WO2004104192A1

WO2004104192A1 - Method for isolating or purifying staphylococcus ump kinase

Info

Publication number: WO2004104192A1
Application number: PCT/GB2004/002158
Authority: WO
Inventors: Peter Clifford Doig
Original assignee: Astrazeneca Ab; Astrazeneca Uk Limited
Priority date: 2003-05-22
Filing date: 2004-05-19
Publication date: 2004-12-02
Also published as: EP1629094A1; GB0311789D0; JP2007500512A

Abstract

An isolated or purified Staphylococcus UMP kinase characterised in that during the process of isolation or purification the enzyme is stabilized by addition of a stabilisation species such that it can be stored for at least one week at between -20 and 4 degrees centigrade without more than 50% loss of specific enzyme activity when compared with its initial stabilised activity.

Description

M_ETHO_{D F}OR ISOLATING OR PURIFYING STAPHYLOCOCCUS UMP KINASE

The present invention relates to nucleoside monophosphate kinases and in particular to their purification, stabilisation and use in drug screening assays to identify kinase inhibitors.

Nucleoside monophosphate kinases are required for the biosynthesis of nucleic acids, of various key nucleotidyl intermediates and in energy metabolism. One member of this kinase family, uridine 5 '-monophosphate (UMP) kinase (PyrH) catalyzes the transfer of the γ- phosphate of adenosine 5'-triphosphate (ATP) to UMP to produce the adensosine 5'~ diphosphate (ADP) and uridine 5 '-diphosphate (UDP) and this function is essential for cellular survival. A unique aspect in bacteria is that they possess separate UMP and cytidine 5'-monphosρhate (CMP) kinases whereas mammals and many higher eukaryotes possess a single enzyme that carries out both functions. These enzymes share little sequence homology and PyrH enzymes from bacteria possess unique structural and enzymological characteristics. The enzyme is ubiquitous, being found in all major pathogenic bacteria. The essential nature of this enzyme and its wide distribution make it an ideal target for intervention with an antibacterial agent.

WO-99/55729, WO-2001/12678 and corresponding US patent no. 6403337 disclose pyrH gene sequences from Staphylococcus aureus (S. aureus). We have cloned and purified S. aureus UMP kinase (PyrH) and the amino acid sequence is set out in Seq ID No. 2 hereinafter. When compared with the published amino acid sequence of this gene, it lacks Methionine as the first amino acid. Whilst we don't wish to be bound by theoretical considerations, this is not believed to have any material effect on the properties of the enzyme, for example when used in drug screening assays. Whilst purifying S. aureus UMP kinase we found that it was necessary to add nucleosides or nucleotides to the purification procedure to provide a soluble, stable or active enzyme. Specifically we found that the addition of uridine 5'-triphosphate (UTP) results in stable protein that is soluble at high concentrations. We also found that guanosine 5'- triphosphate (GTP) and other nucleotides such as ATP are important to stabilize and/or activate the enzyme as well as acting as allosteric-like regulators of activity.

These discoveries are not anticipated in the prior art. US patent application 2002/0119506 discloses the cloning and isolation of UMP kinase from three bacillus microorganisms, namely B. subtilis (Gram negative), M. tuberculosis (acid-fast) and H. influenzae (Gram negative). It reports GTP activation and UTP inactivation of the B. subtϊlis enzyme. The degree of GTP activation for all three species is variable, a factor of 2 for M. tuberculosis, 3 for H. influenzae and between 10 and 20 for B. subtilis. However such effects are believed to be species specific ie. there is no anticipation that other enzymes will show significant activation. In particular there is no indication as to the properties of Gram positive staphylococcus UMP kinases.

Therefore in a first aspect of the invention we provide an isolated or purified

Staphylococcus UMP kinase characterised in that during the process of isolation or purification the enzyme is stabilized by addition of a stabilisation species such that it can be stored for at least one week at between -20 and 4 degrees centigrade without more than 50% loss of specific enzyme activity when compared with its initial stabilised activity.

Stabilization is conveniently effected by protecting the protein from degradation, modification or aggregation. The enzyme is conveniently stabilized by use of a stabilisation species such as UTP or GTP or a functional analogue thereof. The concentration of stabilisation species such as UTP is conveniently at least 100 micromolar and the kinase is present at a concentration of up to 200% equivalent concentration.

Higher concentrations of UTP may be used although for practical purposes there may be no need to exceed 5 millimolar. Whilst we do not wish to be limited by theoretical considerations we believe that for example UTP stabilises the UMP kinase enzyme by occupying a binding site distinct from that of the substrate (UMP and ATP) binding sites.

By "functional analogue" of UTP or GTP we mean a species that recognises and binds to the same binding site. Such species may include, by way of non-limiting example, nucleotide analogues such as 2'-Fluoro-uridine 5'-triphosphate, Guanosine 5'[gamma-thio]-triphosphate, and 273 ' -O-(N-Methyl-anthraniloyl)-guanosine-5 ' -triphosphate.

The enzyme is any convenient Staphylococcus UMP kinase, such as any one of S. aerogenes, S. auricularis, S. arlettae, S. capitis, S. capitis subsp. ureolyticus, S. caprae, S. carnosus subsp. carnosus, S. chromogenes, S. cohnii subsp. cohnii, S. cohnii subsp. urealyticum, S. condimenti, S. delphini, S. epidermidis, S. equorum, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis subsp. hominis, S. hominis subsp. novobiosepticus, S. hyicus, S. intermedius, S. kloosii, S. lentus, S. lugdunensis, S. lutrae, S. muscae, S. ovis, S. pasteuri, S. piscifermentans, S. pulvereri, S. saccharolyticus, S. saprophyticus, S. schleiferi, S. schleiferi subsp. coagulans, S. sciuri subsp. carnaticus, S. sciuri subsp. rodentium, S. sciuri subsp. sciuri, S. simulans, S. succinus, S. vitulus, S. warned, S. xylosus and in particular is from S. aureus.

By "stabilized" we mean that the UMP kinase enzyme is provided in a form such that its specific activity over time is not unduly compromised and remains suitable for use in for example drug screening assays. Specifically, it can be stored for at least one week at between -20 and 4 degrees centigrade without more than 50% loss of specific enzyme activity when compared with its initial stabilised activity.

We have observed a stabilised UMP kinase enzyme from S. aureus as a hexamer with an approximate molecular weight of 156000. Therefore in a further aspect of the invention we provide a stabilized UMP kinase characterised as a hexamer of the kinase.

The stabilized enzyme is conveniently provided as storage solution made up of one or more of: a buffer, a nucleotide, glycerol, and a reducing agent. The solution may contains a convenient buffer, preferably HEPES, PIPES or Tris-HCl, and a neutral or alkaline pH, preferably pH 8.0 through 8.5. In addition the solution contains a nucleotide, preferably UTP at a concentration of 0.5 to 2 millimolar, corresponding to a enzyme to nucleotide ratio of at least 50%. The solution also contains glycerol (10% to 50% vol/vol), sodium chloride or similar salt at a concentration of 100 to 250 millimolar and a reducing agent such as dithiothreitol at a concentration between 100 micromolar to 5 millimolar.

With knowledge of the above properties we have devised assays to identify and characterize inhibitors of UMP kinase enzymes. Such assays use an activated form of a UMP kinase enzyme.

Therefore in a further aspect of the invention we provide an isolated or purified Staphylococcus UMP kinase characterised in that it is activated by addition of an activation species to provide a kinase with a specific activity that is at least two fold greater when compared with the specific activity of the corresponding non-activated kinase.

The activation species is conveniently GTP or ATP or is a functional analogue or either species. The nucleoside triphosphate(s) is conveniently present at a concentration of 100-1000 micromolar and the kinase is present at no more than 50% equivalent concentration.

The enzyme is any convenient Staphylococcus UMP kinase, such as any one of S. aerogenes, S. auricularis, S. arlettae, S. capitis, S. capitis subsp. ureolyticus, S. caprae, S. carnosus subsp. carnosus, S. chromogenes, S. cohnii subsp. cohnii, S. cohnii subsp. urealyticum, S. condimenti, S. delphini, S. epidermidis, S. equorum, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis subsp. hominis, S. hominis subsp. novobiosepticus, S. hyicus, S. intermedius, S. kloosii, S. lentus, S. lugdunensis, S. lutrae, S. muscae, S. ovis, S. pasteuri, S. piscifermentans, S. pulvereri, S. saccharolyticus, S. saprophyticus, S. schleiferi, S. schleiferi subsp. coagulans, S. sciuri subsp. carnaticus, S. sciuri subsp. rodentium, S. sciuri subsp. sciuri, S. simulans, S. succinus, S. vitulus, S. warneri, S. xylosus and in particular is 5 from S. aureus.

The activated enzyme is conveniently provided as a working solution. The working solution may contain one or more of: a buffer, salts, nucleotides, and surfactants. The working solution may contain any convenient buffer MES, PIPES, MOPS, HEPES, Tricine, Tris-HCl or Glycine and preferably in HEPES or PIPES buffer, at any convenient pH (pH 6

10 through pH 9), preferably between pH 7 and 8. The solution may contain one or more salts such as, but not limited to NaCl, NH₄CI, KC1, and/or MgCl₂. The working solution may also contain a surfactant, preferably a non-ionic surfactant, such as Brij 35, Triton X-100 or Tween 20 at a concentration near or below the critical micellar concentration. In addition a nucleotide, preferably GTP and/or ATP, are added conveniently present at a concentration of

15 100-1000 micromolar and the kinase is present at no more than 50% equivalent concentration. In a further aspect of the invention we provide a drug discovery assay using the activated Staphylococcus UMP kinase of the invention.

The activated UMP kinase is conveniently prepared from the stabilized UMP kinase of this invention, for example by displacement of the stabilisation species.

20 By way of non-limiting example, the enzyme is diluted into a working solution containing 5 μM to 5 mM GTP or 5 μM to 5 mM ATP or any combination of these nucleotides. This may provide for example 4000-fold to 4,000,000-fold molar excess over PyrH enzyme. Use of at least an 80,000-fold excess of GTP is preferred. Stabilization at -80 to -30 degrees centigrade may be achieved by modifying the storage solution components.

25 The assay conveniently comprises a pyruvate kinase/lactate dehydrogenase

(PK/LDH) coupling reaction. In this pyruvate kinase converts the products of the UMP kinase reaction, ADP and UDP, along with phosphoenolpyruvate, to ATP, UTP and pyruvate. The second coupling enzyme, lactate dehydrogenase, converts pyruvate to lactate and NADH to NAD⁺. The decrease in NADH concentration as it is converted to NAD⁺ may be monitored

30 spectrophotometrically by either absorbance or fluorescence. The consumption of one mole of UMP results in the production of two moles of NAD⁺.

The assay is conducted at any convenient pH (pH 6 through pH 9), and preferably conducted at a pH between 7 and 8. The assay is conducted in any convenient buffer such as MES, PIPES, MOPS,

HEPES, Tricine, Tris-HCl or Glycine and preferably conducted in HEPES or PIPES buffer. A surfactant (Brij-35, Triton X-100 or Tween 20) is added to prevent protein from sticking to the reaction vessel or pipette tips. Brij-35 at a concentration of 0.01% v/v is the preferred surfactant.

The assay is conducted at any convenient temperature (15-30 degrees centigrade) and preferably conducted at a temperature between 20 and 30 degrees centigrade, especially room temperature (18 to 25 degrees Centigrade such as about 22 degrees centigrade).

The assay is conducted at any convenient UMP kinase concentration, such as between 0.1 and 300 nM, for example about 1.3 nM

The UMP kinase used in the assay is conveniently stabilized by GTP at a concentration between 50 uM - 10 mM GTP, preferably about 5 mM GTP.

The invention will now be illustrated but not limited by reference to the following

Example, Sequence Listing and Figures wherein:

Figure 1 shows the reaction pathway catalysed by UMP kinase

Figure 2 shows that GTP is required for full enzyme activity. The graph shows the effect of pre-incubation of enzyme with varying concentration of GTP.

Figure 3 shows buffer and pH profiles. The graph shows a comparison of buffers at various pH.

Figure 4 shows the temperature dependence of enzyme activity between 15 and 30 degrees centigrade.

Figure 5 shows the effects of salts on enzyme activity. Salts used were Na⁺, NH and SO ^2".

Figure 6 shows the optimisation of MgCl₂ concentration. Figure 7 shows the effect of DMSO on enzyme activity- Figure 8 shows the activity of various concentrations of enzyme.

Figure 9 shows the optimisation of coupler enzyme concentration in the UMP kinase assay.

Figure 10 shows enzyme stability over time with added GTP and Brij-35

Figure 11 shows enzyme stability in assay buffer. Figure 12a shows the effect of varying concentrations of the UMP kinase inhibitor EDTA.

Figure 12b shows the effect of varying concentrations of the UMP kinase inhibitor AMP-para nitro phenol. Figure 13a shows a time course for the UMP kinase assay in 384 well plates. NADH fluorescence was measured using an excitation/emission wavelength pair of 340/465nm. Figure 13b shows a time course for the UMP kinase assay in 384 well plates. Absorbance was measured at 340nm

Example

Cloning of S. aureus UMP kinase (pyrH)

S. aureus pyrH was cloned from strain 601055. Primers INF 5174 (5') and INF 5 5183 (3') were used to obtain the S. aureus pyrH PCR product from the a cell lysate of S. aureus 601055.

INF 5174 CCCATATGGCTCAAATTTCTAAATATAAACG INF 5183 CGGGATCCATTATTTTGTAATTAACGTACCTATC 0 The PCR product was purified using the Qiagen purification kit and ligated into the TOPO Invitrogen cloning vector. The ligated product was transformed into TOPO cells according to the manufacturers recommended method. Transformants were selected and the plasmid recovered using the Qiagen miniprep kit, according to the recommended method. Presence of the correct size DNA insert was verified by digestion with BamHl and Ndel 5 restriction enzymes. Clones with the correct size insert, containing S. aureus PyrH, were selected and DNA prepared using the Qiagen Maxiprep kit. Presence of the insert was rechecked by BamHl/Ndel digestion. The insert was sequenced to assure its identity.

The S. aureus PyrH insert was cloned into E. coli pT73.3 and transformed into BL21 (DE3). These cells were grown in LB media at 30°C, containing tetracycline (10 0 μg/ml) and expression induced by addition of ITPG to a final concentration of 1 mM. Induced cells expressed a protein with an apparent molecular mass of 26,000 as determined by SDS- polyacrylamide gel electrophoresis, consistent with the size expected for the polypeptide. Cell paste was collected by centrifugation and stored at -20°C until use.

5 Purification of 5. aureus UMP kinase (PyrH)

The frozen cell paste was suspended in 50 ml of Lysis Buffer [50 mM Tris-HCl, pH 8.5, 2 mM EDTA, 2 mM DTT, 2 mM UTP, 1 mM PMSF, 1 Protease inhibitor cocktail tablet (Roche Molecular Biochemical)]. Cells were disrupted by passing them twice through a French press operated at 18,000 psi, and the crude extract was centrifuged at 20,000 rpm 0 (45Ti rotor, Beckman) for 30 min at 4°C. The supernatant was loaded at a flow rate of 1.5 rnl/min onto a 20 ml Q-Sepharose HP (HR16/10) column (Pharmacia) pre-equalibrated with Buffer A (50 mM Tris-HCl, pH 8.5, 2 mM EDTA, 2 mM DTT, 2 mM UTP). The column was then washed with Buffer A, and the protein was eluted by a linear gradient from 0 to 1 M NaCl in Buffer A. Fractions containing PyrH were pooled, and solid (NH₄)₂SO₄ (0.4 g/ml) was added to precipitate all the proteins and mixed on ice for 1 hour. The sample was centrifuged at 11,000 rpm for 30 min at 4°C (JA12 rotor, Beckman), the pellet was then dissolved in 5 ml of Buffer A. The 5 ml sample was applied at a flow rate of 0.5 ml/min to a 5 320 ml Sephacryl S-300 (HR 26/60) (Pharmacia) pre-equalibrated with Buffer B (50 mM Tris-HCl, pH 8.5, 2 mM EDTA, 2 mM DTT, 2 mM UTP, 150 mM NaCl). The fractions containing PyrH were pooled and dialyzed against 1 L Storage Buffer (50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA, 1 mM UTP, 150 mM NaCl, 2 mM DTT, 20% Glycerol.). The protein was characterized by SDS-PAGE analysis and analytical LC-MS. The determined mass of 0 the protein indicated that the N-terminal methionine of the polypeptide predicted from the DNA sequence was not present [expected MW = 26013.0 Da (-Met), observed = 26014.4 Da (-Met)]. The protein was stored at -80°C.

Assay for Staphylococcus aureus UMP kinase (PyrH) 5 UMP kinase catalyzes the transfer of the gamma phosphate of ATP to UMP, resulting in the formation of two products, ADP and UDP. This activity can be monitored using a pyruvate kinase/lactate dehydrogenase (PK/LDH) coupling reaction. Pyruvate kinase converts the products of the UMP kinase reaction, ADP and UDP, along with phosphoenolpyruvate, to ATP, UTP and pyruvate. The second coupling enzyme, lactate 0 dehydrogenase, converts pyruvate to lactate and NADH to NAD⁺. The decrease in NADH concentration as it is converted to NAD⁺ can be monitored spectrophotometrically by either absorbance or fluorescence. It must be noted that the consumption of one mole of UMP results in the production of two moles of NAD⁺.

To perform the assay, a 1.67X assay buffer stock solution was prepared such that 5 the reaction would contain final concentrations of 50 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl₂, 6.6 units/mL PK/LDH, 0.4 mM ATP, 0.2 mM NADH, 1.29 μM S. aureus UMP kinase, 0.5 mM GTP and 0.001% Brij-35. Prior to addition to this mixture, a 10X enzyme working stock solution containing 12.9 nM solution of S. aureus UMP kinase in 50 mM HEPES pH 8.5, 100 M KCl, 5 mM GTP, 0.01% Brij-35 was prepared and incubated for 15 0 minutes at room temperature. Assay plates were prepared by dispensing an appropriate volume of the 1.67X assay buffer stock to reaction vessels, adding compound DMSO stock solutions and incubating for at least 15 minutes at room temperature to allow equilibration of reagents before initiating reaction with a 3.33X UMP stock solution. For example, for a reaction volume of 100 μL, 2 μL of 50X compound in DMSO was added to 60 μL 1.67X buffer/enzyme mixture and mixed either by rotary shaking or pipetting. After appropriate incubation time, reaction was initiated with the addition of 40 μL 0.67 mM UMP (final concentration was 0.2 mM). Controls used were 2% DMSO (no inhibition) and 10 mM EDTA (100% inhibition). Assay mixtures were mixed by rotary shaking for -20 seconds following addition of UMP. Blank reads were performed and reaction progress was monitored by following the decrease in NADH absorbance or fluorescence over time by either endpoint or continuous read methods. To facilitate testing large numbers of compounds, in some instances, reactions were quenched after 30 minutes with 10 mM EDTA and the final spectrophotomeric measurements made up to several hours later.

The assay was optimized to yield maximum specific activity within the constraints of desirable kinetic parameters. Assay parameters addressed included enzyme handling, substrate concentrations, salt requirements and effects, pH and buffer selection, temperature, stability of the reaction over time, linearity of the reaction over time, response of the assay to DMSO, coupler enzyme concentration and activity, and robustness of the assay.

S. aureus UMP kinase was found to have a pH optimum between 7.0 and 8.0. The pH selected for the assay was 7.5 as changes in pH of less then 0.5 should not have a significant effect on the assay rate. Buffer effects were small but significant, with Tris and MOPS resulting in the lowest activities and HEPES and PIPES the highest enzyme activities. The specific activity of the S. aureus UMP kinase was monitored over the temperature range 15-30°C. A generally linear increase in activity was observed with increase in temperature, with an overall increase in activity of 40% at 30°C.

A number of salts were tested in the assay to see if they resulted in enhancement or reduction of the UMP kinase activity. There was little change in activity in response to concentrations of either NaCl or NH₄CI up to -100 mM or KCl up to 320 mM. However, ammonium sulfate was observed to seriously inhibit enzyme activity. Increasing MgCl₂ concentrations had little effect in the assay at concentrations above those required for enzyme activity.

NADH fluorescence (excitation emission wavelengths 340nm/465nm) was observed to be linear up to -200 μM. An increased linear range of up to -500 μM could be obtained using the Tecan Ultra in absorbance mode at 340 nm. An assay concentration of 200 μM NADH was chosen to ensure linearity of the monitored change in signal in the assay. A concentration of the coupling enzymes PK/LDH that would ensure their excess was chosen. The assay was observed to be under saturating conditions of PK/LDH at concentrations above 1.3 units/mL in a reaction mixture containing 150 μM UMP, 400 μM ATP and 0.000036 mg/mL PyrH.

Specific activity of S. aureus UMP kinase did not change in response to changes in enzyme concentration. This confirmed that the assay rate was UMP kinase dependant. A concentration of 1.3 nM UMP kinase was found to yield optimal activity. Km's for UMP and ATP were found to be in the range of 40 μM and 550 μM, respectively. For the assay, it was found that 200 μM UMP and 400 μM ATP were sufficient to give good activity and would allow the identification of inhibitors. It was found that the protein was stable when stored at -80°C for several months. For use, enzyme was thawed on ice and diluted immediately into the working stock solution buffer. Once thawed, the stock could be stored on ice for at least 7 hours without significant loss of activity. Stability and solubility of the enzyme was enhanced in the presence of NTPs (ATP, GTP, or UTP). GTP was found to be required for full enzyme activity. Stability at room temperature of S. aureus UMP kinase was investigated under conditions of varying concentrations of GTP and Brij-35 in the working solution containing a concentration of enzyme that was 10X that of the assay concentration. It was found that 5 mM GTP and a concentration of Brij-35 between 0.01 and 0.02% (wt/vol) resulted in maximum stability and highest activity of the enzyme. The working solution conditions chosen were 50 mM HEPES pH 8.5, 100 mM KCl, 5 mM GTP and 0.01% Brij-35. Stability of S. aureus UMP kinase in the assay buffer was also investigated. The higher pH for the working stock solution further increased the stability of the enzyme. Working stock solution enzyme was added to the assay buffer and incubated at room temperature for up to 6 hours before initiating the reactions with UMP. It was found that the enzyme retained >90% activity after incubation in assay buffer for 6 h. References

1. Landais, S., et al., Immunochemical analysis of UMP kinase from Escherichia coli. Journal of Bacteriology, 1999. 181(3): p. 833-40. 2. Bucurenci, N, et al., Mutationάl analysis of UMP kinase from Escherichia coli. Journal of Bacteriology, 1998. 180(3): p. 473-7. 3. Serina, L., et al., Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP. Biochemistry, 1996. 35(22): p. 7003-11. 4. Serina, L., et al., Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP. Biochemistry, 1995. 34(15): p. 5066-74.

5. Schneewies, L. A. et al (2001). Structural studies on the mechanism of GTP as a positive effector of UMP kinase from Streptococcus pneumoniae. Abstract presented at the Protein Society annual meeting. Philadelphia, PA.

Claims

1. An isolated or purified Staphylococcus UMP kinase characterised in that during the process of isolation or purification the enzyme is stabilized by addition of a stabilisation species such that it can be stored for at least one week at between -20 and 4 degrees centigrade without more than 50% loss of specific enzyme activity when compared with its initial stabilised activity.

2. A UMP kinase as claimed in claim 1 which is a Staphylococcus aureus kinase.

3. A UMP kinase as claimed in claim 1 or claim 2 and wherein the stabilisation species selected from UTP or GTP or a functional analogue thereof.

4. A UMP kinase as claimed in claim 3 and wherein the stabilisation species is present at a concentration of at least 100 micromolar and the kinase is present at a concentration of up to

200% equivalent concentration.

5. An isolated or purified Staphylococcus UMP kinase which is stabilised such that it is soluble at a concentration of at least 200 micromolar in a buffer.

6. A stabilized Staphylococcus UMP kinase characterised as a hexamer of the kinase.

7. A method of stabilizing a UMP kinase enzyme which method comprises adding UTP or GTP to the enzyme.

8. An isolated or purified Staphylococcus UMP kinase characterised in that it is activated by addition of an activation species to provide a kinase with a specific activity that is at least two fold greater when compared with the specific activity of the corresponding non-activated kinase.

9. A UMP kinase as claimed in claim 8 and wherein the kinase is activated to provide a kinase with at least three fold greater specific activity when compared with the specific activity of the non-activated kinase.

10. A UMP kinase as claimed in claim 8 or claim 9 which is a S. aureus UMP kinase.

11. A UMP kinase as claimed in any one of claims 8-10 and wherein the activation species is GTP or ATP or a functional analogue thereof.

12. A UMP kinase as claimed in claim 8 or claim 9 and wherein the activation species is GTP or a functional analogue thereof.

13. A UMP kinase as claimed in any one of claims 8-12 and wherein the GTP or ATP is present in a concentration of 100 - 2000 micromolar and the kinase is present at no more than

50% equivalent concentration.

14. A method of activating a UMP kinase enzyme which method comprises adding GTP or ATP to the enzyme.

15. A storage solution comprising a UMP kinase according to any one of claims 1-6 together with one or more of a buffer, nucleotide, glycerol, and a reducing agent and which solution can be stored, after constitution, for at least one week at between -20 and 4 degrees centigrade without more than 50% loss of specific enzyme activity.

16. A working solution comprising a UMP kinase according to any one of claims 8-13 together with one or more of a buffer, salts, nucleotides, and surfactants and which solution can be stored, after constitution, at between 4 and 25 degrees centigrade for 12 hours without more than 50% loss of specific enzyme activity.

17. A working solution as claimed in claim 16 and wherein there is no more than 20% loss of specific enzyme activity.

18. A drug discovery assay comprising the use of an activated UMP kinase as claimed in any one of claims 8-13 or 16-17 to catalyze the transfer of the γ-phosphate of ATP to UMP to produce ADP and UDP.

19. An assay as claimed in claim 18 and wherein the ADP and UDP products of the assay are detected directly.

20. An assay as claimed in claim 19 and comprising a pyruvate kinase/lactate

5 dehydrogenase (PK/LDH) coupling reaction and measurement of conversion of NADH to NAD⁺.

21. An assay as claimed in claim 20 and conducted at a pH between 7 and 8.

10 22. An assay as claimed in claim 20 and conducted in HEPES, PIPES buffer or Brij-35.

23. An assay as claimed in claim 20 and conducted at between 20 and 30 degrees centigrade.

15 24. An assay as claimed in claim 20 and conducted at a UMP kinase concentration of 0.1 - 300 nM.

25. A storage solution as claimed in claim 15, which solution is liquid phase at -20 degrees centigrade. 20