragment 1). pi50 (line 4) was digested with Kpnl and EcoRI and the 4.0 Kb fragment containing the 3' half of the subtilisin gene and vector sequences was purified (fragment 2). Fragments 1 and 2 (line 5), and duplex DNA cassettes coding for mutations desired (shaded sequence, line 6) were mixed in a molar ratio of 1:1:10, respectively. For the particular construction of this example the DNA cassette contained the triplet .J 'j TIT for codon 50 which encodes Phe. This plasmid was designated pF50. The mutant subtilisin was designated F50 . 3 0 B. Construction of Mutation Between Codons 122 and 127 The procedure of Example 2A was followed in substantial detail except that the mutagenesis primer of Figure 11, line 7 was used and restriction-purification for the EcoRV site in pAl24 was used. In addition, the DNA cassette (shaded sequence, Figure L 'v -Sill, iir.e 6) contained the triplet ATT for codon 124 which encodes lie and CTT for Leu. Those plasmids which contained the substitution of lie for Metl24were desigr.eated pI124. The mutant subtilisin was designated 1124.

C. Construction of Various F50/I124/Q222 Multiple Mutants The triple mutant, F50/I124/Q222, was constructed from a three-way ligation in which each fragment contained one of the three mutations. The single mutant Q222 (pQ2 2 2 ) was prepared by cassette mutagenesis as described in EPO Publication No. 0130756. The F50 nutation was contained on a 2.2kb Avail to PvuII fragment from pF50; the 1124 mutation was contained on a 260 bp PvuII to Avail fragment from pI124; and the Q2 2 2 mutation was contained on 2.7 kb Avail to Avail fragment from pQ222. The three fragments were ligated together and transformed into E. coli MM294 cells. Restriction analysis of plasmids from isolated transfcrmants confirmed the construction. To analyze the final construction it was convenient that the Avail site at position 798 in the wild-type subtilisin ger.e was eliminated by the 1124 construction.

The F5C/Q222 and I124/Q222 mutants were constructed in a similar manner except that the appropriate fragment from pS4.5 was used for the final construction.

D. Oxidative Stability of Q222 Mutants The above mutants were analyzed for stability to peracid oxidation. As shown in Fig. 12, upon incubation with diperdodecanoic acid (protein 2mg/mL, oxidant 75ppm[0]), both the I124/Q222 and the F53/H24/Q222 are completely stable whereas the F5C/Q222 and the Q222 are inactivated. This indicates that conversion of Metl24 to 1124 in subtilisin Q222 is sufficient to confer resistance to organic peracid oxidants.

EXAMPLE 3 Subtilisin Mutants Having Altered Substrate Specificity-Hydrophobic Substitutions at Residues 166 Subtilisin contains an extended binding cleft which is hydrophobic in character. A conserved glycine at residue 166 was replaced with twelve non-ionic amino acids which can project their side-chains into the S-l subsite. These mutants were constructed to determine the effect of changes in size and hydrophobicity on the binding of various substrates.

A. Kinetics for Hydrolysis of Substrates Having Altered P-l Amino Acids by Subtilisin from B. Amvlol icruefaciens Wild-type subtilisin was purified from B. subtilis culture supernatants expressing the B. amvlolicrue-faciens subtilisin gene (Wells, J.A., et <al. (1983) Nucleic Acids Res. 11, 7911-7925) as previously described (Estell, D.A., et al.. (1985) J. Biol. Chem. 2_6_0, 6518-6521). Details of the synthesis of tetrapeptide substrates having the form succinyl-L-AlaL-AlaL-ProL-[X]-p-nitroanilide (where X is the PI amino acid) are described by DelMar, E.G., et al. (1979) Anal. Biochem. 99, 316-320. Kinetic parameters, Km(M) and kcat(s-1) were measured using a modified progress curve analysis (Estell, D.A., et a_l. (1985) J. Biol. Chem. 260, 6518-6521). Briefly, plots ^40670 of rate versus product concentration were fit to the differential form of the rate equation using a nor.-linear regression algorithm. Errors in kcat and Km for all values reported are less than five percent. The various substrates in Table VIII are ranged in order of decreasing hydrophobicity. Nozaki, Y. (1971), J. Biol. Chem. 246 , 2211-2217; Tanford C. (1978) Science 200, 1012).

TABLE VIII PI substrate Amino Acid kcat (S-1) 1/Km(M-1) kcat/Km (s-1M-l) Phe 50 7 ,100 360 , 000 Tyr 28 40,000 1 ,100,000 Leu 24 3,100 75,000 Met 13 9,400 120,000 His 7.9 1,600 13,000 Ala 1.9 ,500 11,000 Gly 0.003 8 ,300 21 Gin 3.2 2 ,200 7,100 Ser 2.8 1 ,500 4 ,200 Glu 0.54 32 16 The ratio of kcat/Km (also referred to as catalytic efficienty) is the apparent second order rate constant for the conversion of free enzyme plus substrate (E+S) tc enzyme plus products (E+P) (Jencks, W.P., Catalysis in Chemistry and Enzymoloqy (McGraw-Hill, 1969) pp. 321-436; Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287). The log (kcat/Km) is proportional to transition state binding i / ol l-:; energy, A plot of the log kcat/Xn versus the hydrophobicity of the PI side-chain (Figure 14) shows a strong correlation (r = 0.98), with the exception of the glycine substrate which shows evidence for r.cn-productive binding. These data show that relative differences between transition-state binding energies can be accounted for by differences in P-l side-chain hydrophobicity. When the transition-state binding energies are calculated for these substrates and plotted versus their respective side-chain hydrophobicities, the line slope is 1.2 (not shown). A slope greater than unity, as is also the case for chymotrypsin (Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287; Harper, J.W., et al. (1984) Biochemistry. 23 . 2355-3002), suggests that the PI binding cleft is more hydrophobic than ethanol or dioxane solvents that were used to empirically determine the hydrophobicity of amino acids (Nozaki, Y., et a_l. J. Biol. Chem. (1971) 24 6. 2211-2217 ; Tanford, C. (1978) Science 200. 1012).

For amide hydrolysis by subtilisin, kcat can be interpreted as the acylation rate constant and Km as the dissociation constant, for the Michaelis complex (E-S), Ks. Gutfreund, H., et al (1956) Biochem. J. 63, 656. The fact that the log kcat, as well as log 1/Km, correlates with substrate hydrophobicity is consistent with proposals (Robertus, J.D., et al. (1972) Biochemistry 11 . 24 39-2 4 49 ; Robertus, J.D., et al. (1972) Biochemistry 11, 4293-4303) that during the acylation step the P-l side-chain moves deeper into the hydrophobic cleft as the substrate advances from the Michaelis complex (E-S) to the tetrahedral transition-state complex (E-S7). However, these data can also be interpreted as the hydrophobicity of the PI side-chain effecting the orientation, and thus the susceptibility of the scissile peptide bond to nuclecphilio attack by the hydroxyl group of the catalytic Ser221. 3 The dependence of kcat/Km on P-l side, chain hydrophobicity suggested that the kcat/Km for hydrophobic substrates may be increased by increasing the hydrophobicity of the S-l binding subsite. To test this hypothesis, hydrophobic amino acid substitutions of GlylSS were produced.

Since hydrophobicity of aliphatic side-chains is directly proportional to side-chain surface area (Rose, G.D., et al.. (1985) Science 229. 834-838; Reynolds, J.A., et al. (1974) Proc. Natl. Acad. Sci. USA 71. 2825-2927), increasing the hydrophobicity in the S-l subsite may also sterically hinder binding of larger substrates. Because of difficulties in predicting the relative importance of these two opposing effects, we elected to generate twelve non-charged mutations at position 166 to determine the resulting specificities against non-charged substrates of varied size and hydrophobicity. 2 5 B. Cassette Mutagenesis of the PI Binding Cleft The preparation of mutant subtilisims containing the substitution of the hydrophobic amino acids Ala, Val ^ and Phe into residue 166 has been described in New Zealand Patent Specification No. 208612. The same method was used to produce the remaining hydrophobic mutants at residue 166. In applying this method, two unique and silent restriction sites were introduced in the subtilisin ] ^ genes to closely flank the target codon 166. As can be seen in Figure 13, the wild type sequence (line 1) 1 4 o« was altered by site-directed mutagenesis in K13 using the indicated 37mer mutagenesis primer, to introduce a 13 bp delection (dashedline) and unique SacI and Xmal sites (underlined sequences) that closely flank codon 166. The subtilisin gene fragment was subcloned back into the E. coli - B. subtilis shuttle plasmid, p3S42, giving the plasmid p^l66 (Figure 13, line 2). pil66 was cut open with SacI and Xmal. and gapped linear molecules were purified (Figure 13, line 3). Pools of synthetic oligonucleotides containing the mutation of interest were annealed to give duplex DNA cassettes that were ligated into gapped p£l66 (underlined and overlined sequences in Figure 13, line 4). This construction restored the coding sequence except over position 166(NNN; line 4). Mutant sequences were confirmed by dideoxy sequencing. Asterisks denote sequence changes from the wild type sequence. Plasmids containing each mutant B. amvlol icruefaciens subtilisin gene were expressed at roughly equivalent levels in a protease deficient strain of B. subtil is. BG2036 as previously described. EPO Publication No. 013 07 56; Yang, M., et al. (19 84) J. Bacteriol. 160, 15-21; Estell, D.A., et al (1985) J. Biol. Chem. 260. 6518-6521.

C. Narrowing Substrate Specificity bv Steric Hindrance To probe the change in substrate specificity caused by steric alterations in the S-l subsite, position 166 mutants were kinetically analyzed versus PI substrates of increasing size (i.e., Ala, Met, Phe and Tyr). Ratios of kcat/Km are presented in log form in Figure 15 to allow direct comparisons of transition-state binding energies between various enzyme-substrate pairs. •> L -■? According to transition state theory, the free enery difference between the free enzyme plus substrate i£ (E + S) and the transition state complex (E-S') can be calculated from equation (1) , (1) = -RT In kcat/Km + RT In kT/h in which kcat is the turnover number, Km is the Michaelis constant, R is the gas constant, T is the temperature, k is Boltzmann's constant, and h is Planck's constant. Specificity differences are ezpressed quant itatively as differences between transition state binding energies (i.e., aaG^), and can be calculated from equation (2). (2) = -RT In (kcat/Km) /(kcat/Km) A and B represent either two different substrates assayed againt the same enzyme, or two mutant enzymes assayed against the same substrate.

As can be seen from Figure 15A, as the size of the side-chain at position 166 increases the substrate preference shifts from large to small P-l side-chains. Enlarging the side-chain at position 166 causes kcat/Km to decrease in proportion to the size of the P-l substrate side-chain (e.g., from Glyl66 (wild-type) through W166, the kcat/Km for the Tyr substrate is decreased most followed in order by the Phe, Met and Ala P-l substrates).

Specific steric changes in the position 166 side-chain, such as he presence of a £-hydroxyl group, p- or 7-aliphatic branching, cause large decreases in kcat/Km for larger PI substrates. Introducing a 0-hydroxyl group in going from A166 (Figure 15A) to v S166 (Figure 15B), causes an 8 fold and 4 fold reduction in kcat/Km for Phe and Tyr substrates, respectively, while the values for Ala and Met substrates are unchanged. Producing a £-branched structure, in going from S166 to T166, results in a drop of 14 and 4 fold in kcat/Kra for Phe and Tyr, respectively. These differences are slightly magnified for V166 which is slightly larger and iscsteric with T166. Enlarging the ^-branched substituents from V166 to 1166 causes a lowering of kcat/Km between two and six fold toward Met, Phe and Tyr substrates. Inserting a 7-branched structure, by replacing M166 (Figure 15A) with L166 (Figure 15B) , produces a 5 fold and 18 fold decrease in kcat/Km for Phe and Tyr substrates, respectively. Aliphatic 7-branched appears to induce less steric hindrance toward the Phe P-l substrate than £-branching, as evidenced by the 100 fold decrease in kcat/Km for the Phe substrate in going from L166 to 1166.

Reductions in kcat/Kra resulting from increases in side chain size in the S-l subsite, or specific structural features such as p- and 7-branching, are quantitatively illustrated in Figure 16. The kcat/Km values for the position 166 mutants determined for the Ala, Met, Phe, and Tyr P-l substrates (top panel through bottom panel, respectively), are plotted versus the position 166 side-chain volumes (Chothia, C. (19S4) Ann. Rev. Biochem. 53, 537-572). Catalytic efficiency for the Ala substrate reaches a maximum for 1166, and for the Met substrate it reaches a maximum between VI6 6 and LI 6 6. The Phe substrate shows a broad kcat/Km peak but is optimal with A166. Here, the ^-branched position 166 substitutions form a line that is parallel to, but roughly 50 fold lower in kcat/Km than side-chains of similar size [i.e., C166 versus £ 4 u 6 T166, L166 versus 1166]. The Tyr substrate is most efficiently utilized by wild type enzyme (Glyl66), and there is a steady decrease as one proceeds to large position 166 side-chains. The p-branched and 7-branched substitutions form a parallel line below the other non-charged substitutions of similar molecular volume.

The optimal substitution at position 166 decreases in volume with increasing volume of the PI substrate [i.e., 1166/Ala substrate, L166/Ket substrate, A166/Phe substrate, Glyl66/Tyr substrate]. The combined volumes for these optimal pairs may approximate the volume for productive binding in the S-l subsite. For the optimal pairs, Glyl66/Tyr substrate, A166/Phe substrate, L166/Ket substrate, V166/Met substrate, and 1166/Ala substrate, the 3 combined volumes are 266,295,313,339 and 261 A , respectively. Subtracting the volume of the peptide backbone from each pair (i.e., two times the volume of 3 glycine), an average side-chain volume of 160r32A for productive binding can be calculated.

The effect of volume, in excess to the productive binding volume, on the drop in transition-state binding energy can be estimated from the Tyr substrate curve (bottom panel, Figure 16), because these data, and modeling studies (Figure 2), suggest that any substitution beyond glycine causes steric repulsion. A best-fit line drawn to all the data (r = 0.87) gives a slope indicating a loss of roughly 3 kcal/mol in 3 transition state binding energy per 100A of excess volume. (100A3 is approximately the size of a leucyl side-chain.) D. Enhanced Catalytic Efficiency Correlates with Increasing Hydrophobicity of the Position 166 Substitution Substantial increases in kcat/Km occur with enlargement of the position 166 side-chain, except for the Tyr P-l substrate (Figure 16) . For example, kcat/Km increases in progressing from Glyl66 to 1166 for the Ala substrate (net of ten-fold) , from Glyl66 to L166 for the Met substrate (net of ten-fold) and from Glyl66 to A166 for the Phe substrate (net of two-fold). The increases in kcat/Km cannot be entirely explained by the attractive terms in the van der Waals potential energy function because of their strong distance dependence (1/r ) and because of the weak nature of these attractive forces (Jencks, W.P., Catalysis in Chemistry and Enzvmolocrv (McGraw-Hill, 1969) pp. 321-436; Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-2S7; Levitt, M. (1976) J. Mol. Biol. 104. 59-107). For example, Levitt (Levitt, M. (1976) J. Mol. Biol. 104. 59-107) has calculated that the van der Waals attraction between two methionyl residues would produce a maximal interaction energy of roughly -0.2 kcal/mol. This energy would translate to only 1.4 fold increase in kcat/Km.

The increases of catalytic efficiency caused by side-chain substitutions at position 166 are better accounted for by increases in the hydrophobicity of the S-l subsite. The increase kcat/Km observed for the Ala and Met substrates with increasing position 166 side-chain size would be expected, because hydrophobicity is roughly proportional to side-chain surface area (Rose, G.D., et al.. (1985) Science 229. 834-838; Reynolds, J.A., et al. (1974) Proc. Natl.

Another example that can be interpreted as a hydrophobic effect is seen when comparing kcat/Km for isosteric substitutions that differ in hydrophobicity such as S166 and C166 (Figure 16) . Cysteine is considerably more hydrophobic than serine (-1.0 versus + 0.3 kcal/mol) (Nozaki, Y., et al. (1971) J. Biol. Cheir.. 246. 2211-2217; Tanford, C. (1978) Science 200. 1012) . The difference in hydrophobicity correlates with the observation that C166 becomes more efficient relative to SerI66 as the hydrophobicity of the substrates increases (i.e., Ala < Met < Tye < Phe). Steric hindrance cannot explain these differences because serine is considerably smaller than cysteine (99 versus 118A3). Paul, I.e., Chemistry of the -SH Group (ed. S. Patai, Wiley Interscience, New York, 1974) pp. 111-149.

E. Production of an Elastase-Like Specificity in Subtilisin The 1166 mutation illustrates particularly well that large changes in specificity can be produced by altering the structure and hydrophobicity of the S-l subsite by a single mutation (Figure 17). Progressing through the small hydrophobic substrates, a maximal specificity improvement over wild type occurs for the Val substrate (16 fold in kcat/Km). As the substrate side chain size increases, these enhancements shrink to near unity (i.e., Leu and His substrates). The 1166 enzyme becomes poorer against larger aromatic substrates of increasing size (e.g., 1166 is over 1,000 fold worse against the Tyr substrate than is Glyl66). We interpret the increase in catalytic efficiency toward the small hydrophobic substrates for 1166 compared to Glyl66 to the greater hydrophobicity of isoluecine (i.e., -1.8 kcal/mol versus 0). Nozaki, 24067 — / o 'i-, et a_I . (1971) J. Biol. Cher.. 246. 2211-2217; Tanfora, C. (1978) Science 2 00. 1012. The decrease in catalytic efficiency toward the very large substrates for 1166 versus Glyl66 is attributed to steric repulsion.

The specificity differences between Glyl66 and 1166 are similar to the specificity differences between chymotrypsin and the evolutionary relative, elastase (Harper, J.W., et al (1984) Biochemistry 23. 2995-3002). In elastase, the bulky amino acids, Thr and Val, block access to the P-l binding site for large hydrophobic substrates that are preferred by chyraotrypsin. In addition, the catalytic efficiencies toward small hydrophobic substrates are greater for elastase than for chymotrypsin as we obeseve for 1166 versus Glyl66 in subtilisin.

Substitution of Ionic Amino Acids for Glyl66 The construction of subtilisin mutants containing the substitution of the ionic amino acids Asp, Asn, Gin, Lys and Ang are disclosed in NZ 208612. construction of the mutant subtilisin containing Glu at position 166 (E166) and presents substrate specificity data on these mutants. Further data on position 166 and 156 single and double mutants is presented infra. pj166, described in Example 3, was digested with SacI and Xmal. The double strand DNA cassette (underlined and overlined) of line 4 in Figure 13 contained the EXAMPLE 4 The present example describes the 2 4 0 6 7 0 -7 9- triplet GAA for the codon 166 to encode the replacement of Glu for Glyl66. This mutant plasmid designated pQ166 was propagated in BG2036 as described. This mutant subtilisin, together with the other mutants containing ionic substituent amino acids at residue 166, were isolated as described and further analyzed for variations in substrate specificity.

Each of these mutants was analyzed with the tetrapeptide substrates, succinyl-L-AlaL-Ala?roL-X--p-nitroanilide, where X was Phe, Ala and Glu.

The results of this analysis are shown in Table IX. 1 5 TABLE IX P-l Substrate :o (kcat/Km x "4) Posi tion 166 Phe Ala Glu Gly (wild type) 36.0 1.4 0. 002 Asp (D) 0.5 0.4 <0.001 Glu (E) 3.5 0.4 <0.001 Asn (N) H 03 O 1.2 0.004 Gin (Q) 57.0 2.6 0.002 Lys (K) 52.0 2.8 1.2 Arg (R) 42.0 .0 0.08 These results indicate that charged amino acid substitutions at Glyl66 have improved catalytic efficiencies (kcat/Km) for oppositely charged P-l substrates (as much as 500 fold) and poorer catalytic efficiency for like charged P-l substrates. 240 6 7 EXAMPLE 5 Substitution of Glycine at Position 169 The substitution of Glyl69 in B. amvlol icruefaciens subtilisin with Ala and Ser is described in NZ 208612. The sane method was used to make the remaining 17 mutants containing ail other substituent amino acids for position 169.

The construction protocol is summarized in Figure 18. The overscored and underscored double stranded DNA cassettes used contained the following triplet encoding the substitution of the indicated amino acid at residue 169.

GCT A ATG M TGT C AAC N GAT D CCT P GAA E CAA Q TTC F AGA R GGC G AGC S CAC H ACA T ATC I GTT V AAA K TGG W CTT L TAC Y Each of the plasmids containing a substituted Glyl69 was designated pX169, where X represents the substituent amino acid. The mutant subtilisins were simialrly designated.

Two of the above mutant subtilisins, A169 and S169, were analyzed for substrate specificity against synthetic substrates containing Phe, Leu, Ala and Arg in the P-l position. The following results are shown in Table X.

TABLE X Effect of Serine and Alanine Mutations at Position 169 on P-l Substrate Specificity Position 169 P-l Substrate (kcat/Km x 10 ^1 Phe Leu Ala Ara Gly (wild type) A165 S169 120 40 50 1 1 1 0.6 0.4 0.9 These results indicate that substitutions of Ala and Ser at Glyl69 have remarkably similar catalytic efficiencies against a range of P-l substrates compared to their position 166 counterparts. This is probably because position 169 is at the bottom of the P-l specificity subsite.

Sufcstitution at Position 104 Tyrl04 has been substituted with Ala, His, Leu, Met and Ser. The method used was a modification of the site directed mutagenesis method. According to the protocol of Figure 19, a primer (shaded in line 4) introduced a unique HindiII site and a frame shift mutation at codon 104. Restriction-purification for the unique HindiII site facilitated the isolation of the mutant sequence (line 4). Restriction-selection against this Hindlll site using pimers in line 5 was used to obtain position 104 mutants.

EXAMPLE 6 The following triplets were used in the primers of Figure 19, line 5 for the 104 codon which substituted the following amino acids.

GCT A TTC I? j.

ATG M CCT P CTT L ACA T AGC S TGG W CAC H TAC Y CAA Q GTT V GAA E AGA R GGC G AAC N ATC I GAT D AAA K TGT C 1 5 The substrates in Table XI were used to analyze the substrate specificity of these mutants. The results obtained fo H104 subtilisin are shown in Table XI.

TABLE XI kcat Km Kcat/Km Subs tra te WT H104 KT H104 WT H104 sAAPFoNA 50.0 22.0 1. -4 4x10 7 .1x10" -4 3. 6xl05 3. , lxlO4 sAAPAsJCA 3.2 2.0 2. -4 3x10 1 .9x10" ■3 1. 4 4x10 lxl 03 sFAPFDMA 26.0 38.0 1. -4 8x10 4 . 1x10 ■4 1. 5xl05 9. lxlO4 sFAPApKA 0.32 2.4 7. 3x10"5 1 .5x10" ■4 4. 4xl03 1. 6x104 From these data it is clear that the substitution of His for Tyr at position 104 produces an enzyme which is more efficient (higher kcat/Km) when Phe is at the 35 P-4 substrate position than when Ala is at the P-4 substrate position. £ 4 0 D EXAMPLE 7 Substitution of AIal52 Alal52 has been substituted by Gly and Ser to determine the effect of such substitutions on substrate specificity.

The wild type DNA sequence was mutated by the V152/P153 primer (Figure 20, line 4) using the above restriction-purification approach for the new Kpr.I site. Other mutant primers (shaded sequences Figure 20; S152, line 5 and G152, line 6) mutated the new Km I site away and such mutants were isolated using the restriction-selection procedure as described above for loss of the Kpnl site.

The results of these substitutions for the above synthetic substrates containing the P-l amino acids Phe, Leu and Ala are shown in Table XII.

TABLE XII P-l Substrate Position 152 (kcat/KmxlO 4) Phe Leu Ala Gly (G) Ala (wild type) Ser (S) 40.0 0.2 1.0 . 0 0 . 4 0.5 <0. 04 1.0 0.2 These results indicate that, in contrast to positions 166 and 169, replacement of Alal52 with Ser or Gly causes a dramatic reduction in catalytic efficiencies across all substrates tested. This suggests Alal52, at the top of the S-l subsite, may be the optimal amino acid because Ser and Gly are homologous Ala substitutes.

EXAMPLE 8 Substitution at Position 156 Mutants containing the substitution of Ser and Gin for Glul56 have been constructed according to the overall method depicted in Figure 21. This method was designed to facilitate the construciton of multiple mutants at position 156 and 166 as will be described hereinafter. However, by regenerating the wild type Glyl66, single mutations at Glul56 were obtained.

The plasmid pil66 is already depicted in line 2 of Figure 13. The synthetic oligonucleotides at the top right of Figure 21 represent the same DNA cassettes depicted in line 4 of Figure 13. The plasmid pl66 in Figure 21 thus represents the mutant plasmids of Examples 3 and 4. In this particular example, pl66 contains the wild type Glyl66.

Construction of position 156 single mutants were prepared by ligation of the three fragments (1-3) indicated at the bottom of Figure 21. Fragment 3, containing the carboxy-tenninal portion of the subtilisin gene including the wild type position 166 codon, was isolated as a 610 bp SacI-BamKI fragment. Fragment 1 contained the vector sequences, as well as the amino-terminal sequences of the subtilisin gene through codon 151. To produce fragment 1, a unique Kpnl site at codon 152 was introduced into the wild type subtilisin sequence from pS4.5. site-directed mutagenesis in M13 employed a primer having the sequence 51-TA-GTC-GTT-GCG-GTA-CCC-GGT-AAC-GAA-3' to produce the mutation. Enrichment for the mutant sequence was accomplished by restriction with Kpnl, purification and self ligation. The mutant sequence containing the Kpnl site was confirmed by direct plasmid sequencing to give pV152. pV152 (-1 ^g) was digested with Kpnl and treated with 2 units of DNA polymerase I large fragment (Klenow fragment from Boeringer-Mannheim) plus 50 /jM deoxynucleotide triphosphates at 37 *C for 30 min. This created a blunt end that terminated with codon 151. The DNA was extracted with 1:1 volumes phenol and CHC13 and DNA in the aqueous phase was precipitated by addition of 0.1 volumes 5M ammonium acetate and two volumes ethanol. After centrifugation and washing the DNA pellet with 7 0% ethanol, the DNA was lyophilized. DNA was digested with BamHI and the 4. 6kb piece (fragment 1) was purified by acrylamiae gel electrophoresis followed by electroelution. Fragment 2 was a duplex synthetic DNA cassette which when ligated with fragments 1 and 3 properly restored the coding sequence except at codon 156. The top strand was synthesized to contain a glutamine codon, and the complementary bottom strand coded for serine at 156. Ligation of heterophosphorylated cassettes leads to a large and favorable bias for the phosphorylated over the non-phosphorylated oligonucleotide sequence in the final segrated plasmid product. Therefore, to obtain Q156 the top strand was phosphorylated, and annealed to the non-phosphorylated bottom strand prior to ligation. Similarly, to obtain S156 the bottom strand was phosphorylated and annealed to the non-phosphorylated top strand. Mutant sequences were isolated after ligation and transformation, and were confirmed by restriction analysis and DNA sequencing as before. To express variant subtilisins, plasmids were transformed into a subtilisin-neutral protease deletion mutant of B. subtilis. BG2036, as previously described. Cultures were fermented in shake flasks 5 for 24 h at 37*C in LB media containing 12.5 mg/mL chloramphenicol and subtilisin was purified from culture supernatants as described. Purity of subtilisin was greater than 55% as judged by SDS PAGE.

I-j These mutant plasmids designated pS156 and pQ156 and mutant subtilisins designated S156 and Q156 were analyzed with the above synthetic substrates where P-l comprised the amino acids Glu, Gin, Met and Lys. The results of this analyses are presented in Example 9. 1 5 EXAMPLE 9 2 0 Multiple Mutants With Altered Substrate Specificity - Substitution at Positions 156 and 166 Single substitutions of position 166 are described in Examples 3 and 4. Example 8 describes single substitutions at position 156 as well as the protocol 2 5 of Figure 21 whereby various double mutants comprising the substitution of various amino acids at positions 156 and 166 can be made. This example describes the construction and substrate specificity of subtilisin containing substitutions at position 156 and 166 and summarizes some of the data for single and double mutants at positions 156 and 166 with various substrates.

K166 is a common replacement amino acid in the 156/166 mutants described herein. The replacement of Lys for 2 4 u 5 Glyl66 was achieved by using the synthetic DNA cassette at the top right of Figure 21 which contained the triplet AAA for NNN. This produced fragment 2 with Lys substituting for Glyl66.

The 156 substituents were Gin and Ser. The Gin and Ser substitutions at Glyl56 are contained within fragment 3 (bottom right Figure 21) .

The multiple mutants were produced by combining fragments 1, 2 and 3 as described in Example 8. The mutants Q156/K166 and S156/K166 were selectively generated by differential phosphorylation as described. Alternatively, the double 156/166 mutants, c.f. Q156/K166 and S156/K166, were prepared by ligation of the 4. 6)cb SacI-BamHI fragment from the relevant pl56 plasmid containing the 0.6kb SacI-BamHI fragment from the relevant pl66 plasmid.

These mutants, the single mutant K166, and the S156 and Q156 mutants of Example 8 were analyzed for substitute specificity against synthetic polypeptides containing Phe or Glu as the P-l substrate residue. The results are presented in Table XIII.

TAHU Subs trato Enzymes Compared^ Residue kcat Glul56/Glyl66 (WT) Phe 50.00 Glu 0.54 Kl66 Phe 20.00 Glu 0.70 Q156/K166 Phe 30.00 Glu 1.60 S156/K166 Phe 30.00 Glu 0.60 SI 56 Phe 34.00 Glu 0.40 El 56 Phe 48.00 Glu 0.90 XI r I Km 1.4X10-4 3.4 x10-2 4.0x10~5 5.6xl0~5 1.9xl0~5 3.lxl0~5 1.8xlO~5 3.9x10~5 4.7xl0~5 1.8xl0~3 4.5x10~5 3.3xl0~3 kca t; / Km 3. 6xl05 1.6x101 5.2x105 1.2xl04 1.6xl06 5.OxlO4 1.6xl06 1.6x104 7.3xl05 l.lxlO2 1.lx10 6 2. 7xl02 kcat/Km (mutant) kca t/Km(wt) (1) (1) 1.4 750 4 . 4 3100 4 . 4 1000 2.0 6.9 3.1 17 As can be seen in Table XIV, either of these single nutations improve enzyme performance upon substrates with glutamate at the P-l enzyme binding site. When these single mutations were combined, the resulting multiple enzyme mutants are better than either parent. These single or multiple mutations also alter the relative pH activity profiles of the enzymes as shown in Figure 23.

To isolate the contribution of electrostatics to substrate specificity from other chemical binding forces, these various single and double mutants were analyzed for their ability to bind and cleave synthetic substrates containing Glu, Gin, Met and Lys as the P-l substrate amino acid. This permitted comparisons between side-chains that were more sterically similar but differed in charge (e.g., Glu versus Gin, Lys versus Met) . Similarly, mutant enzymes were assayed against homologous P-l substrates that were most sterically similar but differed in charge (Table XIV).

A TABU-: XIV Kinetics of Position 156/1 6 6 Subti li;; ins Determined for Di fforont P1 Substrates (c) Ul 1 t. J » I Pos i t ion Charge ^^ Glu V. i 1 1 n 1 V./M r>'- n v / urn \ Mnt: Lys 156 166 Glu Asp -2 n • d. 3 .02 (2. 56) 3 .93 (2.74) 4 . 23 (3.00) Glu Glu -2 n .d. 3 .06 (2. 91) 3 . 86 (3.28) 4 . 48 (3.69) Glu Asn 1 .62 (2. 22) 3 . 85 (3. 14) 4 .99 (3.85) 4 . 1 5 (2.88) Glu Gin -1 1 . 20 (2. 12) 4 .36 (3. 64) .43 (4.36) 4 . 1 0 (3.15) Gin Asp -1 1 . 30 (1 . 79) 3 .40 (3. 08) 4 . 94 (3.87) 4 . 41 (3.22) Ser Asp -1 1 .23 (2. 13) 3 .41 (3. 09) 4 . 67 (3.68) 4 . 24 (3.07) Glu Met -1 1 . 20 (2. ) 3 .89 (3. 19) . 64 (4.83) 4 . 70 (3.89) Glu Ala -1 n .d. 4 .34 (3. 55) :65 (4.46) 4 . 90 (3.24) Glu Gly(wt) -1 1 . 20 (1 . 47) 3 .85 (3. ) ,07 (3.97) 4 . 60 (3.13) Gin Gly 0 2 .42 (2. 48) 4 .53 (3. 81) " 11 (4.61) 3. 76 (2.82) Ser Gly 0 2 .31 (2. 73) 4 .09 (3. 68) .61 (4.55) 3. 46 (2.74) Gin Asn 0 2 .04 (2. 72) 4 .51 (3. 76) .79 (4.66) 3. 75 (2.74) Ser Asn 0 1 .91 (2. 78) 4 . 57 (3. 82) . 72 (4.64) 3. 68 (2.80) Glu Arg 0 2 .91 (3. ) 4 . 26 (3. 50) . 32 (4.22) 3. 19 (2.80) Glu Lys 0 4 . 09 (4. ) 4 .70 (3. 88) 6.15 (4.45) 4. 23 (2.93) Gin Lys +1 4 . 70 (4. 50) 4 .64 (3. 68) .97 (4.68) 3. 23 (2.75) Ser Lys 4-1 4 .21 (4 . 40) 4 .84 (3. 94) 6.16 (4.90) 3. 73 (2.84) Maximum difference: log kcat/Km (log l/Km) 3 . 5 (3. 0) 1 . 8 (1. 4) 2.3 (2.2) -1 . 3 (-1.0) i VP 0 1 IV? ■35 "si Footnotes to Table XIV: ( fi. ) • v ' B. subtil is. BG 2036, expressing indicated variant subtilisin were fermented and enzymes purified as previously described (Estell, et a_l. (1985) J. Biol. Chem. 260. 6518-6521). Wild type subtilisin is indicated (vt) containing Glul56 and Glyl66.

^ Net charge in the P-l binding site is defined as the sum of charges from positions 156 and 166 at pH S . 6 . (c) -1 v ' Values for kcat(s ) and Km(M) were measured in 0. 1M Tris pH 8.6 at 25*C as previously described against P-l substrates having the form suceinyl-L-AlaL-AlaL-ProL-[X]-p-nitroanilide, where X is the indicated P-l amino acid. Values for log l/Km are shown inside parentheses. All errors in determination of kcat/Km and l/Km are below 5%. (d) v ' Because values for Glul56/Aspl66(D166) are too small to determine accurately, the maximum difference taken for GluP-1 substrate is limited to a charge range of +1 to -1 charge change. n.d. = not determined The kcat/Km ratios shown are the second order rate constants for the conversion of substrate to product, and represent the catalytic efficiency of the enzyme. These ratios are presented in logarithmic form to scale the data, and because log kcat/Km is proportional to the lowering of transition-state activation energy (aG„.). Mutations at position 156 and 166 produce changes in catalytic efficiency toward Glu, Gin, Met and Lys P-l substrates of 3100, 60, 200 and 20 fold, respectively. Making the P-l binding-site more positively charged [e.g., compare Glnl56/Lysl6 6 (Q156/K166) versus Glul5 6/Met166 (Glul56/M166)] dramatically increased kcat/Km toward the Glu P-l substrate (up to 3100 fold), and decreased the catalytic efficiency toward the Lys P-l substrate (up to 10 fold) . In addition, the results show that the catalytic efficiency of wild type enzyme can be 24 0 6 7 0 greatly improved toward any of the four P-l substrates by mutagenesis of the P-l binding site.

The changes in kcat/Km are caused predominantly by changes in l/Km. Because 1/Kai is approximately equal to 1/Ks, the enzyme-substrate association constant, the mutations primarily cause a change in substrate binding. These mutations produce smaller effects on kcat that run parallel to the effects on l/Km. The changes in kcat suggest either an alteration in binding in the P-l binding site in going from the Hichaelis-complex E-S) to the transition-state complex (E-S/) as previously proposed (Robertus, J.D., et al. (1972) Biochemistry 11, 2439-2449; Robertus, J.D., et al. (1972) Biochemistry 11. 4293-4303), or change in the position of the scissile peptide bond over the catalytic serine in the E*S complex.

Changes in substrate preference that arise from changes in the net charge in the P-l binding site show trends that are best accounted for by electrostatic effects (Figure 28). As the P-l binding cleft becomes more positively charged, the average catalytic efficiency increases much more for the Glu P-l substrate than for its neutral and isosteric P-l homolog, Gin (Figure 28A). Furthermore, at the positive extreme both substrates have nearly identical catalytic efficiencies.

In contrast, as the P-l site becomes more positively charged the catalytic efficiency toward the Lys P-l substrate decreases, and diverges sharply from its neutral and isosteric homolog, Met (Figure 28B) . The similar and parallel upward trend seen with increasing positive charge for the Met and Glu P-l substrates probably results from the fact that all the substrates are succinylated on their anino-teminal end, and thus carry a formal negative charge.

The trends observed in log kcat/Kin are dominated by changes in the Km term (Figures 28C and 28D) . As the pocket becomes more positively charged, the log l/Km values converge for Glu and Gin P-l substrates (Figure 23C) , and diverge for Lys and Met P-l substrates (Figure 28D). Although less pronounced effects are seen in log kcat, the effects of P-l charge on log kcat parallel those seen in log l/Km and become larger as the P-l pocket becomes more positively charged. This may result from the fact that the transition-state is a tetrahedral anion, and a net positive charge in the enzyme may serve to provide some added stabilization to the transition-state.

The effect of the change in P-l binding-site charge on substrate preference can be estimated from the differences in slopes between the charged and neutral isosteric P-l substrates (Figure 2SB). The average change in substrate preference (Alog kcat/Km) between charged and neutral isosteric substrates increases roughly 10-fold as the complementary charge or the enzyme increases (Table XV). When comparing Glu versus Lys, this difference is 100-fold and the change in substrate preference appears predominantly in the Km term.

Differential Effect on Binding Site Charge on log kcat/Km or (log l/Km) . . for P-l Substrates that Differ in Charge "i n P — 1 Ri nr i nc Alog kcat/Km (Alog l/Km) Site Charqe GluGln MetLvs GluLvs -2 to -1 n.d. 1.2 (1.2) n.d. -1 to 0 0.7 (0.6) 1.3 (0.8) 2.1 (1.4) 0 to +1 1.5 (1.3) 0.5 (0.3) 2.0 (1.5) change in log kcat/K or (leg i/'Krr.)rnper .ir.it charge change 1.1 (1.0) 1.0 (0.8) 2.1 (1.5) The difference in the slopes of curves were taken between the P-l substrates over the charge interval giver, for log (kcat/Km) (Figure 28A, B) and (log l/Km) (Figure 28C, D) . Values represent the differential effect a charge change has in distinguishing the substrates that are compared.

^ Charge in P-l binding site is defined as the sum of charges from positions 156 and 166. 2 4 0 6 The free energy of electrostatic interactions in the structure and energetics of salt-bridge formation depends on the distance between the charges and the 5 microscopic dielectric of the media. To dissect these structural and microenvironmental effects, the energies involved in specific salt-bridges were evaluated. In addition to the possible salt-bridges shown (Figures 2 9A and 29B), reasonable salt-bridges C can be built between a Lys P-l substrate and Asp at position 166, and between a Glu P-l substrate and a Lys at position 166 (not shown). Although only one of these structures is confirmed by X-ray crystalography (Poulos, T.L., et al. (1976) J. Mol. Biol. 257 5 1097-1103), all models have favorable torsion angles (Sielecki, A.R., et al. (1979) J. Mol. Biol. 134, 781-804), and do not introduce unfavorable van der Kaals contacts. (j The change in charged P-l substrate preference brought about by formation of the model salt-bridges above are shown in Table XVI.

D TAHI.K XVI Effect of Salt Hridcjc Formation Between Knzyrnn and Substrate on Tl Substrate Pro ference ' Enzymes Compared (b) 1 Glul56/Aspl66 Glul56/Asnl66 Glul56/Glyl66 Glul56/Lsy-166 Glnl56/Aspl66 Glnl56/Asnl66 Glnl56/Glyl66 Glnl56/Lysl66 flnzymo P-l Position Substrates Changed Compa rod 1 5 6 156 156 156 LysMc t LysMet LysMot LysMet Substrate c Preference A log (kcat/Km) . . + 0.30 -0 .84 -0.47 -1.92 -0.53 -2.04 -2.10 -2 .74 Change in Substrate F're fcrencr AAlog (kcat/Km) (1-2) 0 .03 1 . 20 1.63 0.82 Ave AAlog (kcat/Km) 1.10 ± 0.3 Glul56/Aspl66 Glul56/Asn16 6 166 LysMet + 0 .30 -0 . 84 1 .14 Glul56/Glul66 Glul56/Glul66 166 LysMet + 0 .62 -1 .33 1.95 Glnl5 6/Aspl6 6 Glnl56/Asnl66 166 LysMet -0 . 53 -2 .04 1. 51 Serl56/Aspl66 Serl56/Asnl66 166 LysMet -0 .43 -2 .04 1.61 Glul56/Lysl66 Glul56/Metl66 166 GluGln -0 .63 -2 .69 2/06 Ave AAlog (kcat/Km) 1.70 + 0.3 Foctr.otes to Table XVI : f ClN ' Molecular modeling shows it is possible to forn a salt bridge between the indicated charged P-l substrate and a complementary charge in the P-l binding site of the enzyme at the indicated position changed.

^ Er.ziT.es compared have sterically similar amino acid substitutions that differ in charge at the indicated position.

( C ) v ' The P-l substrates compared are structurally similar but differ in charge. The charged P-l substrate is complementary to the charge change at the position indicated between enzymes 1 and 2.

^ Date from Table XIV was used to compute the difference in log (kcat/Km) between the charged and the non-charged P-l substrate (i.e., the substrate preference). The substrate preference is shown separately for enzyme 1 and 2.

( G ) The difference in substrate preference between er.zpe 1 (more highly charged) and enzyme 2 (more neutral) represents the rate change accompanying the electrostatic interaction.

The difference between catalytic efficiencies (i.e., A log kcat/Km) for the charged and neutral P-l substrates (e.g., Lys minus Met or Glu minus Gin) give the substrate preference for each enzyme. The change in substrate preference (AAlog kcat/Km) between the charged and more neutral enzyme homologs (e.g., Glul56/Gly166 minus Glnl56(Q156)/Glyl66) reflects the change in catalytic efficiency that may be attributed solely to electrostatic effects.

These results show that the average change in substrate preference is considerably greater when electrostatic substitutions are produced at position 166 (50-fold in kcat/Km) versus position 156 (12-fold in kcat/Km). From these AAlog kcat/Km values, an average change in transition-state stabilization energy can be calculated of -1.5 and -2.4 kcal/mol for 24067 -9 8- itu: :cns at positions 156 ar.c: 166, respectively.

This should represent the stabilization energy contribute- from a favorable electrostatic interaction for the binding of free enzyme and substrate to form the transition-scate complex.

EXAMPLE 10 Substitutions at Position 217 Tyr217 has been substituted by all other 19 amino acids. Cassette mutagenesis as described in NZ 208612 was used according to the protocol of Figure 22. The EcoRV restriction site was 25 used for restriction-purification of pA217.

Since this position is involved ,,in. substrate binding, mutations,; here effect, kinetic , parameters of the enzyme. An example is the substitution of Leu for Tyr at position 217. For the substrate sAAPFpNa, this -4 mutant has a kcat of 277 5' and a Km of 4.7x10 with a kcat/Km ratio of 6x10^. This represents a 5.5-fold increase in kcat with a 3-fold increase in Km.over the wild type enzyme.

In addition, replacement of Tyr217 by Lys, Arg, Phe or Leu results in mutant enzymes which are more stable at pHs of about 9-11 than the WT enzyme. Conversely, replacement of Tvr217 by Asp, Glu, 'Gly or Pro results 3q in enzymes which are less stable at pHs of about 9-11 than the WT enzyme. 3 5 EXAMPLE 11 Multiple Mutants Having Altered Thermal Stability B. amvlol icruef acien subtilisin does not contain any cysteine residues. Thus, any attempt to produce thermal stability by Cys cross-linkage required the substitution of more than one amino acid in subtilisin with Cys. The following subtilisin residues were multiply substituted with cysteine: Thr22/Ser87 Ser24/SerS7 Mutagenesis of Ser24 to Cys was carried out with a 5' phosphorylated oligonucleotide primer having the sequence •-pC-TAC-ACT-GGA-TGC-AAT-GTT-AAA-G-3 ' .

(Asterisks show the location of mismatches and the underlined sequence shows the position of the altered Sau3 A site.) The B. amvlol icruefaciens subtilisin gene on a 1.5 kb EcoRI-BAHHI fragment from pS4.5 was cloned into M13xnpll and single stranded DNA was isolated. This template (M13mpllSUBT) was double primed with the 51 phosphorylated M13 universal sequencing primer and the mutagenesis primer. Adelman, et a_l. (1983) DNA 2, 183-193. The heteroduplex was transfected into competent JM101 cells and plaques were probed for the mutant sequence (Zoller, M.J., et al. (1982) Nucleic Acid Res. 10, 6487-6500; Wallace, et al. (1981) Nucleic Acid Res. 9, 3647-3656) using a tetramethylammonium chloride hybridization protocol (Wood, et al. (1985) Proc. Natl. Acad. Sci. USA 82, 1585-1588). The Ser87 to Cys mutation was prepared in *+ I ! -ICO- I 4 0 a similar fashion using a 5' phosphorylated primer having the sequence '-pGGC—GTT-GCG-CCA—TGC-GCA-TCA-CT-31.

(The asterisk indicates the position of the mismatch and the underlined sequence shows the position of a new MstI site.) The C24 and C87 mutations were obtained at a frequency of one and two percent, respectively. Mutant sequences were confirmed by aideoxy sequencing in M13.

Mutagenesis of Tyr21/Thr22 to A21/C22 was carried out with a 5' phosphorylated oligonucleotide primer having the sequence i f> '-pAC-TCT-CAA-GGC-GCT-TGT-GGC-TCA-AAT-GTT-3' .

(The asterisks show mismatches to the wild type sequence and the underlined sequence shows the position of an altered Sau3A site.) Manipulations for heteroduplex synthesis were identical to those described for C24. Because direct cloning of the heteroduplex DNA fragment can yield increased frequencies of mutagenesis, the EcoRI-BamHI subtilisin fragment was purified and ligated into pBS42. E. coli KM 294 cells were transformed with the ligation mixture and plasmid DNA was purified from isolated transformants. Plasmid DNA was screened for the loss of the Sau3A site at codon 23 that was eliminated by 0 the mutagenesis primer. Two out of 16 plasmid preparations had lost the wild type Sau3A site. The mutant sequence was confirmed by dideoxy sequencing in M13 . ] 5 2 4 0 ^ 7 Double mutants, C22/C87 and C24/C87, were constructed by ligating fragments sharing a common Clal site that separated the single parent cystine codons. Specifically, the 500 bp EcoRI-Clal fragment containing the 5' portion of the subtilisin gene (including codons 22 and 24) was ligated with the 4.7 kb Clal-EcoRI fragment that contained the 3' portion of the subtilisin gene (including codon 87) plus pBS42 vector sequence. E. coli MM 294 was transformed with ligation mixtures and plasmid DNA was purified from individual transformants. Double-cysteine plasmid constructions were identified by restriction site markers originating from the parent cysteine mutants (i.e., C22 and C24, Sau3A minus; Cys87, MstI plus). Plasmids from E. col i were transformed into B. subtil is BG203 6. The thermal stability of these mutants as compared to wild type subtilisin are presented in Figure 30 and Tables XVII and XVIII. 2fi 4 (j () 7 o -102-TABLE XVII Effect of DTT on the Half-Time of Autolytic Inactivation of Wild-Type and Disulfide Mutants of Subtilisin* Er.nvrnp -DTT /-f DTT - -DDT -f DTT ' min Wild-type 9 5 85 1.1 C22/C87 44 25 1.8 C24/C87 92 62 1.5 Purified enzymes were either treated or not treated with 2 5mM DTT and dialyzed with or without lOniV. DTT in 2zr_M CaCl2, 50mM Tris (pH 7.5) for 14 hr. at 4°C. Enzyme concentrations were adjusted to 80ul aliquots were quenched on ice and assayed for residual activity. Half-tiir.es for autolytic inactivation were dotormined from semi-log plots of (residual activity) versus time. These plots were linear for over 901 of the inactivation.

TABLE XVIII Effect of Mutations in Subtilisin on the Kalf-Time of Autolytic Inactivation at 58*C* Enzyme t min Wild-type 120 C22 22 C24 120 C87 104 C22/C87 43 C24/C87 115 (*) v ' Half-times for autolytic inactivation were determined for wild-type and mutant subtilisins as described in the legend to Table III. Unpurified and non-reduced enzymes were used directly from B. subti1 is culture supernatants.

The disulfides introduced into subtilisin did not improve the autolytic stability of the mutant enzymes when compared to the wild-type enzyme. However, the disulfide bonds did provide a margin of autolytic stability when compared to their corresponding reduced couble-cysteine enzyme. Inspection of a highly refined x-ray structure of wild-type B. amvlol icruefaciens subtilisin reveals a hydrogen bond between Thr22 and Ser8 7. Because cysteine is a poor hydrogen donor or acceptor (Paul, I.e. (1974) in Chemistry of the -SH Group (Patai, S., ed.) pp. 111-149, Wiley Interscience, New York) weakening of 22/87 hydrogen bond may explain why the C22 and C87 single-cysteine mutant proteins are less autolytically Btable than either C24 or wild-type (Table XVIII). The fact that C22 is less autolytically stable than C87 may be the result of the Tyr21A mutation (Table XVIII). Indeed, 'i 4 c cor.struct ion and analysis of Tyr21/C22 shovs the mutant protein has an autolytic stability closer to that of CS7. In summary, the C22 and C87 of single-cysteine mutations destabilize the protein - toward autolysis, and disulfide bond formation increases the stability to a level less than or equal to that of wild-type enzyme.

-, EXAMPLE 12 Multiple Mutants Containing Substitutions at Position 222 and Position 166 or 169 Double mutants 166/222 and 169/222 were prepared by , _ ligating together (1) the 2. 3kb Acall fragment from pS4.5 which contains the 5' portion of the subtilisin ger.e and vector sequences, (2) the 200bp Avail fragment which contains the relevant 166 or 165 mutations from the respective 166 or 169 plasaids, and (3) the 2.2kb Avail fragment which contains the relevant 222 mutation 3' and of the subtilisin genes and vector sequence from the respective p222 plasmid.

Although mutations at position 222 improve oxidation stability they also tend to increase the Km. An example is shown in Table XIX. In this case the A222 mutation was combined with the K166 mutation to give an enzyme with kcat and Km intermediate between the two parent enzymes.

L 4 (f • -105-TABLE XIX kcat Km WT 50 1.4X10~4 A222 42 9.9xl0~4 K166 21 3.7xl0~5 K166/A222 29 2.0xl0~4 substrate sAAPFpNa EXAMPLE 13 Multiple Mutants Containing Substitutions at Positions 50, 156, 156, 217 and Combinations Thereof The double mutant S156/A169 was prepared by ligation of two fragments, each containing one of the relevant nutations. The plasmid pS156 was cut with Xmal and treated with SI nuclease to create a blunt end at codon 167. After removal of the nuclease by phenol/chloroform extraction and ethanol precipitation, the DNA was digested with BamHI and the approximately 4kb fragment containing the vector plus the 5' portion of the subtilisin gene through codon 167 was purified.

The pA169 plasmid was digested with Kpnl and treated with DNA polymerase Klenow fragment plus 50 /jM dNTPs to create a blunt end codon at codon 168. The Klenow was removed by phenol/chloroform extraction and ethanol precipitation. The DNA was digested with BamHI and the 590bp fragment including codon 168 through the carboxy terminus of the subtilisin gene -1C6- if n ' was isolated. The two fragments were then ligated to give £156/A169.

Triple and quadruple mutants were prepared by ligating together (1) the 220bp PvulI/HaelI fragment containing the relevant 156, 166 and/or 169 mutations from the respective pl56, pl66 and/or pl69 double of single mutant plasmid, (2) the 55Cbp Haell/BamHI fragment containing the relevant 217 mutant from the respective p217 plasmid, and (3) the 3.9kb PvuII/BamHI fragment containing the F50 mutation and vector sequences.

The multiple mutant F50/S156/A169/L217, as well as B. amylolicuefaciens subtilisin, B. lichenformis subtilisin and the single mutant L217 were analyzed "D with the above synthetic polypeptides where the P-l amino acid in the substrate was Lys, His, Ala, Gin, Tyr, Phe, Met and Leu. These results are shown in Figures 26 and 27. "u These results show that the F50/S156/A169/L217 mutant has substrate specificity similar to that of the _B. licheniformis enzyme and differs dramatically from the wild type enzyme. Although only data for the L217 mutant are shown, none of the single mutants (e.g., F50, S156 or A169) showed this effect. Although B. licheniformis differs in 88 residue positions from EL amylolicue faciens , the combination of only these four mutations accounts for most of the differences in substrate specificity between the two enzymes.

EXAMPLE 14 Subtilisin Mutants Having Altered Alkaline Stability A random mutagenesis technique was used to generate .j 5 single and multiple mutations within the B. 2 4 0 $ "* a arr.y 1 ol icruef ac ier.s subtilisin gene. Such mutants were screened for altered alkaline stability. Clones having increased (positive) alkaline stability and decreased (negative) alkaline stability were isolated and sequenced to identify the mutations within the subtilisin gene. Among the positive clones, the mutants V107 and R213 were identified. These single mutants were subsequently combined to produce the mutant V107/R213.

One of the negative clones (V50) from the random mutagenesis experiments resulted in a marked decrease in alkaline stability. Another mutant (P50) was analyzed for alkaline stability to determine the effect of a different substitution at position 50. The F50 mutant was found to have a greater alkaline stability than wild type subtilisin and when combined with the double mutant V107/R213 resulted in a mutant having an alkaline stability which reflected the aggregate of the alkaline stabilities for each of the individual mutants.

The single mutant R204 and double mutant C204/R213 were identified by alkaline screening after random cassette mutagenesis over the region from position 197 to 228. The C204/R213 mutant was thereafter modified to produce mutants containing the individual mutations C204 and R213 to determine the contribution of each of the individual mutations. Cassette mutagenesis using pooled oligonucleotides to substitute all amino acids at position 204, was utilized to determine which substitution at position 204 would maximize the increase in alkaline stability. The mutation from Lys213 to Arg was maintained constant for each of these substitutions at position 204. 24 0 6 A. Construction of pBOlSO, an E. coli-B. subtilis Shuttle Plasmid The 2.9 kb EcoRI-BamHI fragment from pEP.3 27 (Ccvarrubias, L., et al. (1981) Gene 13, 25-35) was ~ licated to the 3.7kb EcoRI-BamHI fragment of pBD64 (Gryczan, T. , et al. (19S0) J. Bacteriol. , 141, 246-253 ) to give the recombinant plasrr.id pB0153. The unique EcoRI recognition sequence in pBD64 was eliminated by digestion with EcoRI followed by 10 treatment with Klenow and deoxvnucleotide triphosphates (Maniatis, T. , et al. (eds.) (1982) in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Blunt end ligation and transformation yielded pB0154. The 1 f> unique Aval recognition sequence in pE0154 was eliminated in a similar manner to yield pB0171. pB0 171 was digested with Bam.HI and PvuII and treated with Klenow and deoxynucleotide triphosphates to create blunt ends. The 6.4 kb fragment was purified, :-0 licated and transformed into LE392 cells (Enquest, L.W., et al. (1977) J. Mol. Biol. Ill, 97-120), to yield pB0172 which retains the unique BamHI site. To facilitate subcloning of subtilisin mutants, a unique arid silent Kpnl site starting at codon 166 was ."!> introduced into the subtilisin gene from pS4.5 (Wells, J . A . , e t a 1. (1 9 8 3) Nucleic Acids Res. , 1_1_, 7911-7925) by site-directed mutagenesis. The Kpnl+ plasmid was digested with EcoRI and treated with Klenow and deoxynucleotide triphosphates to create a blunt end. 30 The Klenow was inactivated by heating for 20 min at C8°C, and the DNA was digested with BamHI. The 1.5 kb blunt EcoRI-BamHI fragment containing the entire subtilisin was ligated with the 5.8 kb NruI-BamHI from pRO17 2 to yield pB0180. The ligation of the blunt 35 Nrul end to the blunt EcoRI end recreated an EcoRI 11. Proceeding clockwise around pBOlSO frcm the V"' I site at the 5' cnc of the subtilisin gene is the unique BamHI site at the 3' end of the subtilisin aer.c , the chloramphenicol and neomycin resistance cer.es ar.c UEilO gram positive replication origin derived from pBD64, the ampicillir. resistance gene and cram negative replication origin derived from p3R327. 3. Construction of Random Mutacenesis Librarv - -» The 1.5 kb EcoP.I-BamHI fragment containing the B. an,ylo 1 icuefaciens subtilisin gene (Wells et al., 1983) from pB0180 was cloned into Ml3mpll to give M13mpll SL'BT essentially as previously described (Wells, J.A., et al. (1986) J. Biol. Chem., 261,6564-6570). Decxyuridir.e containing template DNA was prepared according to Kunkel (Kunkel, T.A. (1985) Proc. Natl.

Acad. Sci. USA, 82 488-492). Uridine containing template DNA (Kunkel, 1985) was purified by CsCl density gradients (Maniatis, T. et al. (eds.) (1982) in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). A ; r liner (Aval ) having the sequence ' GAAAAAAGACCCTAGCC-TCGCTTA ending at codon -11, was used to alter the unique Aval recognition sequence within the subtilisin gene. (The asterisk denotes the mismatches from the wild-type pequer.ee and underlined is the altered Aval site.) The 5' phosphorylated Aval primer (-320 pmol) and -40 pmol (-120ug) of uridine containing Ml3mpll SUBT template in 1.88 ml of 53 mM NaCl, 7.4 mM MgC12 and 7.4 mM Tris.HCl (pH 7.5) were annealed by heating to * 4 0 o 7 0 9 0CC for 2 min. ar.c cooling 15 r.in at 24°C (Fic. 31).

Primer extension at 24CC was initiated by addition of ICOuL containing 1 mM in ell four deoxynucleotide triphosphates, and 20„1 Klencw fragment (5 units/1). The extension reaction was stepped every 15 seconds over ten min by addition of lOyl 0.25 M EDTA (pH 8) to 5C„i alicucts of the reaction mixture. Samples were pooled, phenol chlorophorm extracted and DNA was precipitated twice by addition of 2.5 vol 100% ethanol, and washed twice with 70% ethanol. The pallet was dried, and redissolved in 0.4 ml 1 mM EDTA, 1C mM Tris (pH 8).

Misincorporation of c-thiodeoxynucleotides onto the 3' er.de of the pool of randomly terminated template was carried out by incubating four 0.2 ml solutions each containing one-fourth of the randomly terminated template mixture (~20ug), 0.25 mM of a given n-thiodeoxynucleotide triphosphate, 100 units AMV polymerase, 50 mM KCL, 10 mM MgCl^, 0.4 mM c: thiothreitol, and 50 mM Tris (pH 8.3) (Champoux, J.J. (1 984) Genetics, _2, 454-464). After incubation at 3 7 0 C for 90 minutes, misincorporation reactions were sealed by incubation for five minutes at 37°C with 50 mM all four deoxynucleotide triphosphates (pH C], and 50 units AMV polymerase. Reactions were stopped by addition of 25 mM EDTA (final), and heated 68 °C for ten min to inactivate AMV polymerase. After ethanol precipitation and resuspension, synthesis of closed circular heteroduplexes was carried out for two days at 14°C under the same conditions used for the timed extension reactions above, except the reactions also contained 1000 units T4 DNA ligase, 0.5 mM ATP and 1 mM 8-mercaptoethanol. Simultaneous restriction of each heteroduplex pool with Kpnl, BamHI, and EcoRI confirmed that the f -1 excer.s ion reactions were nearly quantitative. Heteroduplex DNA in each reaction mixture was methylated by incubation with BCiX S-adencsylir.ethionine and 150 units dam methylase for 1 hour at 37SC. Kethylation reactions were stopped by heating at 68°C for 15 min.

One-haIf of each of the four methylated heteroduplex reactions were transformed into 2.5 ml competent E. coli JM101 (Messing, j. (1979) Recombinant DNA Tech. Bull. , 2 , 43-48). The number of independent trans formants from each of the four transformations ranged from 0.4-2.0 x 10 . After growing out phage pools, RF DNA from each of the four transformations was isolated ar.c purified by centrifugation through CsCl density gradients. Approximately 2ug of RF DNA frrr each of the four pools was digested with EcoRI, Ear.HI and Aval. The 1.5 kb EcoRI-BamHI fragment (i.e., Aval resistant) was purified on low gel temperature agarose and ligated into the 5.5 kb E j oI - S a rr ?! I vector fragment of pB0180. The total number of independent transformants from each .-i-thiodeoxynucleotide ir.isincorporation plasmid library ranged from 1.2-2.4 x 10^. The pool of plasmids from each of the four transformations was grown out in 200 ml LB media containing 12.5ug/ml cmp and plasmid DNA was purified by centrifugation through CsCl density gradients.

C. Expression and Screening of Subtilisin Point Mutants Plasmid DNA from each of the four misincorporation pools was transformed (Anagnostopoulos, C. , et al. (1967), J. Bacteriol. , 81 , 741-746) into BG2036. For each transformation, 5yg of DNA produced approximately 2.5 x 10 independent BG2036 transfcrmants, and liquid culture aliquots from the four libraries were stored in 10% glycerol at 70*C. Thawed aliquots of frozen cultures were plated on LB/5pg/ml cmp/1.6% skim milk plates (Wells, J.A., et al. (1983) Nucleic Acids Res., 11. 7911-7925), and fresh colonies were arrayed onto 96-well microtiter plates containing 150 1 per well L3 media plus 12.5^g/ml cmp. After 1 h at room temperature, a replica was stamped (using a matched 96 prong stamp) onto a 132 mm BA 85 nitrocellulose filter (Schleicher and Scheull) which was layered on a 140 mm diameter L3/cnp/skim milk plate. Cells were grown about 16 h at 30*0 until halos of proteolysis were roughly 5-7 mm in diameter and filters were transferred directly to a freshly prepared agar plate at 37 *C containing only 1.6% skim milk and 50 mM sodium phosphate pH 11.5. Filters were incubated on plates for 3-6 h at 37*0 to produce halos of about 5 mm for wild-type subtilisin and were discarded. The plates were stained for 10 min at 24'C with Coomassie blue solution (0.25% Coomassie blue (R-250) 25% ethanol) and destained with 25% ethanol, 10% acetic acid for 20 min. Zones of proteolysis appeared as blue halos on a white background on the underside of the plate and were compared to the original growth plate that was similarly stained and destained as a control. Clones were considered positive that produced proportionately larger zones of proteolysis on the high pH plates relative to the original growth plate. Negative clones gave smaller halos under alkaline conditions. Positive and negative clones were restreaked to colony purify and screened again in triplicate to confirm alkaline pK results.

D. Identification and Analysis of Mutant Subtilisins Pl2smid DNA from 5 ml overnight cultures cf more alkaline active B.subtilis clones was prepared 5 according to Birnboim and Doly (Birnboim, H.C., et al. (1 579 ) Nucleic Acid Res. 1_, 1513) except that incubation with 2 mg/ml lysozyme proceeded for 5 rain at 37°C to ensure cell lysis and an additional phenol/CHCl^ extraction was employed to remove 10 contaminants. The 1.5 kb EcoRI-BamHI fragment containing the subtilisin gene was ligated into M13 rr.p 11 and template DNA was prepared for DNA sequencing (Messing, J., et al. (1982) Gene, 19 269-276). Three DNA sequencing primers ending at codon 15 26, +95, and +155 were synthesized to match the subtilisin ccding sequence. For preliminary sequence identification a single track of DNA sequence, corresponding to the dNTPaS ir.isincorporation library from which the mutant came, was applied over the 20 entire mature protein coding sequence (i.e., a single cideoxyguanosine sequence track was applied to identify a mutant from the dGTPas library). A complete four track of DNA sequence was performed 200 hp over the site of mutagenesis to confirm and 25 identify the mutant sequence (Sanger, F. , et al. , (1980) J. Mol. Biol., 143 , 161-178). Confirmed positive and negative bacilli clones were cultured in LB media containing 12.5ug/mL cmp and purified from culture supernatants as previously described (Estell, 30 D.A., et al. (1985) J. Biol. Chem., 260, 6518-6521). Enzymes were greater than 98% pure as analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, U.K. (1970), Nature, 227, 680-685), and protein concentrations were calculated from the absorbance at 35 280 nm, E280^ = ^^ (Matur^ara, H., et al. (1965), J. Biol. Chem, 240, 1125-1130). 2 4 o •; p 7 n Er.:p.e activity was measured with 200^g/n:L succinyl-L-AlaL-AlaL-ProL-Phep-nitroanilide (Sigma) in 0.1M Tris pH 8.6 or 0.1 M CAPS pH 10.8 at 25 *C. Specific activity (p moles product/min-mg) was calculated from the change in absorbance at 410 run fron: production of p-nitroaniline with time per mg of enzyme (E410 = 8,480 M-lcm-1; Del Mar, E.G., et al. (1975), Anal. Biochem.. 99, 316-320). Alkaline autolytic stability studies were performed on purified enzymes (200/jg/mL) in 0.1 M potassium phosphate (pH 12.0) at 37*C. At various times aliquots were assayed for residual enzyme activity (Wells, J.A., et al. (19S6) J. Biol. Chem.. 261. 6564-6570).

E. Results 1. Optimization and analysis of mutagenesis frequency A set of primer-template molecules that were randomly 31-terminated over the subtilisin gene (Fig. 31) was produced by variable extension from a fixed 5'-primer (The primer mutated a unique Aval site at codon 11 in the subtilisin gene). This was achieved by stopping polymerase reactions with EDTA after various times of extension. The extent and distribution of duplex formation over the 1 kb subtilisin gene fragment was assessed by multiple restriction digestion (not shown). For example, production of new Hinfl fragments identified when polymerase extension had proceeded past IlellO, Leu233, and Asp259 in the subtilisin gene.

Misincorporation of each dNTPas at randomly terminated 3' ends by AMV reverse transcriptase (Zakour, R.A., et al. (1982), Nature, 295, 708-710; Zakour, R.A., et al. (1984), Nucleic Acids Res.. 12, 6615-6628) used conditions previously described (Champoux, J.J., (19B4) , Genetics, 2, 454-464). The efficiency of each misincorporation reaction was estimated to be greater than 8 0% by the addition of each dNTPas to the Aval restriction primer, and analysis by polyacrylamide gel electrophoresis. Misincorporations were sealed by polymerization with all four dNTP's and closed circular DNA was produced by reaction with DNA ligase.

Several manipulations were employed to maximize the yield of the mutant sequences in the heteroduplex. These included the use of a deoxyuridine containing template (Kunkel, T.A. (1985), Proc. Natl. Acad. Sci. USA. 82. 488-492 ; Pukkila, P.J. et al. (1983), Genetics. 104 . 571-582), in vitro methylation of the mutagenic strand (Kramer, W. et al. (1982) Nucleic Acids Res., 10 6475-648 5), and the use of Aval restriction-selection against the wild-type template strand which contained a unique Aval site. The separate contribution of each of these enrichment procedures to the final mutagenesis frequency was not determined, except that prior to Aval restriction-selection roughly one-third of the segregated clones in each of the four pools still retained a wild-type Aval site within the subtilisin gene. After Aval restriction-selection greater than 98% of the plasmids lacked the wild-type Aval site.

The 1.5 kb EcoRI-BamHI subtilisin gene fragment that was resistant to Aval restriction digestion, from each of the four CsCl purified M13 RF pools was isolated on low melting agarose. The fragment was ligated in situ from the agarose with a similarly cut £. col i-B. subtilis shuttle vector, pB0180, and transformed directly into E coli LE392. Such direct ligation and transformation of DNA isolated from agarose avoided -11 6- Icsgs and allowed large numbers cf recombinants to be obtained (> 100 , 000 per ug equivalent of input Ml 3 pool).

The frequency cf mutagenesis for each of the four dNTPas misincorporation reactions was estimated from the frequency that unique restriction sites were eliminated (Table XX). The unique restriction sites chosen for this analysis, Clal, PvuII, and Kpr.I, were 10 distributed over the subtilisin gene starting at ccdor.s 35 , 104, and 166, respectively. As a control, the mutagenesis frequency was determined at the PstI site located in the 5 lactamase gene which was outside the window of mutagenesis. Because the absolute 15 mutagenesis frequency was close to the percentage of undigested plasmid DNA, two rounds of restriction-selection were necessary to reduce the background of surviving uncut wild-type plasmid DNA below the mutant plasmid (Table XX). The background of surviving 20 plasmid from wild-type DNA probably represents the sum total of spontaneous mutations, uncut wild-type plasmid, plus the efficiency with which linear DNA can transform E. coli. Subtracting the frequency for unmutagenized DNA (background) from the frequency for 25 mutant DNA, and normalizing for the window of mutagenesis sampled by a given restriction analysis (4-6 bp) provides an estimate of the mutagenesis efficiency over the entire coding sequence (-1000 bp). 3 0 TABLE XX a- thiol cLNTP inis incorporated ^ Restriction Site Selection % resistant clones 1st 2nd round round Total % resistant clones over mutants per Background lOOQbp T C None PstI 0.32 0.7 0.002 0 - G PstI 0.33 1.0 0.003 0.001 0.2 T PstI 0.32 <0.5 <0.002 0 0 C PstI 0.43 3.0 0.013 0.011 3 None Cial 0.28 0.014 0 - G Clal 2.26 85 1.92 1.91 380 T Clal 0.48 31 0.15 0.14 C Clal 0.55 0.08 0.066 17 None PvuII 0.08 29 0.023 0 - G PvuII 0.41 90 0.37 0.35 88 PvuII PvuII 0.10 0.76 67 53 0.067 0.40 0.044 0.38 9 95 None G T C Kpnl 0.41 3 0.012 0 - Kpnl 0.98 0.34 0.33 83 Kpnl 0.36 0.054 0.042 8 Kpnl 1.47 26 0.38 0.37 93 Mutagenesis frequency is estimated from the frequency for obtaining mutations that alter unique restriction sites within the mutagenized subtilisin gene (i.e., Clal, PvuII, or Kpnl) compared to mutation frequencies of the PstI site, that is outside the window of mutagenesis.

Plasmid DNA was from wild-type (none) or mutagenized by dNTPas misincorporation as described. (c) Percentage of resistant clones was calculated from the fraction of clones obtained after three fold or greater over-digestion of the plasmid with the indicated restriction enzyme compared to a L 1 ll D / r.on-dicested control. Restriction-resistant plasmid DNA frcrr. the first round was subjected to a second round of restriction-selection. The total represents the product of the fractions of resistant clones obtained from both rounds of selection and gives percentage cf restriction-site mutant clones in the original starting pool. Frequencies were derived from counting at least 20 colonies ana usually greater than 10C .

( G ) Percent resistant clones was calculated by subtracting the percentage of restriction-resistant clones obtained for wild-type DNA (i.e., none) from that obtained for mutant DNA. (o ) This extrapolates from the frequency of mutation over each restriction site to the entire subtilisin gene (-1 kb) . This has been normalized to the number cf possible bases (4-6 bp) within each restriction site that can be mutagenized by a given misincorporation event.

From this analysis, the average percentage cf subtilisin genes containing mutations that result from dGTPas, dCTPas, or dTTPas misincorporation was {J estimated to be 90, 70, and 20 percent, respectively. These high mutagenesis frequencies were generally quite variable depending upon the dNTPas and misincorporation efficiencies at this site. Misincorporation efficiency has been reported to be ^ both, dependent on the kind of mismatch, and the context of primer (Champoux, J.J., (1984); Skinner, J.A., et al. (1986) Nucleic Acids Res., 14, 6945-6964). Biased misincorporation efficiency of dGTPas and dCTPas over dTTPas has been previously 30 observed (Shortle, D., et al. (1985), Genetics, 110, 539-555) . Unlike the dGTPas, dCTPas, and dTTPas libraries the efficiency of mutagenesis for the dATPas misincorporation library coulc not be accurately assessed because 90s of the restriction-resistant plasmids analyzed simply lacked the subtilisin gene insert. This problem probably arose from self-ligation of the vector when the dATPas mutagenized subtilisin gene was subclor.ed from M13 into pBOlSO. Correcting for the vector background, we estimate the mutagenesis frequency around 20 percent in the dATPas misincorporation library. In a separate experiment (not shown) , the mutagenesis efficiencies fcr dGTPas and dTTPas misincorporation were estimated to be around 50 and 30 percent, respectively, based on the frequency of reversion of an inactivating mutation at codon. 16 9.

The location and identity of each mutation was determined by a single track of DNA sequencing corresponding to the misincorporated athiodeoxy-r.ucleotide over the entire gene followed by a complete four track of DNA sequencing focused over the site of mutation. Of 14 mutants identified, the distribution was similar to that reported by Shortle and Lin (1985) except we did not observe nucleotide insertion or deletion mutations. The proportion of AG mutations was highest in the G misincorporation library, and some unexpected point mutations appeared in the dTTPas and dCTPas libraries. 2. Screening and Identification of Alkaline Stability Mutants of Subtilisin It is possible to screen colonies producing subtilisin by halos of casein digestion (Wells, J.A. et al. (1983) Nucleic Acids Res., 11, 7911-7925). However, two problems were posed by screening colonies under high alkaline conditions (>pH 11). First, B. subtilis vill not grow at high pH, and we have been unable to transform an alkylophilic strain of bacillus. This problem was overcome by adopting a replica plating strategy in which colonies were grown on filters at neutral pH to produce subtilisin and filters subsequently transferred to casein plates at pH 11.5 to assay subtilisin activity. However, at pH 11.5 the casein micells no longer formed a turbid background and thus prevented a clear observation of proteolysis halos. The problem was overcome by briefly staining the plate with Coomassie blue to amplify proteolysis zones and acidifying the plates to develop casein micell turbidity. By comparison of the halo size produced on the reference growth plate (pH 7) to the high pH plate (pH 11.5), it was possible to identify mutant subtilisins that had increased (positives) or decreased (negatives) stability under alkaline conditions.

Roughly 1000 colonies were screened from each of the four misincorporation libraries. The percentage of colonies showing a differential loss of activity at pH 11.5 versus pH 7 represented 1.4, 1.8, 1.4, and 0.6% of the total colonies screened from the thiol dGTPas, dATPas, dTTPas, and dCTPas libraries, respectively. Several of these negative clones were sequenced and all were found to contain a single base change as expected from the misincorporation library from which they came. Negative mutants included A36, Z170 and V50. Two positive mutants were identified as V107 and R213. The ratio of negatives to positives was roughly 50:1. ^40 670 3. Stability and Activity of Subtilisin Mutants at Alkaline pH Subtilisin mutants were purified and their autolytic stabilities were measured by the time course of inactivation at pH 12.0 (Figs. 32 and 33). Positive mutants identified from the screen (i.e., V107 and R213) were more resistant to alkaline induced autolytic inactivation compared to wild-type; negative mutants (i.e., E170 and V50) were less resistant. We had advantageously produced another mutant at position 50 (F50) by site-directed mutagenesis. This mutant was more stable than wild-type enzyme to alkaline autolytic inactivation (Fig. 33) At the termination of the autolysis study, SDS-PAGE analysis confirmed that each subtilisin variant had autolyzed to an extent consistent with the remaining enzyme activity.

The stabilizing effects of V107, R213, and F50 are cumulative. See Table XXI. The double mutant, V107/R213 (made by subcloning the 920 bp EcoRI-Kpnl fragment of pB0180V107 into the 6.6 kb EcoRI-Kpnl fragment of p30180R213) , is more stable than either single mutant. The triple mutant, F50/V107/R213 (made by subcloning the 735 bp EcoRI-PvuII fragment of pF50 (Example 2) into the 6.8 kb EcoRI-PvuII fragment of p30180/V107, is more stable than the double mutant V107/R213 or F50. The inactivation curves show a biphasic character that becomes more pronounced the more stable the mutant analyzed. This may result from some destablizing chemical modification(s) (eg., deamidation) during the autolysis study and/or reduced stabilization caused by complete digestion of larger autolysis peptides. These alkaline autolysis studies have been repeated on separately purified enzyme batches with essentially the same results. Rates of autolysis should depend both on the conformational stability as well as the specific activity of the subtilisin variant (Wells, J.A., et al. (1986), J. Biol. Chem.. 2 61. 6564-6570) . It was therefore possible that the decreases in autolytic inactivation rates may result from decreases in specific activity of the more stable mutant under alkaline conditions. In general the opposite appears to be the case. The more stable mutants, if anything, have a relatively higher specific activity than wild-type under alkaline conditions and the less stable mutants have a relatively lower specific activity. These subtle effects on specific activity for V107/R213 and F50/V107/R213 are cumulative at both pH 8.6 and 10.8. The changes in specific activity may reflect slight differences in substrate specificity, however, it is noteworthy that only positions 17 0 and 107 are within 6A of a bound model substrate (Robertus, J.D., et al. (1972), Biochemistry 11, 2438-2449). 2 4 0 6 7 -123-TA3LE XXI Relationship between relative specific acitivity at pK 8.6 or 10.8 and alkaline autolytic stability Er.zvme Relative PH 8.6 specific activity PH 10.8 Alkaline autolysis half-time (min)b Wild-type 100±1 100±3 86 Q170 4 6± 1 28±2 13 V107 126±3 99±5 102 R213 97r 1 102±1 115 V107/R213 116±2 106±3 130 V5 0 66±4 61±1 58 F50 123±3 157 + 7 131 F50/V107/ R213 126±2 152± 3 168 Relative specific activity was the average from triplicate activity determinations divided by the wild-type value at the same pH. The average specific activity of wild-type enzyme at pH 8.6 and 10.8 was 70/jmoles/min-mg and 37pmoles/min-mg, respectively.

^ Time to reach 50% activity was taken from Figs. 32 and 33. 24 0 6 7 0 F. Random Cassette Mutagenesis of Residues 197 through 228 Plasmid pA2 2 2 (Wells, et al. (1985) Gene 14 , 315-323) was digested with PstI and BanKI and the 0.4 kb , PstI/BamHI fragment (fragment 1, see Fig. 34) purified from a polyacrylamide gel by electroelution.

The 1.5 kb EcoRI/BamHI fragment from pS4.5 was cloned into K13mp9. Site directed mutagenesis was performed to create the A197 mutant and simultaneously insert a silent SstI site over codons 195-196. The mutant EcoRI/BamHI fragment was cloned back into pBS42. The pA197 plasmid was digested with BamHI and SstI and the 5.3 kb BamHI/SstI fragment (fragment 2) was purified from low melting agarose.

Complimentary oligonucleotides were synthesized to span the region from SstI (codons 195-196) to PstI (codons 226-230) . These oligodeoxynucleotides were designed to (1) restore codon 197 to the wild type, (2) re-create a silent Kpnl site present in pA222 at codons 219-220, (3) create a silent Smal site over codons 210-211, and (4) eliminate the PstI site over codons 228-230 (see Fig. 35) . Oligodeoxynucleotides were synthesized with 2% contaminating nucleotides at each cycle of synthesis, e.g., dATP reagent was spiked with 2% dCTP, 2% dGTP, and 2% dTTP. For 97-mers, this 2\ poisoning should give the following percentages of non-mutant, single mutants and double or higher mutants per strand with two or more misincorporations per complimentary strand: 14% non-mutant, 28% single mutant, and 57% with >2 mutations, according to the general formula n p 3 5 f - — e-> . n! \ * r (A. '* where ^ is the average number of mutations and n is a number class of mutations and f is the fraction of the total having that number of mutations. Complimentary oligodeoxynucleotide pools were phosphorylated and annealed (fragment 3) and then ligated at 2-fold molar excess over fragments 1 and 2 in a three-way ligation.

E. coli MM294 was transformed with the ligation reaction, the transformation pool, grown up over night ar.d the pooled plasmid DNA was isolated. This pool 4 represented 3.4 x 10 independent transformants. This plasmid pool was digested with PstI and then used to retransform E. coli. A second plasmid pool was prepared ar.d used to transform B. subtilis (BG2036) . Approximately 40% of the BG2036 transformar.ts actively expressed subtilisin as judged by halo-clearing on casein plates. Several of the non-expressing transformants were sequenced and found to have insertions or deletions in the synthetic cassettes. Expressing BG2036 mutants were arrayed in microtiter dishes with 150^1 of LB/12.5^g/mL chloramphenicol (cmp) per well, incubated at 37*C for 3-4 hours and then stamped in duplicate onto nitrocellulose filters laid on LB 1.5% skim milk/5pg/mL cmp plates and incubated overnight at 33 'C (until halos were approximately 4-8 mm in diameter). Filters were then lifted to stacks of filter paper saturated with 1 x Tide commercial grade detergent, 50 mM Na^CO^, pH 11.5 and incubated at 65'C for 90 min. Overnight growth plates were Commassie stained and destained to establish basal levels of expression. After this treatment, filters were returned to pH7/skim milk/2Optg/mL tetracycline plates and incubated at 37*C for 4 hours to overnight.

I Mutants identified by the high pH stability screen to be more alkaline stable were purified and analyzed for autolytic stability at high pH or high temperature. The double mutant C204/R213 was more stable than wild ^ type at either high pH or high temperature (Table XXII).

This mutant was dissected into single mutant parents (C204 and R213) by cutting at the unique Smal restriction site (Fig. 35) and either ligating wild type sequence 3' to the Smal site to create the single C204 mutant or ligating wild type sequence 5' to the Smal site to create the single R213 mutant. Of the two single parents, C204 was nearly as alkaline stable 15 as the parent double mutant (C04/R213) and slightly more thermally stable. See Table XXII. The R213 mutant was only slightly more stable than wild type under both conditions (not shown).

Another mutant identified from the screen of the 197 to 228 random cassette mutagenesis was R204. This mutant was more stable than wild type at both high pH and high temperature but less stable than C204. -127-TABLE XXII 2 4 0 6 7 Stability of subtilisin variants Purified enzymes (200^g/mL) were incubated in O.IK phosphate, pK 12 at 30'C for alkaline autolysis, or in 2mM CaCl2, 50mM MOPS, pH 7.0 at 62'C for thermal autolysis. At various times samples were assayed for residual enzyme activity. Inactivations were roughly 1C pseudo-first order, and t 1/2 gives the time it took to reach 50% of the starting activity in two separate experiments. t 1/2 t 1/2 (alkaline (thermal autolysis) autolysis) Subtilisin variant Exp. #1 Exp. a2 Exp. #1 Exp. *2 wild type 23 F50/V107/R213 49 41 18 23 R204 32 24 27 C204 43 46 38 40 C204/R213 50 52 32 36 L204/R213 32 21 G. Random Mutagenesis at Codon 204 Based on the above results, codon 204 was targeted for random mutagenesis. Mutagenic DNA cassettes (for codon at 204) all contained a fixed R213 mutation which was found to slightly augment the stability of the C204 mutant.

I k 0 0 Flasnid DNA encoding the subtilisin mutant C204/R213 was digested with SstI and EcoRI and a 1.0 kb EcoRl/SstI fragment was isolated by electro-elution from polyacrvlamide gel (fragment 1, see Fig. 35).

C204/R213 was also digested with Smal and EcoRI and the large 4.7 kb fragment, including vector sequences and the 3' portion of coding region, was isolated from low melting agarose (fragment 2, see Fig. 36).

Fragments 1 and 2 were combined in four separate three-way ligations with heterophosphorylated fragr.er.ts 3 (see Figs. 36 and 37) . This hetero-phosphorylation of synthetic duplexes should !-J preferentially drive the phosphorylated strand into the plasmid ligation product. Four plasmid pools, corresponding to the four ligations, were restricted with Smal in order to linearize any single cut C204/R213 present from fragment 2 isolation, thus 2 0 reducing the background of C2 04/R213. E. col i was then re-transformed with Smal-restricted plasmid pools to yield a second set of plasmid pools which are essentially free of C204/R213 and any non-segregated heterduplex material. 1") These second enriched plasmid pools were then used to transform B. subtil is (BG2036) and the resulting four mutant pools were screened for clones expressing subtilisin resistant to high pH/temperature 30 inactivation. Mutants found positive by such a screen were further characterized and identified by sequencing.

The mutant L204/R213 was found to be slightly more stable than the wild type subtilisin. See Table XXII. 2 k 0 6 1 Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention.

Claims (5)

1. What wc claim Is:

   . .-\ re rr.-rbinant subtilisin ccnta-ning the substitut i on of at le?.:3L one amino acid residue of a precursor subtilisin with a different naturally occurring amino acid, wherein said at least or.e selected amino -scid residue is selected from the group of equivalent art.ir.c acid residu&s of subtilisin naturally produced by Baci llus ar.viol icruef aciens consisting of Tyr2i, rhr22, Ser2 4, Asp2 6. Gly4 6, Ala46, Ser-iS, MetSO, As.i77, 3er87, Lys=4, Val95, Leu96, Ilel07, Glyi:0, Met!24, Lys:7C, Tyrl71, Frol72, Aspl97, Metl99, Ser204, Lys2l3, His67, Leul35, Gly97, SerlOl, Glyl02, Trpl03, Leul26, Glyl27, Glyl2S, Prsl29, Tyr2l4 and Gly2l5.
2. 2. Ihc recombinant subtilisin of Claim 1 wherein said group cf equivalent residues consists of Tyr21, Thr22, Ssr24, Aap3 6, Gly46, Ala48, Ser49, Met5C, Asn77, Ser87, Lys94 , Val95 , Leu96, rlel07, GlyllO, Metl24, Lyei70, Tyrl7.1., ?ro!72, Aspl97, Metl99, Ser204 and Lys2l3.
3. 3. The recombinant subtilisin of Claim 1 wherein said selected s-ino acid residue of said precursor ie substituted by ar. amino acid residue selected from those listed in Tables 1 and II herein for said selected residues.
4. A . Th?> recombinant subtilisin of Claim 2 wherein saic selected ani no acid residue of said precursor is substituted by u;i a:uino acid residue selected from those lisned in Tabic 1 ;i-jrcin for said selected residue.
5. 5. A recombinant subtilisin having an amino acid sequence derived from the anino acid sequence of a precursor aubr.il is in by the substitution of h different amino acid for at least it first c.r.c a second ami::o acid rseid';e of said atr.ino acid sequence oL said precursor subtilisin, said f.irsc atr.ino a.\ i- residue tc;;ng selected from the group of .

   ^ - 1 " /"

   * O

   \\

   *■ c* •

   ' - >134 ;'j /

   -131 -

   equivalent, arr.ino acid residues cf subtilisin naturally produced by Bacillus amy!o_icruef aciens ccnsisting ot Tyr21, r:hr22, Ser24, Asp3 6, Gly4 6, Aia4 3, Se r49, MetSO, Asn77, Ser57, Ly<?94, Val95, Leu9G, Tie!07, GlyllO, Metl24/ Lysi70, 3 Tyrl71, pre:72, Asp:37, Ketl99, Ser204, Lys2l3, His67, l&ul2 6, Leul3 S, Gly97, SerlOl, Glyl02, Trpl03, Leul26, Glyi27, Glyi28, ?ro!29, Tyr214 and Gly2l5 ana said second arr.ino acid residue being selected from the group of equivalent amino acid residues of subtilisin naturally 10 produced by Bacillus anvlolicruefaciena consisting of Asp32, Ser33, His64, TyrlC4, Al«l52, Asnl55, Glul56, Glyl66, Glyi69, Phei89, Tyr217 ar.d yet222.

   6. The recombinant subtilisin cf Claim 5 wherein said first selected amino acid residue in said precursor is

   "j 5 substituted by an amino acid residue selected from those listed in Tables I and II herein for said selected residue.

   7. The recombinant subtilisin of Claim 5 wherein said first selected amino acid residue in said precursor is substituted by an amino acid residue selected from those

   20 listed in Table I herein for said selected residue.

   8. A recombinant subtilisin derived by the replacement of a: leaat one amino acid residue of a precursor subtilisin with a different amino acid, said subtilisin being modified in at least substrate specificity as compared to said

   25 precursor, said at least one amino acid residue being sclectec from the group cf equivalent: ar;.ino acid residues of subtilisin naturally produced by Bacillus amvlolioruefaciens consisting ci His67, IielG7, Leul35, Gly97, Ala9e, Gly 100, Ser 1C1, Gly 102, Gin 103, Leul26, Gly 127, Gly 128, Prol29, 30 Lys213, Tyr 214, Gly215, Glyl53, Asnl54, Glyl£>7, Thrl58, Serl59, Gly 160, Ser 161, Serl62, Serl63, Thrl64, Vall65, Tyrl67, proiee, Lysl70, Tyr 171 and Prol72.

   9. The recombinant subtilinin cf Claim 9 further comprising the substitution of at least a second amino acid

   •132

   240670

   residue selected from the group of equivalent amino acid residues of subtil is ir. naturally produced by Baclll ua anyloliauef acier.s consisting of Tyrl04, Alai52, Glul56, GlylfcS, GlylS9, Phel39 and Tyr217.

   10. A recombinant subtilisin containing the substitution of at least one selected amino acid residue of a precursor subtilisin with a different naturally occurring amino acid, said recombinant subtilisin being altered in at least alkaline stability as compared to said precursor subtilisin, wherein said at least one selected amino acid residue is selected from the group of equivalent amino acid residues of subtilisin naturally produced by Bacillus amyloliauefaciens consisting of Asp36, Ilel07, Lysl70, Aspl97, Ser204, Lye213, Ser24y and Met50.

   15 11. A recombinant subtilisin of Claim 10 further comprising the substitution of a second amino acid residue selected from the group of equivalent amino acid residues of subtilisin naturally produced by Bacillue amvloliauefaoiens consisting of Glul56, Glyl66, Glyl69, Tyr217 and Met222.

   20 12. A recombinant subtilisin derived by the replacement of at least one amino acid residue of a precursor subtilisin with a different amino acid, said subtilisin being modified in at lease thermal stability as compared to said precursor, said at least one amino acid residue being selected from the 2b group of equivalent amino acid residues of subtilisin naturally produced by 3acillug amylol icruefaciens consisting of Asp3S, Ilel07, Lys170, Ser204, Lys2l3, Metl99 and Tyr21.

   13. A recombinant subtilisin derived by the replacement cf at least one amino acid residue of a precursor subtilisin 30 with a different amino acid, said subtilisin being modified in at. least oxidative stability as compared to said precursor, said ac least one amino acid residue being selected from the group of equivalent amino acid residues of

   ! .

   -133-

   i-ufcniliEir. r:&tur.aliy produced by Bacillus amvlol icruefaciens consisting of MetSO and Metl24.

   14 . The recombinant: subtilisin cf Claim 13 furcher comprising the substitution of a second amino acid residue 5 comprising Ket222.

   15. A recombinant subtilisin containing the combined substitution of at least two selected amino acid residues in said precursor subtilisin with different naturally occurring amino acids, wherein said recombinant subtilisin

   10 is altered in at least thermal stability as compared to said precursor subtilisin and said selected combined amino acid residues are selected from the group of equivalent amino acid residues of subtilisin naturally produced by Bacillus amvloliquefaciens consisting of 7hr22/SerB7, Ser24/Ser87 and 15 Tyr21/Thr22/Ser87.

   16. The recombinant subtilisin of Claim 15 wherein said Thr22, Ser24 and Ser67 are substituted with cysteine.

   17. A recombinant subtilisin having amino acid sequence derived from the amino acid sequence of a precursor

   20 subtilisin by a combination of substitutions of at least two arr.ino acid residues in said precursor equivalent to amino acid residues of subtilisin naturally produced by Bacillus amvloliquefaciens, said subtilisin having at least modified oxidative stability as compared to said precursor, said

   2 5 corrbir.ation of substituted equivalent residues being selected from the group consisting of Met50/Metl24, Met50/Ket222, Metl24/Met222 and Met50/Mscl24/Met222.

   ia. The recombinant subtilisin of Claim 17 wherein said MetSO is substituted with Phe, said Metl24 is substituted

   3 0 with lie or Leu ana said Met222 is substituted with Gin.

   19. A recombinant, subtilisin derived from v.he amino acid sequence of a precursor subtilisin by a combination of

   -13'. -

   uuost i Lu::one of at ieani two emino acid residues in said precursor equivalent to amino acid residues of subtilisin r.auuraiiy produced by Baci Uus arr.vlol icruefaciens. said subtilisin having at least altered oxidative stability and r> substrate specificity as compared to said precursor, wherein said combination of subat:ituted equivalent residues is selected from the group consisting of Glyl66/Met222 and Glyl69/Met222.

   25, The recombinant subtilisin of Claim 19 wherein said 10 Givl66 is substituted with Ala, Phe, Lys or Val, and said Met222 is substituted with Ala or Cys.

   21. A recombinant subtilisin derived from the amino acid sequence of a precursor subtilisin by a combination of substitutions of at least two amino acid residues in said 15 precursor equivalent to amino acid residues of eubtilisin naturally produced by Bacillus amvlol 1 crue fac iena. said eubtilisin having at least improved enzyme performance as compared to said precursor, wherein said combination of substituted equivalent residues comprises Glul56 and Glyl66.

   20 22. The recombinant subtilisin of Claim 21 wherein said Glul56 is substituted wich Gin or Ser and said Glyl66 is substituted with Lys.

   23. A recombinant subtilisin derived from the amino acid sequence of a precursor subtilisin by a combination of 75 substitutions of at least two amino acid residues in said precursor equivalent to amino acid residues of subtilisin naturally produced by Bar, illus amvlol iouefaci ens. said subtilisin having at least altered substrate specificity and kinetics as compared to said precursor, wherein said 30 combination of substituted equivalent residues is selected from the group consisting of Glul56/Glyl69/Tyr2l7, G1 yib6/Glyl66/Tyr2l7 and GlulSb"/Tyr217 .

   240 67

   2'"i . T:i- recombinant subtiliseof Clairr. 23 further con:- vi = :.r.^ the substitution of V.cz:>0 with Phe.

   25. The subtilisin of Claim 23 wherein said Glul56 is substituted wich Ser or Gin, said G"_yl6 9 is substituted with r. Ala and said Tyr2i7 i« substituted with Leu.

   26. 7i recombinant eubtilisin derived from the amino acid Deque.-.ce of a precursor subtilisin by a combination of substitutions of at least two amino acid residues in said precursor equivalent to amino acid residues of subtilisin

   10 naturally produced by Bacillue amvioliauefaciens. said subtilisin having at least modified alkaline or thermal stability ae compared to said precursor/ wherein said combination of substituted equivalent residues is selected from the group consisting of Ilel07/Lys2l3, Ser204/Lys213, lb Glul56/Glyl66, Met50/Glul56/Glyl69/Tyr217 and Met50/Ilel07/Lys213.

   27. The recombinant subtilisin of Claim 26 wherein said Ilel07 is substituted with Val, said Lys213 is substituted with Arg, said Glui56 is substituted with Gin or Ser, said

   20 Glyl66 is substituted with Lys or Asn, and said Glyl69 is substituted v/ith Ala.

   28. A recombinant subtilisin derived frcm the amino acid sequence of a precursor subtilisin by a combination of substitutions of at least two amino acid residues in said

   .'5 precursor equivalent to amino acid residues of subtilisin naturally produced by Bacillus amvlol icruefaciens, wherein said combination of substituted equivalent residues is selected from the group consisting ot Thr22/Ser87, Ser24/Ser87, A].a45/Ala48, Ser4 9/Lys94, Ser49/Val95, 30 KetSO/Val9 5, Met50/Glyll0, Met50/Meti24, Met50/Met222, Met124/Met222, Glul56/Glyl6S, Glul5G/Glyl69, G]yiG6/Met222, G1y16 9/M e t 2 2 2, Tyr2I/Thr22, Me 150/Me1324/Me1222 , Tyr21/Tnr22/Ser87, Met50/Glul56/Glyl66/Tyr217,

   'vA

   '•1 e t : C / G ^ 1 b 6 /' T y ; 2 1 7 , V. G t 51 0 / G i u 1 5 6 / G 1 y 1 6 S / T y r 2 1 7 , MetSC/11 e:: 7/Lys213 , £e-204/Ly£;2i:: , and IIsi 07/lys213 .

   29. The recombinant yubti- -1 sir. r/ Claim 28 wherein said selected cc.tbinatior. of residues are substituted by an amino acid residue selected from those listed in Table IV herein.

   3C. A. recombinant subtilisin derived from the amino acid sequence of a precursor subtilisin by the deletion cf at least or.e amino acid residue ir. said precursor equivalent to amino acid residues of subtilisin naturally produced by Bacillue amvlol iquef acifens. wherein said at least: one deleted residue is selected from the group consieting of Serl61, Serl62, Serl63 ana Thrl64.

   31. The recombinant subtilisin of Claim 30 wherein said deletion comprises A161-164.

   32. The recombinant subtilisin of Claims 1 or 5 wherein said precursor is a Bacillus subtilisin.

   33. The recombinant subtilisin of Claim 32 wherein said Bacillus subtilisir. is Bacillus amvlol iaue.f aciens eubtilisin.

   34. The recombinant subtilisin of any of claims 1, 5, 8, 10, 12, 13, 15, 17, 23, 26, 28, or 30 wherein said recombinant subtilisin is substantially pure.

   3 5. The recombinant nubtilisin of any o£ clainf) 1, 5, 8, "0, 12, 13, 15, l"7, 23, 26, 28, or 30 wherein said recombinant oubtilisir. is enzymatically active.

   36. The reconibinant subtilisin of Claim 33 resulting from the expression of DNA encoding said subtilisin.

   37. DNA encoding the subtilisin of ar.v of the Claims 1 to

   3b

   T E

   » (

   '/J

   -137- — f 0 8

   L'xpreGoion vccccr containing the DNA of Claim 28.

   . Host ceU transferred with the expression vector of .aim 33.

   40. A recombinant subtilisin as defined in any one of claims 1, 5, 8, 10, 12, 13, 15, 17, 19, 21, 23, 26. 28 and 30 substantially as herein described with reference to any example thereof and/or to the accompanying drawings.

   41. DNA as claimed in claim 37 substantially as herein described with reference to any example thereof.

   42. Expression vector as claimed in claim 38 substantially as herein described with reference to any example thereof.

   43. Host cell as claimed in claim 39 substantially as herein described with reference to any example thereof.

   (x£N£MCcg j NTfcWTlDMflL} I l\M

   By tho authorised acjents A J PARK & SON

   Per

   \ jS-VGi/ jgg4 £!j

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