L6 Nucleotide Metabolism PDF

Purine and Pyrimidine Metabolism Marks’ Basic Medical Biochemistry , 6th Ed chapter 39 (pages: 867-884) Biochemistry, Cell and Molecular Biology, and Genetics Part V Chapter 37 (313-323) Zeynep Gromley, Ph.D. MANS 429...

Purine and Pyrimidine Metabolism Marks’ Basic Medical Biochemistry , 6th Ed chapter 39 (pages: 867-884) Biochemistry, Cell and Molecular Biology, and Genetics Part V Chapter 37 (313-323) Zeynep Gromley, Ph.D. MANS 429 [email protected] 1  Learning Objectives Outline the differences between de novo purine and pyrimidine biosynthesis and the differences of salvage pathway. Describe how FH4 (folate coenzyme) functions in purine and pyrimidine biosynthesis. Describe the metabolic basis and the signs and symptoms of gout and deduce the outcomes of uric acid accumulation. Explain the outcomes of HGPRT enzyme deficiency and explain the signs and symptoms of Lesch-Nyhan syndrome. Describe the SCID due to ADA deficiency. Describe metabolic conditions (UMP synthase and OTC deficiency) that results in accumulation of orotic acid in urine. Differentiate orotic aciduria due to UMP synthase deficiency and OTC deficiency. Differentiate megaloblastic anemia due to UMP synthase deficiency and megaloblastic anemia due to B12 and/or folate deficiency 2  Functions of Nucleotides Polymerize to make DNA and RNA Energy currency of the cell e.g., ATP, GTP Act as carriers of active intermediates in various metabolic pathways e.g., UDP- glucose in glycogen synthesis, SAM Component of coenzymes e.g., CoA, FAD, NAD, NADP Act as 2nd messengers e.g., cAMP and cGMP Allosteric regulation of various metabolic pathways e.g., ATP inhibits PFK-1 3  Ribose Sugar – Ribose – Deoxyribose Nitrogenous Bases – Purines – Pyrimidines Nucleoside – Base + ribose Nucleotide – Base + ribose + phosphate – AMP, GMP, CMP, UMP – dAMP, dGMP, dCMP, dTMP 4  Nucleotide Synthesis Nucleotides can be made using two processes: – By salvaging nucleotides from degradation of nucleic acids or available trough the diet. Non-hepatic tissues rely on salvage pathway. – By synthesizing them from scratch (De Novo) 5 De Novo Purine Synthesis 6 Biosynthesis of purine nucleotides begins with the synthesis of 5-Phosphoribosyl 1- pyrophosphate (PRPP) by the PRPP synthetase enzyme. 7 8  AMP and GMP Synthesis IMP is the starting point for AMP and GMP To form AMP, Aspartate donates an amine, Fumarate leaves forming AMP To form GMP, first the C-2 is oxidized, oxidation is followed by replacement of the O by N from Glutamine 9  How cells make dGDP and dADP? RR = ribonucleotide reductase reduces the 2’-OH, producing dADP and dGDP 10  Ribonucleotide Reductase (RR) is a target for cancer treatment Hydroxyurea is a potent inhibitor of RR It is widely used for treatment of CML 11 De Novo Purine Synthesis 12  Humans Catabolize Purines to Uric Acid Humans convert adenosine and guanosine to uric acid. Adenosine first converted to inosine by adenosine deaminase (ADA). Purine nucleotide phosphorylase converts inosine to hypoxanthine and guanosine to guanine. Xanthine oxidase and guanine deaminase route everything to xanthine. Xanthine oxidase converts xanthine to uric acid, which excreted in the urine. Final product of purine catabolism is uric acid. 13  Catabolism of Purines and Gout Uric acid is poorly soluble in plasma especially at lower temperatures. The accumulation of uric acid causes gout. When serum urate levels exceed the solubility limit, Na-Urate crystalizes in soft tissues and joints causes an inflammatory reaction, gouty arthritis. Hyperuricemia can be caused by either: underexcretion (lactic acidosis and alcohol, some drugs e.g., thiazide diuretics) or overproduction of uric acid due to enhance tissue turnover (cancer, psoriasis). A gain of function mutation in PRPP synthetase present clinically as gout. 14 The drug allopurinol, structural analog of hypoxanthine, inhibits xanthine oxidase. Hypoxanthine and xanthine is more soluble than uric acid therefore easy to excrete in urine. Acute attacks of gout treated with colchicine or indomethacin to reduce the inflammation If hyperuricemia is due to under excretion of uric acid, these patients are treated with uricosuric drug (probenecid) If gout is due to overproduction of uric acid, it is treated with allopurinol or febuxostat, non-purine inhibitor of xanthine oxidase 15  Purine Salvage pathway When free purines released during nucleotide catabolism, they can be salvaged by reattachment of the bases to PRPP. For certain cell types such as lymphocytes, the salvage of purines is the major form of nucleotide generation. 16 An important enzyme of the salvage pathway is hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Unlike guanosine, adenosine can be phosphorylated directly back to AMP by adenosine kinase. 17  HGPRT deficiency: Lesch-Nyhan Syndrome Lesch-Nyhan Syndrome is X-linked recessive, caused by genetic mutations that decrease or abolish the HGPRT activity. PRPP levels increased, and purines are synthesized in excess therefore increases the degradation of purines resulting high plasma and urine uric acid. Affected children suffer from intellectual disability and self mutilation. 18  ADA deficiency Adenosine first converted to inosine by adenosine deaminase (ADA) If ADA is deficient adenosine accumulates. Adenosine can be phosphorylated directly back to AMP by adenosine kinase. AMP is converted ADP Patients with ADA deficiency have 100-fold increase in the concentration of dATP, which is a potent inhibitor of ribonucleotide reductase 19 Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency disease (SCID) Patients with ADA deficiency have 100-fold increase in the concentration of dATP, which is a potent inhibitor of ribonucleotide reductase The inhibition of ribonucleotide reductase results in decrease dGTP, dCTP, dTTP Therefore, inhibits the DNA synthesis and cell division. Immune cells have short-term half life, and they have to divide fast. Therefore, ADA deficiency causes SCID. 20 21  Pyrimidine Synthesis Pyrimidine ring assembles first and then combines with PRPP to form initial nucleotide UMP. Carbamoyl phosphate synthetase II (CPS-II) in the cytosol catalyzes carbamoyl phosphate formation from CO2 and nitrogen of glutamine. Aspartate transcarbamoylase catalyzes the formation of N- carbamoyl aspartate. Next, dehydration and dehydrogenation produces orotate. Oratate is then transferred to PRPP by UMP synthase The initial pyrimidine nucleotide is UMP. 22  UMP UDP Ribonucleotide Reductase (RR) catalyze dUDP formation dUDP dUMP Thymidylate synthase methylates dUMP to form dTMP dUMP is converted to dTMP by receiving a methyl group from methylene-FH4 23  Antimetabolite, 5-Fluorouracil, inhibits thymidylate synthase It is used in cancer chemotherapy. 24  Deficiency of UMP Synthase causes: Orotic aciduria Megaloblastic anemia Low activities of UMP synthase results in poor growth, megaloblastic anemia, and excretion of large amount of orotate in the urine. Treatment is uridine administration. Administration of uridine results in improvement of the anemia and decreased excretion of UMP Synthase orotate. 25  Two Orotic Acidurias 1. OTC deficiency 2. UMP Synthase deficiency Q. What laboratory test would help in distinguishing an orotic aciduria caused by ornithine transcarbamylase deficiency from that caused by UMP synthase deficiency? Blood ammonia level would be expected to be elevated in ornithine transcarbamylase deficiency but not in UMP synthase deficiency. UMP synthase deficiency causes megaloblastic anemia 26  OTC UMP-synthase Orotate Ammonia BUN Anemia 27 Catabolism of Pyrimidines In contrast to purines, pyrimidine rings can be cleaved and degraded to highly soluble end products. Salvage of Pyrimidines Salvage of pyrimidines is a minor pathway in humans. The pyrimidine nucleosides can be salvaged by nucleoside kinases. nucleoside + ATP → nucleotide + ADP 28