nucleic acid metabolism robert f. waters, phd
DESCRIPTION
Nucleic Acid Metabolism Robert F. Waters, PhD. Nucleotides Essential for all cells Carriers of activated intermediates in carbohydrate, lipids and proteins CoA FAD NAD NADP Energy Carriers ATP Inhibiting or activating enzymes DNA RNA. Nucleotide Structure. Ribose Sugar Ribose - PowerPoint PPT PresentationTRANSCRIPT
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Nucleic Acid MetabolismRobert F. Waters, PhD
Nucleotides– Essential for all cells– Carriers of activated intermediates in carbohydrate, lipids and proteins
• CoA• FAD• NAD• NADP
– Energy Carriers• ATP
– Inhibiting or activating enzymes– DNA– RNA
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Nucleotide Structure
Ribose Sugar– Ribose – Deoxyribose
Base– Purines– Pyrimidines
Nucleoside– Base plus sugar
Nucleotide– E.g., AMP, ADP, ATP
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NomenclatureDNA Purine Bases– Adenine– Guanine
Purine Nucleosides– Adenosine – Guanosine
DNA Nucleotides (Purine)– dAMP (deoxyadenylate)– dGMP (deoxyguanylate)
RNA Nucleotides (Purine)– Adenylate (AMP)– Guanylate (GMP)
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Nomenclature ContinuedDNA Pyrimidine Bases– Thymine– Cytosine (Also RNA)
DNA Pyrimidine Nucelosides– Thymidine– Cytidine
DNA Pyrimidine Nucleotides– (dTMP) deoxythymidylate– (dCMP) deoxycytidylate
RNA Pyrimidine Nucleotides– (CMP) cytidylate– (UMP) uridylate
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PRPP 5-Phosphoribosyl 1-Pyrophosphate
•Addition of the ribose sugar component
•HMP
•ATP Required
•Mg++
•Pi activates and nucleosides inhibit
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Pyrimidine Synthesis
UMP (Uridine 5-monophosphate) to UTP– Precursor to CTP
Occurs on mitochondria inner membraneCarbamoyl phosphate synthetase II– Different from CPS I
• CPS I uses free ammonia• CPS II uses glutamine for amino source
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Carbamoyl Phosphate Synthetase II
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Formation of Uridine 5’-phosphate
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Enzymes of Pyrimidine Biosynthesis
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UTP to CTP Conversion
CTP Synthetase Reaction
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Conversion of Ribonucleotides to Deoxyribonucleotides
Ribonucleotide reductaseNADPThioredoxin reductaseExample is production of dCDP
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Allosteric Inhibition of Ribonucleotide Reductase
ATP activates dATP inhibits
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Thymidylate Biosynthesis
Substrates and Vitamins– dUMP– Folate (N5, N10,-Methylene-THF)– Glycine/Serine– NADP
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Conversion of dUMP to dTMP:Overall
5-fluorouracilMethotrexate
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Thymidylate Pathway:Specific
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Thymidylate Synthesis and Cancer Chemotherapy
Thymidylate synthase is target for fluorouracil– Action is 5-fluorouracil (5-FU)is converted to 5-fluoro-2’-deoxyuridylate
(dUMP structural analog)– Then 5-fluoro-2’-deoxyuridylate binds to the enzyme Thymidylate
Synthase and undergoes a partial reaction where part of the way through 5-fluoro-2’-deoxyuridylate forms a covalent bridge between Thymidylate Synthase and N5, N10-Methylene THF and is an irreversible inhibition.
• Normally, the enzyme, Thymidylate Synthase and the vitamin would NOT be linked together permanently
– This type of inhibition is called “suicide-based enzyme inhibition” because the inhibitor participates in the reaction causing the enzyme to react with the compound producing a compound that inactivates the enzyme itself.
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Fluorouracil Pathway
Suicide inhibition because Flurouracil does not directly inhibit enzyme.
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Methotrexate
Competitive inhibitor of Dihydrofolate Reductase– Used in,
• Acute lymphoblastic leukemia• Osteosarcoma in children
– Solid tumor treatment• Breast, head, neck, ovary, and bladder
Prevents regeneration of tetrahydrofolate and removes activity of the active forms of folate
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Leucovorin Rescue Strategy in Methotrexate Chemotherapy
Patients given sufficient methotrexate that if were not followed by Leucovorin (N5-methenyl-THF) would be fatal.– All neoplastic cells are killed
Patients are “rescued” (6-36 hours) by the Leucovorin (Folate) otherwise would die due to permanent tetrahydrofolate shutdown.Tumor resistance to methotrexate can occur in patients who have “gene amplification” of dihydrofolate reductase (in tumor cells)– More dihydrofolate reductase is produced by more than the normal
active genes usually present in normal cells.
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Purine BiosynthesisIMP (Inosine Monophosphate)– Precursor to
• GMP and AMP
Utilizes (Substrates)– Glycine– Glutamine– ATP– Folate (N10-formyl-THF)– Aspartate– CO2
PRPP amidotransferase is rate limiting– Inhibited by AMP and GMP
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IMP Pathway
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IMP to AMP and GMP
Glutamine, NAD, ATP used in GMP productionAspartate, GTP used AMP production
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AMP and GMP Pathway
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Nucleotide Pyrimidine Catabolism
Degradation of pyrimidine metabolitesUMP, CMP, TMPEnd products are acetyl-CoA and Propionyl-CoARibose sugar component may be converted to ribose-5-phosphate which is a substrate for PRPP SynthetaseRibose sugar component may be further catabolized in HMP pathway
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Pyrimidine Catabolic Pathway
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Purine Catabolism
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Regulation of Nucleotide Metabolism
Pyrimidine Regulation– Primary regulatory step is Carbamoyl Phosphate via
Carbamoyl Phosphate Synthetase II
Purine Regulation
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Action of Allopurinol
Allopurinol is purine base analogThree mechanisms– Allopurinol is oxidized to alloxanthine by xanthine dehydrogenase– Then Allopurinol and alloxanthine are inhibitors of xanthine
dehydrogenase– This inhibition decreases urate formation
Then concentrations of Allopurinol and alloxanthine increase but do not precipitate as urate does.Allopurinol and alloxanthine are excreted into the urine
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Action of Allopurinol:Pathway
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Biosythesis of Nucleotide Coenzymes
CoA– OTC is pantothenate– Uses ATP, CTP, Cysteine
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Coenzyme A Pathway
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FMN and FAD
OTC is riboflavin– Consumes ATP