Nucleotide Metabolism

C483 Spring 2013

1. A ribose sugar is added to ________ rings after their synthesis and to ________ rings during their synthesis.A) purine; pyrimidineB) pyrimidine; purineC) purine; purineD) pyrimidine; pyrimidine 2. The first nucleotide product in the de novo biosynthetic pathway of purines is A) AMP.B) GMP.C) IMP.D) XMP.

3. Which of the following statements is false concerning purine synthesis?A) N7 is from glycineB) C2 is from carbon dioxide (bicarbonate)C) N3 is from glutamineD) C8 is from 10-formylTHF.

4. Which is a precursor in the de novo synthesize CTP? A) CMP.B) GMP.C) TMP.D) UMP.

5. Which of the following is not a role of a catalytic sulfur atom in ribonucleotide reductase?

A) Proton donorB) Radical stabilizationC) Redox reactionD) Covalent catalysis

6. Dihydrofolate reductase and thymidylate synthetase are major targets for anticancer drugs because A) these enzymes are unique in cancer cells. B) cancer cells lack sufficient amounts of these enzymes.C) cancer cells grow rapidly and are very dependent upon the activities of these enzymes. D) they donate one-carbon groups.E) All of the above.

Terminology of Nucleic Acids

• Nucleotide• Nucleoside• Nucleobase• AMP• ADP• ATP• dAMP

Some Examples of Nucleotides

ATP

GTP

S-AM

FADNAD+

UDP-Glucose

CoA

De Novo Synthesis

De Novo Synthesis of Purines

• Form activated ribose

• Form 5-phospho ribosylamine

• Build IMP from precursors

• Synthesis of AMP and GMP

PRPP

• Pentose phosphate pathway

• 2 ATP equivalents• Over production of

PRPP is one cause of gout because PRPP stimulates the next step…

5-phosphoribosylamine

• First step of purine biosynthesis

• Glutamine is N donor• Regulated– Activation by PPRP– Increased purine levels– Degradation of purines

leads to compound which can cause gout

Purine Pathway

• Don’t need to know details, order

• Know precursors– N from Asp, Gln– C from THF, Gly, CO2

• Cost– 2 ATP eq for PRPP– 5 more ATP steps Know this figure!

Purines

• Two distinct strategies for amination– Mechanisms

• Regulation– Feedback to 5-phospho

ribosylamine– Branchpoint regulation

Compare/Contrast• Purine biosynthesis– Salvage is a major

pathway– Base synthesized while

attached to ribose– IMP is common

intermediate for AMP and GMP, but itself is not a typical nucleotide

• Pyrimidine biosynthesis– De novo is a major

pathway– Base is synthesized, then

attached to ribose– UMP, a typical nucleic

acid, is converted into other pyrimidines

De novo Pyrimidine Synthesis

• First step regulated (compare to urea cycle)

• Asp is different than purine—whole molecule is incorporated

Further Modifications

• Interconversion of nucleotides (mono, di, tri phosphates)

• Reduction to form deoxynucleotides• Methylation to form dTMP

Nucleotide Interconversions

• Fast, reversible, driven by high [ATP]• NMPNDP catalyzed by specific nucleoside

monophosphate kinase• NDPNTP catalyzed by nonspecific kinase• AMP + ATP ADP + ADP important in energy

balance

NMP NDP NTP

ATP ADP ATP ADP

Deoxyribonucleotides• Deoxygenation occurs on diphosphates• One enzyme affects all transformations

[dUDP]

Ribonucleotide Reductase

• Sulfur does amazing chemistry!– Stable radical– Proton donor– Redox reagent

• NADPH is ultimate source of reducing

Regulation of Reductase

• One enzyme balances needs of cell via regulation of activity and selectivity

• Be able to explain why this table makes sense

Methylation• dTMP is made from dUMP• Key step in replicating cells• Therapeutic target for anti-cancer drugs• Two key enzymes

Thymidylate Synthase

• Methylene-THF acts as a “methyl” donor– Donates methylene– And hydride

• Fascinating chemistry!– Sulfur is covalent

catalyst– Internal 1,3-hydride

shift

• THF is left as DHF

5-Fluorouracil• Incorporated into

monophosphate nucleotide in body

• Mechanism based inhibitor (Trojan Horse)

• Forms covalent link to enzyme like normal

• No elimination possible because proton replaced with fluorine

NH

O

ONO

OHOH

HH

HH

OP-O

O-

O

NH

O

ONO

OHOH

HH

HH

OP-O

O-

O

F

DHF reductase

• DHF must be reduced back to THF to be a viable cofactor

• Second chemotherapy target

• Competitive inhibitor that is structurally similar to THF would end methylation process

Review of Purines• Knowing blue in figure will help with chapter summary

Review of Pyrimidines• Knowing blue in figure will help with chapter summary

Catabolism

• Less important than other catabolic processes– not a major energy source– Lots of salvage– Serves to clear excess

• In humans, purines uric acid (excreted)• In humans, pyrimidines acetyl CoA, succinyl

CoA for some energy gain

Severe Combined Immunodeficiency Syndrome (SCIDS)

• Deficiency of adenosine deaminase

• First step in catabolism• High levels of dATP

lead to low levels of dNTP

• No DNA kills fast growing T-cells

*

Answers

1. B2. C3. B4. D5. D6. C