BIOC Dr. Tischler Lecture 20 – February 10, 2006 METABOLISM: NUCLEOTIDE SYNTHESIS & DISORDERS

CLINICAL PREMISE S.G., a 45-year-old Caucasian male, presented to your office complaining of foot pain. The pain began approximately one week ago when he noticed one morning that his right big toe was swollen and painful to touch. He attributed the pain to “stubbing” his toe two days earlier on a coffee table. He initially took aspirin and Tylenol with some minimal improvement in the pain, but over the past week the pain has increased and now the big toe is red. Further history reveals that S.G. is an accountant, has had a weight problem “most of his life”, doesn’t exercise and is a wine connoisseur. Physical examination reveals an obese, middle-aged white male. His vital signs are normal with the exception of elevated BP. His right toe is swollen and the skin over the joint is inflamed and tender to touch. His left big toe is swollen but is not inflamed. A complete blood count is normal. His blood chemistry values were normal except for uric acid of 14.5 mg/dl (nl: 3-9 mg/dL). Urinalysis reveals a crystalluria. Analysis of RBC enzymes relevant to uric acid metabolism shows normal activity and normal regulation of the metabolic pathway.

building blocks for DNA and RNA “second messengers” in signal transduction cascades energy “currency” of the cell ROLES OF NUCLEOTIDES

Ribose-5-phosphate 5-phospho-  -ribosylamine Glu PRPP amidotransferase Gln inosine monophosphate (IMP) Amino acids: Gly + Gln + Asp Cofactors: N 10 -formyl THF Glucose-6-P pentose phosphate pathway PRPP synthetase ATPAMP 5-phosphoribosylpyrophosphate (PRPP) Figure 1. Synthesis of inosine monophosphate (IMP)

NAD + NADH Gln + ATP Glu+AMP+PP i guanosine monophosphate (GMP) GTP GDP adenosine monophosphate (AMP) aspartate + GTP ATP ADP fumarate GDP + P i IMP Figure 2. Formation of AMP and GMP from IMP

allosteric inhibition PRPP synthetase PRPP amido- transferase IMP GMPAMP GDP ADP Figure 3. Allosteric inhibition of purine biosynthesis; also ATP stimulates stimulates formation of AMP.

GMP, GDP or GTPAMP, ADP or ATP Guanosine Adenosine Guanine Inosine Hypoxanthine adenosine deaminase purine nucleoside phosphorylase Ribose-1-P Xanthine Uric acid xanthine oxidase xanthine oxidase HGPRT +PRPP IMP +PRPP HGPRT GMP NH 3 Figure 4. Degradation of purines to uric acid and salvage of purine bases via hypoxanthine-guanine phosphoribosyl transferase (HGPRT).

Ribose-5-phosphate 5-phospho-  -ribosylamine Glu PRPP amidotransferase Gln inosine monophosphate (IMP) Amino acids: Gly + Gln + Asp Cofactors: N 10 -formyl THF Glucose-6-P pentose phosphate pathway PRPP synthetase ATPAMP 5-phosphoribosylpyrophosphate (PRPP) Figure 1. Hyperuricemia: Gout: X 1a = PRPP synthetase defects associated with a superactive enzyme characterized by an increased Vmax or an enzyme with a reduced Km for ribose-5-P. X 1a

Figure 3. Gout: X 1b = PRPP synthetase defect associated with resistance to feedback inhibition. PRPP synthetase PRPP amido- transferase IMP GMPAMP GDP ADP X 1b

+PRPP GMP, GDP or GTPAMP, ADP or ATP Guanosine Adenosine Guanine Inosine Hypoxanthine adenosine deaminase purine nucleoside phosphorylase Xanthine Ribose-1-P Uric acid xanthine oxidase xanthine oxidase +PRPP HGPRT IMP GMP NH 3 Figure 4. X 2 = moderate defect (>50% activity) leading to gout; severe defect (very low activity) leads to Lesch-Nyhan syndrome. X2X2 X2X2 Inhibited by allopurinol

Gout = hyperuricemia due to a variety of causes GOUT AND LESCH-NYHAN SYNDROME Lesch-Nyhan syndrome = excessive hyperuricemia; leads to self-mutilation Urate crystals appearing in a diaper – often found in synovial fluid of joints

Incan Mask Depicting An Individual Presumably with Lesch-Nyhan as Evidenced by the Self-Mutilation

Figure 5. Biosynthesis of the pyrimidine nucleotides UTP and CTP. carbamoyl phosphate carbamoyl- aspartate aspartate aspartate transcarbamoylase orotate +PRPP OMP orotate phosphoribosyl transferase UMP orotidylic acid decarboxylase Glutamine + 2ATP + CO 2 carbamoyl phosphate synthetase II (gln) Glu + P i + 2 ADP +2 ATP UTP +2 ADP Gln + ATP CTP Glu + P i + ADP - UDP UTP  ATP, PRPP

ADP GDP UDP CDP ribonucleotide reductase reduced thioredoxin dADP dGDP dUDP dCDP oxidized thioredoxin +NADPH + H + NADP + thioredoxin reductase dUMP dihydrofolate thymidylate synthase N 5,N 10 -methylene THF TMP Figure 6. Biosynthesis of deoxyribonucleotides by ribonucleotide reductase and of thymidine monophosphate (TMP) by thymidylate synthase.

THF N 5 -Methyl THF N 5,N 10 - Methylene THF Glycine Serine N 5,N 10 -Methenyl THF N 10 -Formyl THF PRPPPurines: AMP + GMP DHF reductase dUMP thymidylate synthase TMP DIETARY FOLIC ACID Dihydrofolate (DHF) Methyl B 12 OH-B 12 folate trap X3X3 MTX X5X5 F- dUMP 5-F-uracil X4X4 Figure 7. Metabolism of dietary folic acid to its various cofactor forms. X 4 = fluorodeoxyuridylate (F-dUMP), synthesized from 5-flurouracil, acts as chemotherapeutic agent X 5 = site at which methotrexate (MTX) acts as a chemotherapeutic agent; X 3 = site at which cobalamin (B12) deficiency causes folate to be trapped as N5-methyl THF Suicide inhibitor