Nucleotide Metabolism Lecture 20 PDF

Nucleotide Metabolism Lecture 20 Learning Outcomes At the end of these lectures, students should be able to: Describe purine synthesis Describe pyrimidine synthesis Describe synthesis of deoxyribonucleotides Explain regulation of nucleotides synthesis Describe purine breakdown Describe pyrimidine br...

Nucleotide Metabolism Lecture 20 Learning Outcomes At the end of these lectures, students should be able to: Describe purine synthesis Describe pyrimidine synthesis Describe synthesis of deoxyribonucleotides Explain regulation of nucleotides synthesis Describe purine breakdown Describe pyrimidine breakdown Describe purine salvage pathway Nucleotide Biosynthesis Nucleotides can be synthesized de novo (“from the beginning”) from amino acids, ribose-5-phosphate, CO2, and NH3. Nucleotides can be salvaged from RNA, DNA, and cofactor degradation. De Novo Biosynthesis of Nucleotides Approximately the same in all organisms studied Phosphoribosyl pyrophosphate (PRPP) is made from ribose 5-phosphatein (Activated ribose) Gln provides most amino groups Gly is precursor for purines Asp is precursor for pyrimidines and purines Nucleotide pools except ATP are kept low, so cells must continually synthesize them – This synthesis may actually limit rates of transcription and replication Ribose 5-P is made by PPP Several pathways share 5-phosphoribosyl-1pyrophosphate (PRPP) as an intermediate Synthesized from ribose 5-phosphate of pentose phosphate pathway via ribose phosphate pyrophosphokinase (PRPP synthetase). – A highly regulated allosteric enzyme Origin of Ring Atoms in Purines (THF) (THF) De novo biosynthesis of purines begins with PRPP Synthesis begins with reaction of 5-phosphoribosyl 1-pyrophosphate (PRPP) with Gln Committed step: synthesis of phosphoribosylamine Purine ring builds up following addition of two carbons and one nitrogen from glycine The first intermediate with full purine ring is inosinate (Inosine Monophosphate, IMP) Adenine and guanine are synthesized as AMP and GMP from IMP Construction of IMP First amino group is donated by glutamine and attached at C-1 of PRPP. The first committed step of the pathway. Synthesis of AMP and GMP from IMP Note that ATP is used to phosphorylate GMP precursor, while GTP is used to phosphorylate AMP precursor. Regulation of Purine Biosynthesis in E. coli Largely Consists of Feedback Inhibition Four Major Mechanisms 1. Glutamine-PRPP amidotransferase is inhibited by end-products IMP, AMP, and GMP. 2. Excess GMP inhibits formation of xanthylate from inosinate by IMP dehydrogenase. 3. GMP and AMP concentrations inhibit phosphorylation steps. 4. PRPP synthesis is inhibited by ADP and GDP. De Novo Synthesis of Pyrimidines Pyrimidines are made from Asp, PRPP, and carbamoyl phosphate - Carbamoyl phosphate required for pyrimidine synthesis is made by carbamoyl phosphate synthetase II in cytoplasm. Similar to carbamoyl phosphate synthetase I required for urea synthesis in mitochondria. Unlike purine synthesis, pyrimidine synthesis proceeds by first making the pyrimidine ring and then attaching it to ribose 5-phosphate De Novo Synthesis of Pyrimidine Nucleotides (1) First committed step is the reaction between Asp and Ncarbamoyl phosphate, catalyzed by aspartate transcarbamoylase (ATCase) Aspartate and carbamoyl phosphate provide the atoms for the ring structure De Novo Synthesis of Pyrimidine Nucleotides (2) After addition of ribose-5phosphate via PRPP, the resulting nucleotide (orotidylate) is decarboxylated to form uridylate (UMP), the first possible pyrimidine. De Novo Synthesis of Pyrimidine Nucleotides (3) UMP is phosphorylated to UTP. After formation of UTP, amination can convert UTP to CTP. Regulation of pyrimidine biosynthesis is also via feedback inhibition ATCase is inhibited by endproduct CTP and is accelerated by ATP Nucleoside Monophosphates Are Converted to Nucleoside Triphosphates In most cells, phosphorylation of AMP to ADP is catalyzed by Adenylate kinase Then ADP is phosphorylated to ATP by the glycolytic enzymes or through oxidative phosphorylation Other nucleotides (ATP is almost the phosphate donor): Nucleoside monophosphate kinases Nucleoside diphosphate kinase Ribonucleotides Are Precursors to Deoxyribonucleotides Two H atoms are donated by NADPH and carried by proteins thioredoxin or glutaredoxin. - catalyzed by ribonucleotide reductase For example, ADP is reduced to 2′-dADP, and GDP is to dGDP Reduction of Ribonucleotides by Ribonucleotide Reductase NADPH serves as the electron donor. Funneled through glutathione (a) or thioredoxin pathways (b) Substrates are ribonucleoside diphosphates (NDP) dTMP is made from dUTP Roundabout pathway… 1. dUTP is made (via deamination of dCTP or by phosphorylaton of dUDP) 2. dUTP  to dUMP by dUTPase (efficient to keep dUTP pools low and prevent incorporation of uridylate into DNA) 3. dUMP  dTMP by thymidylate synthase - adds a methyl group from tetrahydrofolate Nucleotide Catabolism Catabolism of Purines All purine degradation leads to uric acid (but it might not stop there) Ingested nucleic acids are degraded to nucleotides by pancreatic nucleases, and intestinal phosphodiesterases in the intestine Group-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosides – Direct absorption of nucleosides – Further degradation Nucleoside + H2O  base + ribose (nucleosidase) Nucleoside + Pi  base + r-1-phosphate (n. phosphorylase) NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND EXCRETED. 25 Catabolism of Purines 1. Dephosphorylation (via 5’nucleotidase) 2. Deamination and hydrolysis of ribose lead to production of xanthine. 3. Hypoxanthine and xanthine are then oxidized into uric acid by xanthine oxidase. Conversion of Uric Acid to Allantoin, Allantoate, and Urea Degree of further oxidation of uric acid is organism dependent. Excess uric acid seen in gout Painful joints (often in toes) due to deposits of sodium urate crystals Primarily affects males May involve genetic under-excretion of urate and/or may involve overconsumption of fructose Treated with avoidance of purinerich foods (seafood, liver) or avoidance of fructose Also treated with xanthine oxidase inhibitor allopurinol Catabolism of Pyrimidines: Leads to NH4+ and urea Can produce intermediates of CAC – Example: Thymine is degraded to succinyl-CoA. – Cytosine is deaminated to uracil – Uracil degraded to acetyl CoA Purine and Pyrimidine Bases Are Recycled by Salvage Pathways Free bases, released in metabolism, are reused. – Example: Adenine reacts with PRPP to form the adenine nucleotide AMP. catalyzed by adenosine phosphoribosyltransferase The brain is especially dependent on salvage pathways. The lack of hypoxanthine-guanine phosphoribosyltransferase leads to Lesch-Nyhan syndrome with neurological impairment and fingerand-toe-biting behavior. Purine and Pyrimidine Bases Are Recycled by Salvage Pathways Free purine and pyrimidine bases are constantly released in cells Two key enzymes: 1. Adenosine phosphoribosyltransferase (APRT) 2. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) Adenosine deaminase is important in rapidly dividing cells