Nucleic Acid Metabolism Robert F. Waters, PhD Nucleotides –Essential for all cells –Carriers of...

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

Nucleotide Structure

Ribose Sugar– Ribose – Deoxyribose

Base– Purines– Pyrimidines

Nucleoside– Base plus sugar

Nucleotide– E.g., AMP, ADP, ATP

NomenclatureDNA Purine Bases– Adenine– Guanine

Purine Nucleosides– Adenosine – Guanosine

DNA Nucleotides (Purine)– dAMP (deoxyadenylate)– dGMP (deoxyguanylate)

RNA Nucleotides (Purine)– Adenylate (AMP)– Guanylate (GMP)

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

PRPP 5-Phosphoribosyl 1-Pyrophosphate

•Addition of the ribose sugar component

•HMP

•ATP Required

•Mg++

•Pi activates and nucleosides inhibit

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

Carbamoyl Phosphate Synthetase II

Formation of Uridine 5’-phosphate

Enzymes of Pyrimidine Biosynthesis

UTP to CTP Conversion

CTP Synthetase Reaction

Conversion of Ribonucleotides to Deoxyribonucleotides

Ribonucleotide reductaseNADPThioredoxin reductaseExample is production of dCDP

Allosteric Inhibition of Ribonucleotide Reductase

ATP activates dATP inhibits

Thymidylate Biosynthesis

Substrates and Vitamins– dUMP– Folate (N5, N10,-Methylene-THF)– Glycine/Serine– NADP

Conversion of dUMP to dTMP:Overall

5-fluorouracilMethotrexate

Thymidylate Pathway:Specific

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.

Fluorouracil Pathway

Suicide inhibition because Flurouracil does not directly inhibit enzyme.

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

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.

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

IMP Pathway

IMP to AMP and GMP

Glutamine, NAD, ATP used in GMP productionAspartate, GTP used AMP production

AMP and GMP Pathway

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

Pyrimidine Catabolic Pathway

Purine Catabolism

Regulation of Nucleotide Metabolism

Pyrimidine Regulation– Primary regulatory step is Carbamoyl Phosphate via

Carbamoyl Phosphate Synthetase II

Purine Regulation

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

Action of Allopurinol:Pathway

Biosythesis of Nucleotide Coenzymes

CoA– OTC is pantothenate– Uses ATP, CTP, Cysteine

Coenzyme A Pathway

FMN and FAD

OTC is riboflavin– Consumes ATP