Chapter 8. Nucleotide Metabolism Functions of nucleotites: They are precursors of DNA and RNA ATP is a universal currency of energy Physiological mediators (cAMP, cGMP) They are activated intermediates in many biosynthesis (UDP-glucose, CDP-choline) Adenine nucleotides are components of some coenzymes (NAD+, NADP+ and FAD )

1. Biosynthesis of purine nucleotites: 1) The origins of the atoms in the purine ring: One C unit One C unit

2) Formation of phosphoribosylamine Ribose 5-phosphate PRPP ATP ADP PRPP synthase PRPP: 5-Phosphoribose-1-pyrophosphate

Glutamine glutamate + PPi PRPP 5-phosphoribosyl-1-amine Glutamine glutamate + PPi Gln PRPP Amidotransferase

3) Formation of inosinate (IMP)

4) Formation of AMP and GMP

5) Formation of ATP and GTP AMP ADP ATP GMP GDP GTP Kinase Phosphorylation Pi ATP ADP Kinase Kinase ATP ADP ATP ADP

6) Regulation of purine nucleotite synthesis Feedback inhibitors: AMP, ADP, GMP, GDP, IMP AMP ADP ATP R-5-P PRPP PRA IMP GMP GDP GTP PRA: 5-phosphoribosyl-1-amine . activation inhibition

7) Salvage pathways for purine nucleotite synthesis PRPP Purine ribonucleotide Adenine + PRPP AMP + PPi Hypoxanthine + PRPP IMP + PPi Guanine + PRPP GMP + PPi Adenosine + ATP AMP +ADP

8) Biosynthesis of deoxyribonucleotides (At the NDP level) Ribonucleoside deoxyribonucleoside diphosphate diphosphate Base=purine or pyrimidine

The mechanism for ribonucleotide reduction: NDP dNDP Ribonucleotide SH reductase SH Ribonucleotide S reductase S Thioredoxin S S Thioredoxin SH SH Thioredoxin reductase FADH2 FAD NADPH + H+ NADP+

8) Inhibition of purine nucleotide biosynthesis by some anticancer drugs Reaction Inhibitor PRPP PRA 6-mercaptopurine(6MP) Glycinamide ribonucleotide Methotrexate(MTX) Formylglycinamide ribonucleotide Formylglycinamide ribonucleotide Azaserine Formylglycinamidine ribonucleotide IMP AMP 6MP IMP GMP 6MP, Azaserine Adenine AMP 6MP Guanine GMP 6MP

2. Degradation of purine nucleotides: AMP IMP Hypoxanthine Xanthine Uric acid GMP Guanine Xanthine Uric acid

Gout is induced by high serum levels of urate, a disease that affects the joints and kidneys. Allopurinol is an analog of hypoxanthine. It is extensively used to treat gout. In the body, allopurinol is converted into alloxanthine, which then remains tightly bound to the active site of xanthine oxidase and thus inhibits the production of urate.

3. Synthesis of pyrimidine nucleotides: 1) Origins of the atoms in the pyrimidine ring C N C C C N Carbamoyl phosphate Aspartate

2) Pathway of pyrimidine nucleotide synthesis Formation of carbamoyl phosphate 2ATP + HCO3- + 2ADP+ Pi Gln Glu

Differences between carbamoyl phosphate biosynthesis in the pyrimidine pathway and that in the urea cycle In pyrimidine pathway In urea cycle Location Cytosol Mitochondria -NH2 from: Gln NH4+ Enzyme Carbamoyl phosphate Carbamoyl phosphate synthase-II synthase-I N-acetyl-Glu No effect Activator

B) Formation of orotate

C) Formation of UMP

C) Formation of other pyrimidine nucleotides UMP UDP UTP ATP ADP ATP ADP Kinase Kinase

UDP dUDP dUTP dUTP dUMP NADPH NADP+, H2O ATP ADP Ribonucleotide reductase Kinase H2O PPi dUTPase

- - D) Regulation of pyrimidine nucleotide synthesis CO2 + Gln + ATP Carbamoyl phosphate N-Carbamoylaspartate UMP - Inhibited by UMP - Inhibited by CTP

3) Salvage pathway for pyrimidine nucleotide biosynthesis Pyrimidine Pyrimidine ribonucleoside monophosphate Orotate Orotidylate UMP PRPP PPi Pyrimidine Phosphoribosyl transferase PRPP PPi H+ CO2 Orotate Phosphoribosyl transferase Orotidylate decarboxylase

4) Anticancer drugs that block the pyrimidine nucleotide biosynthesis A) 5-Fluorouracil (5-FU): is converted in vivo into fluorodeoxyuridylate (F-dUMP), which is an analog of dUMP and irreversibly inhibits thymidylate synthase.

B) Aminopterin and methotrexate (MTX): both are analogs of dihydrofolate. They are potent competitive inhibitors of dihydrofolate reductase and thus block the reactions using one-carbon units.

C) Cytosine arabinoside and azaserine: Cytosine arabinoside is an analog of cytosine riboside and thus inhibits the reduction of CDP to dCDP, while azaserine inhibits the conversion of UTP to CTP.

- - - - Azaserine UMP UTP CTP CDP dCDP UDP dUDP dUMP dTMP 5-FU Cytosine arabinoside Azaserine - - 5-FU - - Aminopterin

4. Degradation of pyrimidine nucleotide The pyrimidine ring can be completely degraded in humans. The products include: NH3, CO2, b-alanine, and b-aminoisobutyrate. Both b-alanine, and b-aminoisobutyrate can be further converted into acetyl-CoA and succinyl-CoA, respectively, or are excreted in the urine.

5. Dysmetabolism of nucleotides Disease Deficiency Symptoms Gout PRPP synthetase hyper uricemia Lesch-Nyhan HGPRT purine and uric syndrom acid, cerebral paralysis Immunodeficiency adenosine deaminase deoxyadenosine Diseases uria, dysostosis Kidney stone APRT kidney stone, urodynia, hematuria Xanthinuria xanthine oxidase kidney stone, no symptoms