• PURINES ARE NOT MADE AS FREE BASES

OUTLINE
• PURINES ARE MADE AS NUCLEOTIDES
I. Purine Metabolism
II. Purine Synthesis; PRPP
III. Conversion of Nucleoside
Monophosphates To Nucleoside
Diphosphates And Triphosphates
IV. Conversion of Ribonucleotides to
Deoxynucleotides
Step Enzyme Process Product
V. Antimetabolites of Purine Nucleotides 1 Activation of ATP AMP PRPP
VI. Degradation of Purine Nucleotides ribose-5- Ribose
VII. Diseases Associated with defects in Purine phosphate phosphate
Metabolism pyrophosphoki
VIII. Pyrimidines Are Not Synthesized as nase
2 APA -N9 PRPP Committed Phospho
Nucleotides Amidotransfer step -Beta-D-
IX. Biosynthesis of Pyramidine Nucleotides ase ribosyla
X. Overproduction of Pyramidine Catabolites Glutamine  mine
XI. Summary Glutamate + Pi
3 APA- C4, Glycinamide Condensation GAR
C5, N7 synthetase of glycine
I. PURINE METABOLISM Glycine + ATP
 ADP
Functions of Nucleotides: 4 APA – C8 GAR Addition of FGAR
• Polymerize to make DNA and RNA transformylase formyl group
• Energy currency of the cell e.g. ATP, GTP N10 formyl H4
• Act as carriers of active intermediates in various metabolic folate
5 APA - N3 Glutaminegl Amidation FGAM
pathways e.g. UDP-glucose in glycogen synthesis, utamate +
SAM ATP ADP
• Component of coenzymes e.g. FAD, NADH, NADPH 6 Closing Dehydration + Removal of AIR
• Act as 2nd messengers e.g. cAMP and CGMP imidazole ATP  ADP H20
• Allosteric regulation of various metabolic pathways e.g. ATP ring
inhibits PFK-1 7 APA – C6 AIR Addition of CAIR
Carboxylase CO2
Nitrogenous Bases: ATP, HCO3-
• Pyrimidine 8 APA – N1 Aspartate + Aspartate SAICAR
• Purine ATP condensation
Pentose Sugars: SAICAR
• Ribose Synthetase
• 2-Deoxyribose 9 Fumarate Fumarate Succinyl AICAR
Elimination Adenylosuccin excreted as
Nucleotide Synthesis: ate lyase Fumarate
• De novo pathway - synthesis from low molecular weight 10 APA – C2 Formyl H4 Formyl added FAICAR
precursors (amphibolic intermediates) Folate to AICAR +
• Salvage pathway - synthesis from nucleosides or bases that Folate C2 of purine
become available through the diet or from degradation of nucleic AICAR ring
Transformylas
acids
e
Sources of purine ring components 11 Ring closure IMP
Note: Sources of purine ring components to form AMP,
• Aspartate – N1 IMP Co GMP
• CO2 – C6
parent
• Formyltetrahydrofolate – C2
purine
• Methenyltetrahydrofolate – C8
nucleotide
• Glycine – C4, C5, and N7
• Glutamine – N3, N9 LEGEND:
II.PURINE SYNTHESIS: PRPP SYNTHESIS APA acquisition of purine atom
1
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG
PRPP Phosphoribosyl Pyrophosphate SALVAGE REACTIONS- CONVERT PURINES & THEIR
GAR  Glycinamide Ribonucleotide NUCLEOSIDES TO MONONUCLEOSIDES
FGAR  Formyl Glycinamide Ribonucleotide
FGAM  Formylglycinamine
AIR  5-Aminomidazole ribonucleotide
CAIR  Carboxyaminomidazole ribonucleotide
SAICAR  N-Succinylo-5 aminomidazole-4-carboxamide
ribonucleotide
AICAR  5- Aminoimidazole-4-carboxamide ribonucleotide
FAICAR  N-Formylaminosimidazole-4- carboxamide
ribonucleotide
IMP  Inosine Monophosphate • Purines that result from the normal turnover of cellular nucleic
Piinorganic phosphate acids, or that are obtained from the diet and not degraded, can
be converted to nucleoside triphosphates and used by the body
Conversion of IMP to AMP and GMP - Phosphoribosylation is more important
Conversion of purine bases to nucleotides:
• Two enzymes are involved:
- Adenine phosphoribosyltransferase
- Hypoxanthine-guanine phosphoribosyltransferase
(HGPRT)
• Both enzymes use PRPP as the source of the ribose
5phosphate group
• The release of pyrophosphate and its subsequent hydrolysis
by pyrophosphatase makes these reactions irreversible
AMP
Adenosine & Hypoxanthine-Phosphoribosyl Transferase
•requires guanosine triphosphate (GTP) as an energy source
• catalyze the phosphoryl transfer from ATP in conversion of
•inhibits adenylsuccinate
adenine, hypoxanthine and guanine to their mononucleotides
GMP
• requires ATP Negative feedback:
Adenosine Kinase
• first reaction in each pathway is inhibited by the end product
• catalyse phosphorylation of the purine nucleotides
of that pathway
• converts adenosine and deoxyadenosine to AMP and dAMP
•inhibits IMP dehydrogenase
- Adenine, guanine, and hypoxanthine released during
• this provides a mechanism for diverting IMP to the synthesis
the turnover of nucleic acids are reconverted to
of the species of purine present in lesser amounts
nucleoside triphosphates via so called salvage
• if both AMP and GMP are present in adequate amounts, the
pathways
de novo pathway of purine synthesis is turned off at the
aminotransferase step
SYNTHESIS OF DEOXYRIBONUCLEOTIDES
III.CONVERSION OF NUCLEOSIDE MONOPHOSPHATES TO
NUCLEOSIDE DIPHOSPHATES AND TRIPHOSPHATES
Base-specific nucleoside monophosphate kinases - specific
Nucleoside diphosphate kinases - general

IV.CONVERSION OF RIBONUCLEOTIDES TO
DEOXYRIBONUCLEOTIDES

2
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG
• Deoxyribonucleotide synthesis involves reduction at the 2’- • Reduction of ribonucleoside diphosphates (NDPs) to dNDPs
position of the ribose ring of nucleoside diphosphates is subject to complex regulatory controls that achieve
• Reduction of the 2’-hydroxyl of purine and pyrimidine balanced production of dNTPs for synthesis of DNA
ribonucleotides, catalyzed by the complex that includes
ribonucleotide reductase
- provides the deoxyribonucleoside diphosphates (dDNPs)
needed for both the synthesis and repair of DNA
- diphosphates are reduced not the monophosphate

• Ribonucleotide Reductase
- aka Ribonucleoside diphosphate reductase
- composed of 2 non-identical dimeric units, R1 and R2
specific for the reduction of purine nucleoside diphosphates
(ADP & GDP), and pyrimidine nucleoside diphosphates, cytidine
diphosphate (CDP) and uridine diphosphate (UDP) to their
deoxy forms (dADP, dGDP, dCDP, and dUDP)

• Ribonucleotide Reductase is Regulated by Nucleotide

Binding
- Ribonucleoside reductase is modulated in 2 ways to
• Enzyme complex is only functional when cells are actively maintain a balance of dATP, dGTP, dCTP, and dTTP
synthesizing DNA - First, the catalytic activity of the enzyme must be turned
• Reduction requires: on and off in response to need for dNTPs
- Reduced thioredoxin - Second, the amounts of each NDP substrate transformed
- Thioredoxin reductase must be controlled
- NADPH - ATP = activates
• Immediate reductant ➲ Reduced Thioredoxin - dATP = inhibits the overall activity site
- produced by NADPH-dependent reduction of oxidized - ATP, dATP, dTTP, and dGTP bind at the specificity site to
thioredoxin regulate the selection of substrates and the products made
• Regeneration of reduced enzyme
- in order for ribonucleotide reductase to continue to
produce deoxyribonucleotides, the disulfide bond created
during the production of 2’-deoxy carbon must be reduced
- source of the reducing equivalents for this purpose is
thioredoxin
• Thioredoxin must be converted back to its reduced form in
order to continue to perform its function

3
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG
 • Azaserine (AS) is an analog of Gln

3.Folic Acid Analogs

• Aminopterin (AP) and Methotrexate (MTX)

• The structural analogs of folic acid(e.g. MTX)are widely used

-Reduction and Phosphorylation to control cancer (e.g. leukaemia)

NOTE: These inhibitors also affect the proliferation of normally

growing cells. This causes many side-effects including
anemia, baldness, scaly skin, etc.

V.ANTIMETABOLITES OF PURINE NUCLEOTIDES

• Antimetabolites of purine nucleotides are structural analogs of
purine, amino acids and folic acid
• They can interfere, inhibit or block synthesis pathway of purine
nucleotides and further block synthesis of DNA, RNA, and
proteins
• Widely used to control cancer

1.Purine Analogs
• 6-Mercaptopurine (6-MP) is an analog of hypoxanthine (

VI.DEGRADATION OF PURINE NUCLEOTIDES

1.An amino group is removed from AMP to produce IMP, or from

adenosine to produce inosine (hypoxanthine-ribose) by AMP
Metacarpo- Thiol - "sulfur containing"IMP of de novo deaminase or adenosine deaminase
synthesis 2.IMP and GMP are converted into their nucleoside forms—
inosine and guanosine—by the action of 5′-nucleotidase
3.Purine nucleoside phosphorylase converts inosine and
guanosine into their respective purine bases, hypoxanthine
and guanine
NOTE: A mutase interconverts ribose 1- and ribose 5-
phosphate
4.Guanine is deaminated to form xanthine
2. Acid Analogs
4
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG
5.Hypoxanthine is oxidized by xanthine oxidase to xanthine, HYPERURICEMIA
which is further oxidized by xanthine oxidase to uric acid, the • characterized by plasma urate (uric acid) greater than 7.0
final product of human purine degradation mg/dl
• Uric acid is excreted in the urine • Normal plasma level
- Female = 2.4 – 6 mg/dl
- Male = 3.4 – 7 mg/dl
1.Primary Hyperuricemia
- an innate defect in purine metabolism and/or uric acid
excretion
2.Secondary Hyperuricemia
- increased availability of purines due to medications/
medical conditions or through diet
• Progression of Hyperuricemia to Gout
Stage 1: Asymptomatic hyperuricemia
- at a serum urate concentration greater than 6.8mg/dL,
*Purine – De Novo – Liver – Blood – Salvaged by peripheral urate crystals may start to deposit in the joints
tissue - no evidence that treatment is required
Stage 2: Acute gout
The Purine Nucleotide Cycle in Skeletal Muscle Serves as an - if sufficient urate deposits develop around joints, and if
Anaplerotic Pathway the local environment or some trauma triggers the
release of crystals in to the joint space, an
Deamination of AMP to IMP by AMP deaminase, followed by
inflammatory response occurs
resynthesis of AMP from IMP in de novo purine pathway
- these flares can be self-resolving but are likely to recur
enzymes, constitutes a purine nucleoside cycle (Figure 26.9).
Stage 3: Inter-critical periods
This cycle has the net effect of converting aspartate to - these are the intervals between attacks
fumarate plus NH4+. - during these periods, crystals may still be present at a
low level in the synovial tissue and fluid, resulting in
Skeletal muscle lacks the usual anaplerotic enzymes and future
relies on AMP deaminase for this purpose. attacks
Stage 4: Advanced gout
The Purine Nucleotide Cycle in Skeletal Muscle Serves as - if crystal deposits continue to accumulate, patients may
an Anaplerotic Pathway develop chronically stiff, swollen joints and tophi
- this advanced stage of gout is relatively uncommon
generally avoidable with therapy

This nucleotide cycle is important – fumarate that is

generated replenishes the levels of citric acid intermediates lost
in amphibolic side reactions.

VII.DISEASES ASSOCIATED WITH DEFECTS IN PURINE

METABOLISM

5
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG
 GOUT ๏ Olecranon bursitis

๏ punched out erosions with overhanging spicules

• Exceed solubility limits: Crystals
• causes:
- Under excretion of uric acid
- Diet rich in purines/alcohol; deficient in dairy products
- Increased purine degradation
- Increased PRPP Synthetase activity
- Over production of PRPP ➲ increased purine synthesis ➲
increased purine degradation ➲ increased uric acid
production
• Various genetic defects in PRPP synthetase present
- Decreased/partial HGPRT activity
clinically as GOUT
• Gout is caused by precipitation of sodium urate crystals in the
• diagnosis:
joints resulting in inflammation and pain
- definitive diagnosis is made by the presence of urate
crystals in the synovial fluid of the joint
- Clinical features
- crystals are needle-shaped and negatively birefringent
• treatment:
- Colchicine – reduces inflammation
- Uricosuric agents — increase renal excretion of uric acid
(probenecid)
- Hypoxanthine and xanthine
- Low purine diet
• Foods that are high in purine include:
๏ Swollen MTP joint and ankle -Red meat and organ meats (eg. Liver)
๏ Tophaceous deposits in left ear - Yeasts and yeast extracts (eg. beer and alcoholic
๏ Olecranon bursitis beverages)
๏ Punched out erosions with overhanging spicules - Asparagus, spinach, beans, peas, lentils, oatmeal,
cauliflower and mushrooms
๏ tophaceous deposits in left ear
• Prevention:
- Avoid caffeine and alcohol
- Keep hydrated
- Allopurinol
๏ an analog of hypoxanthine, is a potent inhibitor of xanthine
oxidase
๏ Allopurinol binds tightly to xanthine oxidase, preventing
uric acid formation.

6
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG
 ๏ Hypoxanthine and xanthine do not accumulate to harmful ADENOSINE DEAMINASE & PURINE NUCLEOSIDE
concentrations because they are more soluble and thus more PHOSPHORYLASE DEFICIENCY
easily excreted. • ADENOSINE DEAMINASE DEFICIENCY
- associated with immunodeficiency disease in which both
KIDNEY STONES thymus derived lymphocytes (T cells) and bone-marrow-derived
• when uric acid is presenting high concentrations in the blood, lymphocytes (B cells) are sparse and dysfunctional
it may precipitate as a salt in the kidneys - severe immunodeficiency
• salt can form stones, which can in turn cause pain, infection, - fatal infections in infants: if no enzyme replacement or bone
and kidney damage marrow transplantation
• PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY
LESCH-NYHAN SYNDROME - severe deficiency of T cells but apparently normal B cell
• X-Linked recessive disorder function
• complete HGPRT deficiency - as a result from accumulation of dGTP and dATP, which inhibit
• characterized by mental deficiency, aggression, self- ribonucleotide reductase and thereby deplete cells of DNA
destructive behavior, characterized by lip and finger biting precursors
• since high urate levels are present in the blood, individuals
with this condition are also prone to gout and kidney stones INHERITED DISORDERS OF PURINE METABOLISM & THEIR
ASSOCIATED ENZYME ABNORMALITIES

SEVERE COMBINED IMMUNODEFICIENCY (SCID)

• Adenosine deaminase deficiency
• accumulation of dATP= inhibition of ribonucleotide reductase
• result: B and T cells unable to divide

VIII. PYRIMIDINES ARE NOT SYNTHESIZED AS

NUCLEOTIDES
Adenosine Deaminase catalyzes the conversion of Pyrimidine Biosynthesis
Adenosine into Inosine • in contrast to purines, pyrimidines are not
7
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG
synthesized as nucleotides • STEP 5: Orotate is joined with a ribose-P to form orotidine-
• rather, the pyrimidine ring is completed before a ribose-5-P is 5’phosphate - the ribose-P donor is PRPP
added • STEP 6: Decarboxylation of orotidine monophosphate (OMP)
• CARBAMOYL-P AND ASPARTATE are the precursors of the by OMP decarboxylase makes URIDINE MONOPHOSPHATE
6 atoms of the pyrimidine ring (UMP)
• OMP is the parent pyrimidine mononucleotide

IX.BIOSYNTHESIS OF PYRIMIDINE NUCLEOTIDE

Ribose-5-P is the last to attached in Pyrimidine unlike in Purine

• STEP 1: Catalyst for the initial reaction a cytosolic

CARBAMOYL PHOSPHATE SYNTHASE II
- Carbamoyl phosphate for pyrimidine synthesis is made from
glutamine and CO2, catalyzed by carbamoyl synthetase II (CPS
II)
CPS II is inhibited by Uridine triphosphate (UTP) and is
activated by PRPP
• This is a cytosolic enzyme (whereas CPS I is mitochondrial
and used for the urea cycle)
• Substrates are:
- HCO3- De Novo Pyrimidine Synthesis
- glutamine METABOLIC CHANNELING
- 2ATP • Eukaryotic pyrimidine synthesis involves channeling and
• Compartmentation provides an independent pool of multifunctional polypeptides
carbamoyl phosphate for each process • UDP is made from UMP, and UTP is made from UDP
• PRPP = participates in pyrimidine biosynthesis only • CTP synthetase forms CTP from UTP and ATP
subsequent to assembly of the pyrimidine ring
• As for the biosynthesis of pyrimidines, purine nucleoside
biosynthesis is energetically costly
• Compartmentation provides an independent pool of carbamoyl
phosphate for each process
• PRPP = participates in pyrimidine biosynthesis only
subsequent to assembly of the pyrimidine ring
• As for the biosynthesis of pyrimidines, purine nucleoside Synthesis Of Thymine Nucleotides
biosynthesis is energetically costly
• Thymine nucleotides are made from dUMP, which derives from
DE NOVO PYRIMIDINE SYNTHESIS dUDP, Dcdp
• STEP 2: Aspartate transcarbamoylase (ATCase) catalyzes the • dUDP ➲ dUTP ➲ dUMP ➲ dTMP
condensation of carbamoyl phosphate with aspartate to form • dCDP ➲ dCMP ➲ dUMP ➲ dTMP
carbamoyl-aspartate • Thymidylate synthase methylates dUMP at 5-position to make
NOTE: that carbamoyl phosphate represents an ‘activated’ dTMP
carbamoyl group • N5, N10-methylene THF is 1-C donor
• STEP 3: Ring closure and dehydration — catalyzed by
dihydroorotase
• STEP 4: Removal of hydrogen from carbons 5 and 6 by DHO
dehydrogenase introduces a double bond forming OROTIC
ACID
8
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG
 End-product: Water soluble; less disorders
1.B alanine
2.B amino isobutyrate

Methylation- Thymidilate Synthase

Dihydrofolate Reductase: (NADPH to NADP+)
Synthesis of Thymine Nucleotides
• Thymine nucleotides are made from dUMP, which derives from
dUDP, dCDP X.OVERPRODUCTION OF PYRIMIDINE CATABOLITES IS
ONLY RARELY ASSOCIATED WITH CLINICALLY
• dUDPdUTPdUMPdTMP SIGNIFICANT DISORDERS
Orotic Aciduria
• dCDPdCMPdUMPdTMP
• Orotic aciduria that accompanies REYE’s SYNDROME is a
• Thymidylate synthase methylates dUMP at consequence of the inability of severely damaged mitochondria
5-position to make dTMP to utilize carbamoyl phosphate, which then becomes available
for cytosolic overproduction of orotic acid
• N5,N10-methylene THF is 1-C donor • TYPE I OROTIC ACIDURIA
- deficiency of both OROTATE
PHOSPHORIBOSYLTRANSFERASE & OROTIDYLATE
DECARBOXYLASE
• TYPE II OROTIC ACIDURIA
- rare
- due to deficiency only of OROTIDYLATE DECARBOXYLASE
Deficiency Of A Urea Cycle Enzyme Results In Excretion Of
Pyrimidine Precursors
• deficiency in liver mitochondrial ORNITHINIE
TRANSCARBAMOYLASE
• results to increased EXCRETION of:
- Orotic acid
- Uracil
Control of pyrimidine nucleotide synthesis. Solid lines represent - Uridine
metabolite flow. Dashed lines represent positive (+) and
negative (-) feedback regulation • EXCESS CARBAMOYL PHOSPHATE
- exits to the cytosol, where it stimulates pyrimidine nucleotide
biosynthesis
- results to mild OROTIC ACIDURIA (increased by high-
nitrogen foods)

Drugs That May Precipitate Orotic Aciduria

1. ALLOPURINOL
- alternative substrate for orotate phosphoribosyltransferase ,
competes with OROTIC ACID
- results to a nucleotide product that also inhibits orotidylate
decarboxylase which also results to OROTIC ACIDURIA &
OROTIDINURIA
Catabolism of pyrimidines
9
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG
 2. 6-AZAURIDINE - Lesch-Nyhan syndrome
- following conversion to 6-azauridylate, also competitively - Von Gierke disease
inhibits orotidylate decarboxylase , enhancing excretion of -Hypouricemias
OROTIC ACID & OROTIDINE • Pyrimidine catabolites are water-soluble
• Excretion of pyrimidine precursors can result from a deficiency
of ORNITHINE TRANSCARBAMOYLASE because excess
carbamoyl phosphate is available for pyrimidine biosynthesis
• Drugs which precipitate orotic aciduria
1. Allopurinol
-an alternative substrate for orotate
phosphoribosyltransferase competes with orotic acid. The
resulting nucleotide product also inhibits orotidylate
decarboxylase resulting in orotic aciduria and orotidinuria
2. 6-azauridine
-competitively inhibits orotidylate decarboxylase enhancing
excretion of orotic acid and orotidine

Reference: Harpers, powerpoint presentation of Dr. Quitasol

XI. SUMMARY
• Ingested Nucleic acids are degraded to PURINES &
PYRIMIDINES
• New purines and pyrimidines are formed from AMPHIBOLIC
INTERMEDIATES and thus are DIETARILY NON ESSENTIAL
• Several reactions of IMP biosynthesis requires FOLATE
DERIVATIVES & GLUTAMINE
- antifolate drugs and glutamine analogs INHIBIT purine
biosynthesis
• OXIDATION & AMINATION of IMP
- forms AMP & GMO
• SUBSEQUENT PHOSPHORYL TRANSFER FROM ATP
- forms ADP & GDP

• FURTHER PHOSPHORYL TRANSFER FROM ATP TO GDP

forms GTP
• OXIDATIVE PHOSPHORYLATION
- converts ADP to ATP
• REDUCTION of NDPs forms dNDPs
• Regulation of hepatic purine nucleotide biosynthesis includes:
- Pool size of PRPP
- FB inhibition of PRPP-GLUTAMYL AMIDOTRANSFERASE
by AMP & GMP
• Humans catabolize purines to uric acid, present as the
relatively insoluble acid at acidic pH or as its more soluble
sodium urate salt at a pH near neutrality
• URATE CRYSTALS are diagnostic of GOUT
• Other disorders of PURINE CATABOLISM include:
10
AGGABAO, BUÑI, DIALA, JARDIOLIN, LOCSIN, MOLOWA, WONG