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Purine metabolism

synthase- no high energy compound involved

synthetase – uses high energy compound

Purine biosynthesis

- Ribose-5-P (from pentose phosphate pathway)( PRPP via PRPP synthetase ( 5-P-beta- ribosylamine via PRPP amidotransferase

- PRPP used in synthesis of purine and pyrimidine nucleotides by providing ribose sugar and alpha phosphate

- PRPP amidotransferase is rate determining and committed step of purine synthesis

- remaining reaction require ATP, amino acids (gly, glu, asp), and folic acid-derived cofactor (N10- formyltetrahydrofolate, formyl THF)

- 6 high energy P bonds cleaved in production of inosinic acid (IMP) ( first purine nucleotide (base, sugar, P) formed

- AMP and GMP from IMP (separate pathways), figure 2 on 15-3

- GMP synthesis required redox reaction and nitrogen incorporation from amide side chain of glutamine

- AMP acquires nitrogen from aspartate with production of fumarate; aspartate regenerated from fumarate by formation, in citric acid cycle, of oxaloacetate, which is then aminated to aspartate (fumarate( malate( oxaloacetate( aspartate)

- mono-P products phosphorylated to di- and triphosphates by nucleoside monophosphate kinase and nucleoside diphosphate kinase

- end result is production of purine ribonucleotides

- purine deoxyribionucleotides catalyzed by ribonucleotide reductase

1.

a.

Regulation of purine synthesis

- adenine and guanine nucleotides both inhibit the synthesis of IMP early in pathway at both PRPP synthetase and PRPP amidotransferase; they also feedback inhibit their own product from IMP

- excess ATP activates pathway from IMP ( GMP, while excess GTP activates pathway from IMP ( AMP to maintain proper balance of purine nucleotides

b.

Purine degradation

- starts with removal of phosphate from purine nucleotide forms, yielding nucleoside (base, sugar)

- adenosine ( inosine catalyzed by adenosine deaminase

- sugars removed by purine nucleoside phosphorylase ( purine bases guanine and hypoxanthine, and ribose-1-P

- base products ( xanthine ( oxidized by xanthine oxidase ( uric acid; xanthine oxidase also converts hypoxanthine ( xanthine; xanthine oxidase requires molecular oxygen and molybdenum, non-heme iron, and FAD

Uric acid secretion

- uric acid is the purine degradative product ( weak acid, with pK of 5.8; ionized form more water-soluble than protonated form

- urine at pH 4.8 ( uric acid is 90% protonated ( can dissolve 1/10 as much urate as urine at pH 6.8 (90% ionized)

- normal urine pH below 5.8 ( overproduction of uric acid can lead to formation of stones (not as soluble)

c.

Purine salvage pathway

- besides de novo synthesis, purine nucleotides can be formed directly from purine bases via salvage pathway

- two enzymes: hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and adenine phosphoribosyl transferase

- HGPRT adds PRPP (from PRPP synthetase) to hypoxanthine ( IMP or to guanine ( GMP

- prevents irreversible destruction of hypoxanthine, guanine and adenine (purine bases reutilized)

- salvage pathway saves energy because of high energy demand of de novo synthesis pathway

- important for salvage of dietary nucleotides; salvage purine bases ( uric acid kept low (prevents gout)

- HGPRT defect ( very low activity; Lesch-Nyhan syndrome

d. Disorders associated with defects of enzymes in purine metabolic pathways:

- elevation of uric acid from overproduction of purines or decreased excretion of uric acid

- hyperuricemia ( associated with type I glycogen storage disease (von Gierke’s); glucose-6-Pase defect ( increased oxidation of glucose-6-P ( ribose-5-P ( elevates purine production through saturation of PRPP synthetase

- hyperuricemia ( gout ( excessive accumulation of uric acid in body fluids; arthritis pain in joints ( urate crystals in cartilage around joint; kidney stones

Classification and causes of gout

- primary ( inherited disorder

- secondary ( induced by many disorders, leukemia

- linked to defects in metabolism of purines; PRPP synthetase superactive variant associated with increased Vmax; another variant has an increased affinity (low Km) for ribose-5-P ( leading to overproduction of PRPP

- 3rd defect associated with loss of feedback inhibition of this enzyme by purine nucleotides; when purine nucleotides reach excessive concentration ( no signal for shutting off their further production

- defects of HGPRT ( inability to salvage purine bases from degradation ( overproduction of uric acid gout

Gout treatment

- allopurinol ( competitive inhibitor of xanthine oxidase; hypoxanthine and xanthine excreted during allopurinol therapy since xanthine oxidase uses both of these purines as substrates

- allopurinol (like purine bases) can be converted to ribonucleotide form by HGPRT; treatment uses additional PRPP amidotransferase to reduce purine biosynthesis; analogue ribotide produced may inhibit PRPP amidotransferase

- high [hypoxanthine] results from inhibition of xanthine oxidase ( causes HGPRT to reutilizes this base and further inhibit de no purine synthesis; purine synthesis lowered during allopurinol treatment

- avoid animal products rich in nucleic acid (organ meats)

Lesch-Nyhan syndrome

- tremendous overproduction of uric acid; severe defect in HGPRT; males (X-linked)

- aggressive behavior, mental retardation, self-mutilation

- enzyme has essential role in non-hepatic tissue where de no synthesis of purines is slow ( non-hepatic tissues depend on circulating purine bases or nucleosides from liver

- non-hepatic tissues thought to take up circulating purines and through HGPRT form nucleotides

2. PYRIMIDINE METABOLISM

Pyrimidine biosynthesis and its regulation

a. - glutamine (nitrogen donor) ( carbamoyl phosphate through carbamoyl phosphate synthetase II; urea cycle carbamoyl synthetase I uses ammonia

- genetic defect of ornithine transcarbamoylase, in urea cycle, causes accumulation of carbamoyl phosphate that leaks from mitochondria to cytoplasm ( increase pyrimidine synthesis; increased pyrimidines excreted into urine (diagnose urea cycle defects)

- pyrimidine biosynthesis continues with addition of aspartate via aspartate transcarbamoylase to provide remainder of ring elements ( carbamoyl aspartate

- final product of pyrimidine synthesis is UMP ( can be phosphorylated to UTP ( cytidine triphosphate (CTP)

- pyrimidines and ATP/GTP required for RNA synthesis

b.

- regulation of pyrimidine biosynthesis occurs at carbamoyl phosphate synthetase II ( feedback inhibition by uridine nucleotides (UDP, UTP)

- PRPP in excess (PRPP, ATP)( enzyme stimulated

- to maintain balance of purine and pyrimidine nucleotides ( high amounts of purine nucleotides activate carbamoyl phosphate synthetase II

3. Metabolism of deoxyribionucleotides

Formation of deoxynucleotides

- 2-hydroxyl group of ribonucleotides reduced to form deoxyribonucleotide; catalyzed by ribonucleotide reductase; requires thioredoxin as a reducing source (it is oxidized); thioredoxin must be reduced back to its active form action of thioredoxin reductase (requires NADPH)

- feedback regulation by deoxynucleotide triphosphates, dATP and dGTP

Formation of thymidine

- thymidine nucleotides required for DNA synthesis in place of UTP

- TTP derived from TMP, which is formed from dUMP

- dUMP ( TMP catalyzed by thymidylate synthase; dUMP acquires a carbon from N5,N10-methyleneTHF ( converted to DHF

4. Metabolism of Folic Acid

- folic acid/folate undergoes activation and conversion to various forms used in several biochemical reaction

- folate serves as a donor of one carbon group

- ingested folate ( converted to dihydrofolate ( reduced to tetrahydrofolate (THF) by dihydrofolate reductase

- THF is backbone for production of other active forms of folate; must be in this form to carry carbon

- N5,N10-methyleneTHF gains a carbon from side chain of serine yielding glycine product; required for TMP synthesis from dUMP

- N5,N10-methenylTHF is a precursor for formation of N10-formyl THF ( required in purine biosynthesis (IMP formation); it is also a precursor of N5-methyl THF (in processing vitamin B12, cobalamin) ( provides methyl group for formation of methyl cobalamin; B12 deficiency ( folate trapped at N5-methyl THF because reaction from N5,N10-methyleneTHF is not reversible

- folate supplementation prevents neural tube defects; prevent vascular disease

5. Chemotherapy and inhibition of DNA formation

- cancer cells require DNA synthesis, chemotherapeutic agents inhibit DNA synthesis at level of synthesis of thymidine nucleotides because they are used selectively in DNA synthesis

Flurodeoxyuridylate (F-dUMP) – is the product of metabolism of 5-fluorouracil (form of drug given) via orotate phosphoribosyl transferase; competitive inhibitor of thymidylate synthase

Methotrexate – inhibits dihydrofolate reductase resulting in decreased synthesis of THF (DHF ( THF via DHF reductase normally); inhibitor ultimately decreases production of active forms of folate used in purine and pyrimidine synthesis (DNA synthesis disrupted)

- analogues of folate given during chemotherapy to supply demand of slowly dividing normal cells

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