Menter – Nucleotide Metabolism – Flashcards
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| What is the structure of a nucleotide? |
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| Nucleotide = base + pentose sugar + phosphate Bases: 1. Purines (a 6- and a 5-membered ring) - A, G, Xanthine and Hypoxanthine - 9 atom heterocycle (4 N, 5 C) 2. Pyrimidines (a 6-membered ring) - C, T, U and Orotic Acid - 6 atom heterocycle (2N, 4 C) |
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| How does numbering on purines work? |
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| Purines: Numbering always starts on the heterocyclic (N) atom on the 6-member ring closest to the functional group (NH2 or C = O) and goes around the ring so that the next N has the lower number. In the 5-member ring, N7 is always closer to the functional group on the 6-member ring |
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| How are pyrimidines numbered? |
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| Pyrimidines: The N opposite the NH2 or C=O (usually on the "bottom") is N1 and the numbering goes around the ring so that the next N has the lower number. (N3, not N5) |
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| What is the structure of the nucleoside? |
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| Nucleoside: base + pentose sugar (-ine) A - adenosine G - guanosine C - cytidine T - thymidine U - uridine |
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| What adds the phosphate to the sugar ring? |
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| Phosphate groups are derived from phosphoric acid moieties Kinases are responsible for the addition of phosphate to the pentose sugar ring |
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| What is the structure of a nucleotide? |
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| Nucleotide: base + pentose sugar + phosphate(s) * mono-, di- or triphosphate esters of nucleosides Nucleoside monophosphates: AMP, CMP, GMP, TMP, UMP Nucleoside diphosphates: ADP, CDP, GDP, TDP, UDP Nucleoside triphosphates: ATP, CTP, GTP, TTP, UTP Deoxyribonucleoside triphosphate: dATP, dCTP, dGTP, dTTP * phosphate group attaches by an ester linkage to the 5’ OH of the pentose * 5’-ribonucleotide or 5’-deoxyribonucleotide * nucleotides are joined together to form polynucleotides, like RNA and DNA |
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| Explain the metabolic origin of the nine atoms in the purine ring system |
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| N1 - Aspartate C2 - N10-formyl-THF N3 - glutamine (amide-N) C4, C5, N7 - Glycine C6 - C02 C8 - N10-formyl-THF N9 - glutamine (amide-N) |
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| Explain the very basics of De Novo Purine Nucleotide Synthesis |
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| Ribose-5-Phosphate ---> ATP and GTP a) Ribose-5-Phosphate --> IMP b) IMP --> AMP and GMP --> ATP and GTP * IMP is the first nucleotide formed. * IMP is then converted to either AMP or GMP, which are used to make ATP and GTP, respectively. |
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| What is the rate-limiting step of de novo purine synthesis? |
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| IMPORTANT Ribose-5-Phosphate ->5’ phosphoribosyl-1-pyrophosphate (PRPP) * occurs actively in the cytosol of the liver * get Ribose-5-Phosphate from the Pentose Phosphate Pathway (PPP) when demands for pentoses are greater than the need for NADPH Enzyme = PRPP Synthetase or Ribose Phosphate Pyrophosphokinase enzyme also called ATP phosphoribosyl transferase * activated by Pi * inhibited by purine nucleoside di- or triphosphates - production inhibition * product PRPP also participates in pyrimidine synthesis, salvage pathways and the formation of NAD and NADP - rate-limiting step but NOT committed step |
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| What is the committed step in de novo purine synthesis? |
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| PRPP reacts with glutamine to form 5-phosphoribosylamine (PRA) Enzyme = Glutamine-PRPP Amidotransferase (also known as Amidophosphoribosyl Transferase 1) Reaction Transfers a -NH2 from Glutamine to PRPP. (2) Reaction takes place at anomeric carbon. (3) Reaction involves a Walden inversion. (4) Reaction is essentially irreversible |
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| How is Glutamine-PRPP Amidotransferase regulated? |
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| *controlled by feedback inhibition * small active enzyme molecules aggregate to larger inactive molecules caused by the presence of nucleotides * very high concentrations of PRPP overcome and reverse the nucleotide feedback inhibition |
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| What is the 1st reaction in de novo purine synthesis involving THF? |
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| Phosphoribosylglycinamide formyl transferase transfers a formyl group to the free amino group of the glycyl residue on glycinamide ribonucleotide (GAR) 10-Formyl-THF transfers one-carbon fragment as -CHO producing FGAR and THF |
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| What is the 2nd reaction in de novo purine synthesis that THF is involved in? |
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| Formlytransferase uses tetrahydrofolate as a cofactor to add the final carbon atom. Goes from AICAR to FAICAR (1) Uses methenyl THF to transfer - CHO to free NH2. (2) Free NH2 is more nucleophilic than - NH2CO because of electron-withdrawing effect of carbonyl on amide group |
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| How is IMP formed? |
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| Inosinicase (IMP cyclohydrolase) catalyzes the ring closure of FAICAR and IMP formation (1) Ring Closure to IMP involves intramolecular attack of NH2 on CHO, with loss of H2O. (2) IMP is first intermediate with complete purine ring. (3) IMP is scarce because it gets used up right away. |
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| How is ATP used in de novo purine synthesis? |
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| (1) Four ATP used in purine pathway (2) These ATP are involved in condensation and synthetase reactions, which are endothermic |
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| What effect does azaserine have on purine synthesis? |
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| Azaserine (a glutamine antagonist) inhibits those steps in which glutamine donates a nitrogen (ex. incorporation of N3 and N9 in the purine ring) It is an irreversible inhibitor of glutamine-dependent enzymes It inhibits the committed step |
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| What is an overview of important points in de novo purine synthesis? |
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| - Ribose-5-phosphate is the initial substrate that must be activated; after activation, atoms are successively added. - PRPP is limiting substance for purine nucleotide synthesis. - PRPP is a branch point. Thus, PRPP amidotransferase is the committed step to purine nucleotide synthesis. - IMP is the parent nucleotide produced |
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| What's the importance of tetrahydrofolate (THF)? |
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| * Derived from folic acid; serves as a vital coenzyme in reactions that involve the transfer of single carbon groups * THF and its derivatives play a role in: purine synthesis pathways pyrimidine synthesis pathways amino acid conversions * Deficiencies in THF affect both nucleic acid and protein synthesis. Thus, actively dividing and growing cells tend to be the first affected. |
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| What is the structure of IMP? |
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| Inosine-5'-Monophosphate (IMP): Base = Hypoxanthine; Nucleoside = Inosine; Nucleotide = Inosinate |
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| What are the basics of GMP and AMP synthesis from IMP? |
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| * IMP synthesis is inhibited by ADP and GDP * Conversion of IMP to GMP requires ATP as an energy source, while conversion of IMP to AMP requires GTP as an energy source * The first reaction in each pathway is inhibited by the end product of that pathway. |
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| How is ATP synthesized from IMP? |
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| IMP reacts with aspartic acid to form adenoylsuccinate using adenylosuccinate synthetase as the enzyme Adenylosuccinase catalyzes the departure of fumarate to form AMP Adenylate kinase phosphorylates AMP to ADP Nucleoside diphosphate kinase (kinase w/ broad specificity) phosphorylates ADP to ATP |
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| How is GTP produced from IMP? |
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| IMP reacts with H20 to form Xanthosine monophosphate using NAD+ as an oxidizing agent. Enzyme used is IMP Dehydrogenase This reacts with glutamine to form GMP using the enzyme GMP synthetase. Guanylate kinase phosphorylates GMP to GDP nucleoside diphosphate kinase (kinase w/ broad specificity) phosphorylates GDP to GTP |
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| How is de novo purine synthesis regulated? |
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| ADP and GDP inhibit PRPP synthetase PRPP activates Gln-PRPP amidotransferase (high concentration of PRPP) AMP, ADP, ATP and GMP, GDP, GTP all inhibit Gln-PRPP amidotransferase (committed step for purine synthesis) GMP inhibits IMP dehydrogenase (inhibits formation of its precursor - XMP) AMP inhibits adenolysuccinate synthetase (inhibits formation of its precursor - adenoylsuccinate) |
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| What is the purine salvage pathway? |
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| Purines resulting from normal turnover of nucleic acids or diet can be reconverted into nucleoside triphosphates and used by the body. * Involves two enzymes: 1. Adenine Phosphoribosyl Transferase (APRT) 2. Hypo-xanthine-guanine Phosphoribosyl Transferase (HGPRT) * These reactions are irreversible. |
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| What is the APRT reaction? |
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| Adenine + PRPP --> AMP + PPi |
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| What are the HGPRT reactions? |
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| Hypoxanthine + PRPP --> IMP + PPi Guanine + PRPP --> GMP + PPi |
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| Explain Lesch-Nyhan Syndrome |
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| * Inherited deficiency in HGPRT * Recessive X-linked disorder that occurs primarily in males * Increased PRPP and de novo purine synthesis *Characterized by: Excessive uric acid production Involuntary Movements Neurological defects Mental Retardation Self Mutilation Hyperuricemia |
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| Explain purine degradation |
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| * Purine nucleotides are sequentially degraded * End product in humans is uric acid which gets excreted in the urine; other mammals oxidize uric acid further to allantoin, urea and/or ammonia * Nucleotidases and Nucleosidases release ribose and phosphates to leave free bases. *Xanthine, the last base formed, is a precursor for uric acid synthesis. |
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| What is the xanthine oxidase reaction? |
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| Xanthine to uric acid *enzyme is xanthine oxidase * Humans and other primates excrete uric acid in the urine, but most nitrogen goes out as urea * this reaction is the last step in purine degradation * Xanthine Oxidase is inhibited by allopurinol |
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| Explain gout - causes, treatment, etc. |
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| Gout (hyperuricemia) occurs from accumulation of uric acid crystals in the extremities, which causes acute arthritic joint inflammation It is caused by hyperactivity of xanthine oxidase Treatments 1. Anti-inflammatory drugs: alleviate pain (ex. colchicine) 2. Uricosuric agents: increase renal excretion of uric acid (ex. probenecide) 3.Inhibition of xanthine oxidase: decrease formation of uric acid (ex. allopurinol - structure closely resembles hypoxanthine) 4. Changes in diet: low protein diet; excess of amino acids increases de novo purine synthesis |
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| Explain the adenosine deaminase reaction and how it relates to SCID |
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| Adenosine deaminase catalyzes the reaction of adenosine (deoxyadenosine) to inosine (deoxyinosine) *ADA deficiency causes severe combined Immunodeficiency (SCID) * SCID causes T-cell and some B-cell dysfunction * ADA-deficient children usually die before age two from severe infections * ADA deficiency causes extremely large buildups of dATP, which promotes inhibition of DNA synthesis. |
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| Describe the mechanism of action of sulfanilamide (as an antibacterial agent) |
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| Sulfa drugs (e.g. sulfanilamide) are used to treat bacterial infections are analogues of p - aminobenzoic acid (PABA). They prevent growth and cell division in bacteria by interfering with the synthesis of folate. Since humans connot synthesize folate (it is a vitamin), sulfa drugs do not affect human cells in this way |
