26 - Pyrimidine Metabolism Flashcards
List the purines and pyrimidines
Purines
- Adenosine
- Guanosine
Pyrimidines
- Cytosine
- Uracil/thymine
Describe de novo synthesis of UMP (precursor of CDP and UDP)
- Starts with ATP, glutamine, water and bicarbonate
- THis is converted to carbamoyl phosphate via CPS1 in mitochondria (ammonia) and CPSII in cytosol (glutamine) (this step is inhibited by UDP and UTP and activated by ATP and PRPP)
- Aspartate is used to convert carbamoyl phosphate to carbamoyl aspartate
- Carbamoyl aspartate is converted to dihydroorotate (specific to pyrimidine pathway!)
- Dihydroorotate is converted to orotate
- Orotate is converted to OMP
- OMP is converted to UMP
How is pyrimidine biosynthesis regulated?
Bacteria
- Aspartate transcarbamylase (ATCase) is allosterically stimulated by ATP and inhibited by CTP in E coli or UTP in other bacteria
Animals
- CPSII is activated by ATP and PRPP, inhibited by UDP and UTP
- OMP decarboxylase is competitively inhibited by UMP
What is the difference between CPSI and CPSII?
CPSI
- Mitochondrial
- Urea synthesis
- REquires ammonia
CPSII
- REquires glutamine
- Pyrimidine synthesis
- Cytosolic
How are pyrimidines degraded?
Through a series of reactions that yield amino acid products (eg. beta-alanine) that are taken up by other metabolic processes.
UMP and dTMP are degraded by the same enzyme (pathways for dTMP degradation)
There are no known diseases from deficiencies (unlike in purine degradation with uric acid)
How are pyrimidines degraded?
Through a series of reactions that yield amino acid products (eg. beta-alanine) that are taken up by other metabolic processes.
UMP and dTMP are degraded by the same enzyme (pathways for dTMP degradation)
There are no known diseases from deficiencies (unlike in purine degradation with uric acid)
What is the main endproduct of pyrimidine degradation?
Malonyl-CoA
- Can feed into fatty acid synthesis
How are deoxyribonucleotides formed from the two purines and two pyrimidines?
Purines
- ribonucleotide reductase (RNR) deoxygenates dinucleotides
- ATP phosphorylates them to become d_TPs (for both A and G)
CDP
- RNR deoxygenates
- ATP phosphorylates to make dCTP
UDP
- RNR deoxygenates
- ATP phosphorylates to get dUTP
- dCMP is hydroxylated (NH3 leaves) and interacts with dUTP to form dUMP
- dUMP is methylated by THF to form dTMP
- dTMP is phosphorylated with ATP to form dTDP
- dTDP is phosphorylated with ATP to form dTTP
Describe ribonucleotide reductase
Quaternary structure: 2 R1 subunits, 2 R2 subunits
3 allosteric effector sites in each R1 subunit. Enzyme is more active when substrates are present.
- Specificity site
- Hexamerization site
- Activity site
Each R2 contains a binuclear Fe(III) complex and a phenoxy radical at Tyr122
- At interface between R1 and R2 are two active sites
There are 3 allosteric effector sites in each R1 subunit. Enzyme is more active when substrates are present.
- Specificity site
- Hexamerization site
- Activity site
List the possible effectors for each site
Specificity site
- ATP, dATP, dGTP, dTTP
Hexamerization site
- ATP
Activity site
- ATP, GDP, UDP, CDP
How is ribonucleotide reductase regulated?
d
There are 3 allosteric effector sites in each R1 subunit. Enzyme is more active when substrates are present.
- Specificity site
- Hexamerization site
- Activity site
List the possible effectors for each site
Specificity site
- ATP, dATP, dGTP, dTTP
Hexamerization site
- ATP
Activity site
- ATP, GDP, UDP, CDP
What is thioredoxin and glutaredoxin’s roles in RNR’s reduction of ribose?
Generates reduced structure of RNR by transmitting electrons to it from NADPH
- Electrons transmitted to glutaredoxin from reduced glutathione by glutaredoxin reductase
- Electrons transmitted to thioredoxin from FADH2 via thioredoxin reductase
- The sulfide groups in glutaredoxin reductase are contributed by 2 molecules of bound glutathione (regenerates glutaredoxin)
- Thioreductase is a flavoenzyme
How is dTMP synthesized from dUMP?
dUMP + methylene carrying THF
With thymidylate synthase
dTMP + dihydrofolate (DHF)
The transferred methylene group is reduced to a methyl group on dTMP
Give three salvage pathways which can produce dTMP
dTMP
- Deoxythymidine to dTMP via thymidine kinase
- dTTP inhibits this
dUMP
- deoxyuridine to dUMP via thymidine kinase
- dUMP to dTMP via thymidylate synthase
dCMP
- Deoxycytidine to dCMP via deoxycytidine kinase (dTTP inhibits this)
- dCMP to dUMP via dCMP deaminase
- dUMP to dTMP via thymidylate synthase
Give three salvage pathways which can produce dTMP
Which of these enzymes are sometimes targeted in chemotherapy?
dTMP
- Deoxythymidine to dTMP via thymidine kinase
- dTTP inhibits this
dUMP
- deoxyuridine to dUMP via thymidine kinase
- dUMP to dTMP via thymidylate synthase
dCMP
- Deoxycytidine to dCMP via deoxycytidine kinase (dTTP inhibits this)
- dCMP to dUMP via dCMP deaminase
- dUMP to dTMP via thymidylate synthase
Thymidine kinase and thymidylate synthase are targeted for chemotherapy. Stopping synthesis of dTMP can stop cell replication.
Give the steps of 5,10-methylene-THF regeneration and dTMP production from this molecule.
Which two steps of 5,10-methylenetetrahydrofolate (THF) regeneration are targeted in chemotherapy?
- Conversion of DHF to THF via dihydrofolate reductase (drugs: methotrexate, aminopterin, trimethoprim)
- Conversion of THF to 5,10-methylene-THF via serine hydroxymethyltransferase
- Transfer of methyl group onto dUMP via thymidylate synthase to form dTMP (inhibited by 5-fluorouracil -> FdUMP)
Steps 1 and 3 are targeted as written.
Describe how inhibition of thymidylate synthase is a chemotherapeutic agent
Targets dTMP synthesis and folate metabolism
- Actively growing cancer cells require a steady supply of dTMP to survive. Treatment with 5-fluorouracil ends this by being converted to FdUMP by salvage pathway
- FdUMP then binds to and irreversibly inactivates the enzyme, forming a thymidylate synthase-FdUMP-THF ternary complex
- suicide inhibitor
How can inhibition of dihydrofolate reductase with antifolates act as a chemotherapeutic agent?
How can patients develop resistance to this therapy?
Methotrexate, aminopterin and trimethoprim bind the enzyme 1000x better than DHF. This prevent the regeneration of 5,10-methylene-THF
Treatment for childhood leukemias. Must rescue patient after treating with massive dose of methotrexate (rescue with 5-formyl-THF and/or thymidine)
Resistance can develop if DHFR gene is amplified or if MTX is not taken into cell.
Methotrexate can also inhibit immune system to decrease arthritis inflammation.
Trimethoprim binds more tightly to bacterial DHFR, making it an antibiotic.
How can inhibition of dihydrofolate reductase with antifolates act as a chemotherapeutic agent?
How can patients develop resistance to this therapy?
Methotrexate, aminopterin and trimethoprim bind the enzyme 1000x better than DHF. This prevent the regeneration of 5,10-methylene-THF
Treatment for childhood leukemias. Must rescue patient after treating with massive dose of methotrexate (rescue with 5-formyl-THF and/or thymidine)
Resistance can develop if DHFR gene is amplified or if MTX is not taken into cell.
Methotrexate can also inhibit immune system to decrease arthritis inflammation.
Trimethoprim binds more tightly to bacterial DHFR, making it an antibiotic.
How can inhibition of viral thymidine kinase be used as anti-viral agent?
Herpes virus’ thymidine kinase converts acyclovir to acycloguanosine monophosphate
- Host thymidine kinase will not
- Acyclovir incorporated into viral DNA and results in chain termination
How can inhibition of viral reverse transcriptase by manipulated at pyrimidine level?
Give AZT (dideoxy thymidine), which will be converted to AZT triphosphate, which inhibits viral reverse transcriptase with no effect on host DNA polymerase
