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Nucleotides play key roles in many, many cellular processes 1. Activated precursors of RNA and DNA 2. Adenine nucleotides are components of three major co-enzymes, NAD, FAD, and CoA 3. Nucleotide derivatives are activated intermediates in biosynthetic processes (UDP-glucose, SAM) 4. Serve as metabolic regulators (for example cAMP and the activation of cell signaling. 5. Serve as major currency of energy in all cells (ATP and GTP). 6. Many metabolic diseases have their etiology in nucleotide metabolism. Purine metabolism (Overview) 1. Nomenclature/nucleotide structure 2. De novo synthesis pathways 3. Re-utilization pathways 4. Metabolic diseases of purine Metabolism(Gout, Lesch-Nyham, SCID) Why go through to the trouble to convert Uracil to Thymine? dUMP Thymidylate synthase reduced N5,N10-methylenetetrahydrofolate dTMP oxidized Dihydrofolate NADPH Serine transhydroxymethylase Dihydrofolate reductase Tetrahydrofolate NADP+ Thymidylate Synthase The nomenclature of purines and pyrimidines depends on their linkage to a pentose Cytosine Base Cytidine Nucleoside* Base Cytidine Monophosphate Nucleotide Base (P04 ester) * when the base is purine, then the nucleoside ends in OSINE (AdenOSINE, GuanOSINE, InOSINE) when the base is pyrimidine, then the nucleoside ends in IDINE (UrIDINE, CytIDINE, ThymIDINE) The active forms of nucleotides in biosynthesis and energy conversions are di and triphosphates Nucleoside Monophosphate Kinase CMP + ATP CDP + ADP Nucleoside Diphosphate Kinase XDP + YTP XTP + YDP RIBONUCLEOTIDE REDUCTASE 1. Complex enzymatic reaction whereby electrons are transferred from NADPH through a series of sufhydryl groups at the catalytic site of Ribonucleotide Reductase. NADPH NADP+ 2. Active site of RR contains thioredoxin, a 12 kD protein with two exposed cysteines, which become oxidized. 3. This ultimately allows for the reduction of ribose. REGULATION 1. Based on the response to cellular need for dATPs. dATP is general inhibitor ATP is a general activator Nucleotides are linked by 5’ to 3’ phosphodiester bonds to generate DNA and RNA Structures of Common Purine Bases. H= 6 oxy purine X= 2,6 dioxy purine A= 6 amino purine G= 2 amino, 6-oxy purine Structures of Common Purine Bases. H= 6 oxy purine X= 2,6 dioxy purine A= 6 amino purine G= 2 amino, 6-oxy purine Structures of Common Purine Bases. (N source) Aspartate (N source) Glutamine The common mechanistic them for the conversion of A and G is the conversion of a carbonyl oxygen to an amino group There are two basic mechanisms to generate purines and pyrimidines 1. DE NOVO BIOSYNTHETIC PATHWAYS (building the bases from simple building blocks) 2. SALVAGE PATHWAYS (the reutilization of bases from dietary or catabolic sources) The biosynthesis of purine (A and G) begins with the synthesis of the ribose-phosphate Pentose phosphate pathway Ribose phosphate pyrophosphoKINASE The major regulatory step in purine biosynthesis is the conversion of PRPP to 5-Phosphoribosyl-1-amine * Glutamine PRPP Glutamate PPi Amidophosphoribosyl transferase Amidophosphoribosyl transferase is a important regulatory enzyme in purine biosynthesis. It is strongly inhibited by the end products IMP, AMP, and GMP. This type of inhibition is called FEEDBACK INHIBITION. Several amino acids are utilized in purine biosynthesis, IMP is the precursor for both AMP and GMP, the base is also called hypoxanthine QuickTime™ and a GIF decompressor are needed to see this picture. Structures of Common Purine Bases. (N source) Aspartate (N source) Glutamine The common mechanistic them for the conversion of A and G is the conversion of a carbonyl oxygen to an amino group Purines:where do the atoms come from? Purine intermediates include: 1. Glycine 2. 1 C units of 5,10 mTHF 3. Glutamine 4. Asparate The regulation of purine biosynthesis is a classic example of negative feedback Inhibited by AMP AMP Ribose 5-phosphate PRPP Phosphoribosyl amine IMP GMP Inhibited by IMP, AMP, and GMP Inhibited by GMP QuickTime™ and a GIF decompressor are needed to see this picture. Nucleotidase Phosphorylase Cytosine Base Cytidine Nucleoside* Base Cytidine Monophosphate Nucleotide Base (P04 ester) Salvage pathways for the re-utilization of purines; There are 2 salvage enzymes with different specificities; 1. Adenine phosphoribosyl transferase 2. Hypoxanthine-guanine phosphoribosyl transferase QuickTime™ and a + PPi decompressor + GIF Guanine are needed to see this picture. A-PRT PRPP + Adenine Adenylate HG-PRT PRPP + Guanine Guanylate What happens in gout? Inhibited by AMP AMP Ribose 5-phosphate PRPP Phosphoribosyl amine IMP GMP Inhibited by IMP, AMP, and GMP Inhibited by GMP 1. Negative regulation of PRPP Synthatase & PRPP Amidotransferase is lost 2. PRPP levels are increased because of defects in salvage pathways Therefore, there is net increase in biosynthetic/degradation pathways!! “The Gout” James Gilray, 1799. “By Royal Authority” by George Cruickshank. 19th century. David Wells New York Yankees Purines in humans are degraded to Urate Important points: 1. Nucleotides are constantly undergoing turnover! 2. There are many enzymes involved; Nucleotidases Nucleoside phosphorylases Deaminases Xanthine oxidases 3. the final common intermediate in humans is Urate, which is excreted. 4. there are several metabolic disorders resulting from defects in purine catabolism. GOUT (Gouty Arthritis): A defect of purine metabolism Serum Uric Acid Levels (mg/dl) Incidence of Gout (% of cases) >9.0 7-9 <7.0 ~10% 0.5-3.5% 0.1% Hypoxanthine Guanine xanthine oxidase Xanthine Urate xanthine oxidase Allopurinol: a. decrease urate b. increase xanthine & hypoxanthine c. decrease PRPP SCID-Severe Combined Immunodeficiency Syndrome Autosomal recessive disorder Mutations in ADA AMP H20 Nucleotidase Pi Adenosine Both T and B cells are significantly Reduced (dATP is toxic) H20 Adenine deaminase* NH3 Inosine Infants subject to bacterial, candidiasis, viral, protazoal infections Hypoxanthine 1995-AdV expressing ADA was sucessfullly employed as gene therapy strategy Disorders of Purine Metabolism: Disorder Gout Lesch Nyhan syndrome Defect PRPP synthase/ HGPRT lack of HGPRT SCID ADA von Gierke’s disease glucose -6-PTPase Comments Hyperuricemia Hyperuricemia high levels of dAMP Hyperuricemia Structure of Pyrimidines C= 2 oxy, 4 amino pyrimidine T= 2,4 dioxy 5-methyl pyrmidine U= 2,4 dioxy pyrimidine O= 2,4 dioxy 6 carboxy pyrimidine Pyrimidines: where do the atoms come from? Pyrimidine biosynthesis: (occurs in cytosol) Pyrimidine biosynthesis begins with the assembly of the ring, then linked To ribose phosphate. Precursors are Glutamine (NH2), Bicarbonate (C) , and ATP (PO4). Q. Why is it advantageous to generate carbamoyl phosphate in the cytosol rather than the mitochondria? ATCase is the committed step in pyrimidine biosynthesis committed step in pyrimidine biosynthesis Carbamoyl phosphate synthase II, ATCase, and Dihydrooratase are linked in a single 240 kD polypeptide chain. The enzyme is sometimes referred to as CAD. The second phase of pyrimidine biosynthesis QuickTime™ and a GIF decompressor are needed to see this picture. in pyrimidine biosynthesis, the addition of ribose phosphate *Note, moiety occurs late in the pathway, via its addition of Orotate. * QuickTime™ and a GIF decompressor are needed to see this picture. QuickTime™ and a GIF decompressor are needed to see this picture. ATCase is feedback inhibited by the end-products of pyrimidine biosynthesis C02 + Glutamine + ATP Rate Carbonyl Phosphate ATP CTP Inhibited by CTP [Aspartate] Carbonyl Asparate UMP UTP CTP Why go through to the trouble to convert Uracil to Thymine? dUMP Thymidylate synthase reduced N5,N10-methylenetetrahydrofolate dTMP oxidized Dihydrofolate NADPH Serine transhydroxymethylase Dihydrofolate reductase Tetrahydrofolate NADP+ Thymidylate Synthase QuickTime™ and a GIF decompressor are needed to see this picture. Common chemotherapeutic drugs act at the level of dTMP synthesis. Fluorodeoxyuridylate (5-FU) dUMP Thymidylate synthase reduced N5,N10-methylenetetrahydrofolate dTMP oxidized Dihydrofolate NADPH Dihydrofolate reductase Tetrahydrofolate NADP+ Methotrexate AZT is used to inhibit HIV reverse transcriptase (RNA-dependent DNA pol) N=N=N 3’ azido-2’3’ dideoxythymine (AZT) This class of compounds (chemotherapeutics, viral inhibitors, etc are called nucleoside analogs. Common side effects of DNA inhibitor chemotherapeutics: Diarrhea Skin and eye sensitivity to sunlight Abnormal liver function tests Hair loss Immuno-suppression Skin rashes Fatigue Headache, backache, Spinal cord irritation Peripheral neuropathies Summary: 1. Recognize basic structures of purines and pyrimidines 2. Key regulatory enzymes and feedback networks 3. Targets for clinical interventions