Biochemistry - 45 - Nucleotide Metabolism 2023 PDF

Purine and Pyrimidine Metabolism Lecture 45 Reference: Lieberman and Peet, Chapter 39 Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD Learning Objectives a. Recall the terms nucleotide, nucleoside, nitrogenous base, purine, pyrimidine and recognize by sight the struct...

Purine and Pyrimidine Metabolism Lecture 45 Reference: Lieberman and Peet, Chapter 39 Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD Learning Objectives a. Recall the terms nucleotide, nucleoside, nitrogenous base, purine, pyrimidine and recognize by sight the structures of adenine, guanine, thymine, cytosine and uracil. b. Identify the origin of purine ring atoms, recall inosine monophosphate as the primary goal of purine synthesis, and recall adenine and guanine are derivatives of this initial product. Identify hypoxanthine as the base associated with IMP. c. Relate the role of thioredoxin and the enzymes ribonuclease reductase / thioredoxin reductase in transforming ribonucleotides to deoxyribonucleotides. d. Identify the origin of ring atoms in pyrimidines, recall UDP as the primary goal of pyrimidine synthesis and recall CTP and TTP are derivatives of UTP. e. Compare the enzymes CPS I and CPS II in terms of their substrates and roles in the cell, and the metabolic effect that would result from a deficiency of either enzyme. f. Recognize the general strategy for how purine and pyrimidine synthesis is regulated. g. Recall the major steps of purine degradation and identify hypoxanthine and xanthine as major intermediates in this process. Identify xanthine oxidase as a major enzyme in this process. h. Recall the biochemical basis of gout and relate why allopurinol is an effective treatment. i. Relate the salvage of purines and interpret how salvage of purines can protect humans from gout. Interpret the central role of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and adenine phosphoribosyl-transferase (APRT) in these pathways. j. Recall that pyrimidine degradation leads to normal cellular metabolites. 2 Nucleotide versus Nucleoside Objective A Sugar: Ribose or deoxyribose Nitrogenous Base(s): Purines – Adenine and Guanine Pyrimidines – Cytosine, Uracil, Thymine 3 Purines and Pyrimidines Objective A Why is this important? • Activated precursors of DNA and RNA • Structural moieties of many coenzymes (NADH, FAD) • Critical elements of energy metabolism (ATP, GTP) • Secondary messengers (cAMP, cGMP) • Metabolic allosteric regulators Dietary uptake of purine and pyrimidine bases is minimal therefore de novo synthesis is required. 4 Purine Biosynthesis Objective B De novo pathway of purine synthesis is complex • 11 steps, requiring 6 ATP for every purine synthesized (occurs in liver) • Precursors that donate components to produce purine nucleotides include glycine, ribose 5-phosphate, glutamine, aspartate, carbon dioxide, and N10-formyl-FH4 • Goal of synthesis is inosine monophosphate (IMP), which is modified to AMP or GMP ààà 5 De Novo Synthesis of Purine Nucleotides Objective B Goal of purine synthesis: Inosine Monophosphate (IMP) IMP is the initial product of the 11-step purine nucleotide pathway Starting Point: Synthesis of PRPP Purines are built on an activated form of ribose called 5phosphoribosyl-1-pyrophosphate (PRPP) • Synthesized from ATP and ribose 5’-phosphate • PRPP Synthetase is a regulated enzyme • • Activated by inorganic phosphate Inhibited by purine nucleotides (end-product inhibition) • NOT a committed step 6 De Novo Synthesis of Purine Nucleotides STEP 1: Synthesis of 5-phosphoribosyl 1-amine (1st committed step) • PRPP reacts with glutamine • • Catalyzed by glutamine phosphoribosyl amidotransferase (highly regulated) • • The amide group of glutamine replaces the pyrophosphate group attached to C1 of PRPP. Inhibited by AMP and GMP (end-products of the pathway) Produces nitrogen 9 of the purine ring 7 De Novo Synthesis of Purine Nucleotides STEP 2: Glycine is added to growing precursor • Glycine provides carbons 4 and 5 and nitrogen 7 of the purine ring • Catalyzed by phosphoribosylglycinamide synthetase • This step requires energy in the form of ATP 8 De Novo Synthesis of Purine Nucleotides Objective B Subsequently: • Carbon 8 is provided by N10-formyl-FH4 • Nitrogen 3 by glutamine • Carbon 6 by CO2 • Nitrogen 1 by Aspartate • Carbon 2 by N10-formyl-FH4 • NOTE: 6 high energy bonds of ATP are required (starting with ribose 5phosphate) to synthesize IMP • IMP has a hypoxanthine base joined by an N-glycosidic bond from N-9 • Hypoxanthine is NOT found in DNA, but it’s the precursor for other purine bases. 9 Objective B Synthesis of Adenosine and Guanosine Monophosphate • • IMP can be converted to either AMP or GMP Two step energy requiring pathway • Note: synthesis of AMP requires GTP as an energy source, whereas the synthesis of GMP requires ATP • The 1st reaction of each pathway is inhibited by the end product of that pathway • AMP and GMP can then be phosphorylated to their di- and triphosphates, then used for RNA synthesis. 10 Synthesis of Deoxyribonucleotides Objective C For DNA synthesis: • Ribose is reduced to deoxyribose by the enzyme ribonucleotide reductase which requires the cofactor thioredoxin ADP dADP GDP dGDP CDP dCDP UDP dUDP • Reduction to deoxyribose occurs at the nucleotide diphosphate level • The deoxyribose diphosphates can be phosphorylated to the triphosphate level and used as precursors for DNA synthesis. 11 Regulation of Purine Synthesis Objective F Feedback Inhibition of Purine Synthesis • GDP and ADP inhibit PRPP Synthetase preventing the synthesis of PRPP • GMP, AMP and each of their di- and triphosphate analogs can inhibit glutamine phosphoribosyl amidotransferase (1st committed step) • AMP and GMP each inhibit the first step in their synthesis from IMP. 12 Objective D De Novo Synthesis of Pyrimidine Nucleotides Goal of pyrimidine synthesis: UDP production • • Unlike the synthesis of the purine ring, which is constructed on a pre-existing ribose 5-phosphate, the pyrimidine ring is synthesized before being attached to ribose 5-phosphate (donated by PRPP) The origin of the atoms of the ring (aspartate and carbamoyl phosphate which is derived from CO2 and glutamine) 13 Objective E De Novo Synthesis of Pyrimidine Nucleotides Goal of pyrimidine synthesis: UDP production • UTP is the initial product of the 7-step pyrimidine nucleotide pathway STEP 1: Synthesis of carbamoyl phosphate • Analogous to the first reaction of the urea cycle except glutamine is the source of nitrogen rather than ammonia and it occurs in the cytosol • • CPS II Catalyzed by carbamoyl phosphate synthetase II (CPS II) CPS II is inhibited by uridine triphosphate (UTP) and is activated by PRPP 14 CPS I versus CPS II Objective E 15 De Novo Synthesis of Pyrimidine Nucleotides Objective D STEP 2-7: UDP Production • Asp is added to carbamoyl phosphate (aspartate transcarbamoylase) • Molecule produces a closed ring which is oxidized to form orotic acid (oroate) • Transfer of ribose 5-phosphate from PRPP to oroate to form orotidine 5’phosphate (OMP) by orotate phosphoribosyltransferase • Decarboxylation of OMP by orotidine 5’-phosphate decarboxylase forms UMP • UMP is subsequently phosphorylated to UDP and UTP • CTP (RNA synthesis) and dTTP (DNA synthesis) are derivatives of UTP 16 Hereditary Orotic Aciduria • Very rare disorder caused by the deficiency of one or both activities of the single polypeptide chain UMP synthase • Increased orotic acid (oroate) excreted in the urine. • Defective enzymes: • Orotate phosphoribosyltransferase • Orotidine 5’-phosphate decarboxylase • With no UMP production, pyrimidines cannot be synthesized, therefore normal growth does not occur. • Treatment: Oral administration of uridine • Uridine is converted to UMP to bypass metabolic block Aside: compare orotate produced by ornithine transcarbamoylase (OTC) deficiency vs orotate phosphoribosyltransferase (pyrimidine synthesis). One major difference is high blood ammonia for OTC deficiency. 17 Objective F De Novo Synthesis of Pyrimidine Nucleotides Feedback Inhibition of Pyrimidine Synthesis • CPSII is the regulated step • Inhibited by UTP • Activated by PRPP • As pyrimidines decrease in concentration (indicated by UTP levels), CSPII is activated and pyrimidines are synthesized 18 Synthesis of Cytidine Triphosphate Objective D • Cytidine triphosphate (CTP) is produced by amination of UTP by CTP synthetase, with glutamine providing the nitrogen • Come CTP is dephosphorylated to CDP, which is a substrate for ribonucleotide reductase • The dCDP product can be phosphorylated to dCTP for DNA synthesis or dephosphorylated to dCMP that is deaminated to dUMP 19 Objective D Synthesis of Deoxythymidine Monophosphate • dUMP is converted to dTMP by thymidylate synthase • N5, N10-methylene FH4 is the source of the methyl group, forming FH2. • 5-fluorouracil (5-FU) forms FdUMP which inhibits thymidylate synthase. • Methotrexate inhibits dihydrofolate reductase • These inhibitors are anti-cancer agents: • Blocking the synthesis of purine or pyrimidines is an effective way to inhibit cell proliferation 20 Degradation of Purine Nucleotides • • • • • • • Objective G Purine nucleotides synthesized de novo are degraded primarily in the liver. AMP is first deaminated to produce IMP (AMP deaminase) IMP and GMP are dephosphorylated to their nucleoside forms (inosine and guanosine) by the action of 5’-nucleotidase Purine nucleoside phosphorylase converts inosine and guanosine into their respective purine bases, hypoxanthine and guanine. [Ribose is cleaved from base] Guanine is deaminated by guanase to produce xanthine Hypoxanthine is oxidized by xanthine oxidase to xanthine Xanthine is further oxidized by xanthine oxidase to uric acid 21 Gout • Objective H Gout is a disorder initiated by high levels of uric acid in the blood (hyperuricemia), as a result of either the overproduction or inadequate excretion of uric acid (most common). • Uric acid (pK of 5.4) is ionized in the body to form urate which is relatively insoluble in aqueous environment. • Urate will crystallize in tissues (e.g. synovial lining of joints, particularly that of the big toe) • Inflammatory response to the crystals can cause acute and chronic gouty arthritis • Nodular masses of crystals (tophi) may be deposited in the soft tissues, resulting in tophaceous gout Treatment – Allopurinol Allopurinol (a structural analog of hypoxanthine) inhibits uric acid synthesis and is used in patients who are “overproducers” of uric acid. Allopurinol is converted to oxypurinol, which inhibits xanthine oxidase, resulting in accumulation of hypoxanthine and xanthine, compounds more soluble than uric acid and, therefore, less likely to initiate an inflammatory response. 22 Purine Salvage Pathways Objective I GOAL: Save ATP (special case) …also avoid excess hypoxanthine / xanthine Free bases react with PRPP to form nucleotides • • • Guanine/Hypoxanthine by hypoxanthineguanine phosphoribosyltransferase (HGPRT) • Adenine by adenine phosphoribosyltransferase (APRT) PRPP is used as the source of ribose 5phosphate • Nucleotides are converted to nucleosides by 5’nucleotidase • Free bases are generated from nucleosides by purine nucleoside phosphorylase Adenosine is the only purine nucleoside to be salvaged intact (top right). It is phosphorylated directly back to AMP by adenosine kinase 23 Pyrimidine Salvage Pathways Objective I Pyrimidine bases can be salvaged as nucleosides, which can be phosphorylated to nucleotides. However, the high solubility of pyrimidines makes their salvage less significant clinically than purine salvage. Two-step route: Step 1 – Nonspecific pyrimidine nucleoside phosphorylase converts pyrimidine bases to respective nucleosides Step 2 – More specific nucleoside kinases react with nucleosides to form nucleotides 24 Degradation of Pyrimidine Nucleotides • • • • • • Objective K Unlike the purine ring, which is not cleaved in humans, the pyrimidine ring is opened and degraded to highly soluble products. Pyrimidine nucleotides are hydrolyzed to their nucleosides and Pi Nucleosides are cleaved to produce ribose-1 phosphate and the free pyrimidine bases: thymine, uracil, cytosine. Cytosine is deaminated, forming uracil which is converted to CO2, NH3, and βalanine. These products are excreted in urine or converted to CO2, H2O, and NH3 (forms urea) Catabolism of the pyrimidine bases does not cause problems, in contrast to purine bases (urate can precipitate leading to gout) • Normal cellular metabolites Objective h 25 Sample Question Which of the following is the general strategy of purine synthesis? (Note: PRPP = 5-phosphoribosyl 1-pyrophosphate; UMP = uridine monophosphate; IMP = inosine monophosphate; ATP = adenosine triphosphate) A. Ammonia + CO2 + 2ATP à carbamoylphosphate + ornithine à à à UMP B. Glutamine + CO2 + 2ATP à carbamoylphosphate + aspartate àà orotate + PRPP à UMP C. PRPP + Glutamine + CO2 + 2ATP à orotate + carbamoylphosphoate à UMP D. Ribose-5-P + ATP à PRPP + glutamine à à IMP E. Ribose-5-P + ATP à PRPP + asparagine à à hypoxanthine + ATP à IMP 26 Sample Question Adenine phosphoribosyl-transferase and hypoxanthine-guanine phosphoribosyl-transferase are the key enzymes in which nucleotide pathway? A. purine degradation B. purine synthesis C. purine salvage D. pyrimidine degradation E. pyrimidine synthesis 27 Sample Question Using the figure on the right, which molecule, if it accumulates in tissues, can cause gout? A B C D E 28 Thank You! 29