Biochem Nucleotide Metabolism

What are nucleotides?

phosphate esters of ribose or deoxyribose, with a purine or pyrimidine base attached to the sugar's C1′

What are the building blocks of nucleic acids?

nucleoside triphosphates

What key metabolic cofactors are nucleotides involved in?

FAD, NAD+, coenzyme A

What are the nitrogenous bases?

adenine, thymine, guanine, cytosine, uracil

What are pyrimidines?

cytosine, uracil, thymine

What are purines?

Adenine and Guanine

What is the equation for the pentose phosphate pathway?

3G6P + 6NADP+ + 3H2O → 6NADPH + 6H+ + 3CO2 + 3Ru5P

What is NADPH?

- essential for biosynthetic reactions, such as fatty acid and cholesterol synthesis

- critical for maintaining redox balance (e.g., regenerating

reduced glutathione to protect against oxidative stress in cells like red blood cells)

What does the synthesis of Ribose-5-Phosphate provide?

ribose sugars for the synthesis of nucleotides and nucleic acids (DNA, RNA)

Describe how the pentose phosphate pathway allows for metabolic flexibility.

allows cells to shift between anabolic (biosynthetic) and catabolic (energy-generating) processes depending on cellular needs

What is Inosine monophosphate (IMP)?

the first fully formed purine nucleotide in the de novo synthesis pathway of nucleotides

What is the purpose of IMP?

acts as a branch point for the synthesis of AMP (adenosine monophosphate) and GMP (guanosine monophosphate)

What is the purpose of hypoxanthine base in IMP?

can be modified into either adenine or guanine bases

Where does purine synthesis occur?

de novo (from scratch) mainly in the liver and builds the purine ring on a ribose-phosphate backbone (from PRPP)

What are the steps of purine synthesis for IMP?

1. PRPP (phosphoribosyl pyrophosphate) is produced from ribose-5-phosphate

2. PRPP amidotransferase adds an amino group from

glutamine

3. Initiation of adding a N-glycosyl part

- A series of 10 reactions incorporates atoms from

glycine, glutamine, aspartate, formate (from N¹⁰-formyl-

Tetrahydrofuran, THF), and CO2, gradually building the purine ring, forming IMP as the final product

How is IMP converted to AMP?

IMP reacts with aspartate and catalyzed by adenylosuccinate synthetase (requires GTP), forming adenylosuccinate, which is then converted to AMP and fumarate

How is IMP converted to GMP?

IMP is first oxidized to xanthosine monophosphate (XMP)

by IMP dehydrogenase (requires NAD⁺), which is then converted to GMP using glutamine and ATP (ATP binds to GMP synthetase) by GMP synthetase

How is purine biosynthesis regulated by feedback inhibition?

- AMP, GMP, and IMP inhibit PRPP amidotransferase, the committed step enzyme

- AMP inhibits its own branch (adenylosuccinate synthetase)

- GMP inhibits GMP synthesis (GMP synthetase)

How is purine biosynthesis regulated by feedforward activation?

High levels of PRPP activate PRPP amidotransferase and drive the pathway forward

What is the purine salvage pathway?

recycles free purine bases (adenine, guanine, hypoxanthine) into nucleotides, saving energy

What key enzymes are involved in purine salvage pathway?

mediating N-glycosidic bond formation

• HGPRT (hypoxanthine-guanine phosphoribosyltransferase) converts hypoxanthine → IMP and guanine → GMP

• APRT (adenine phosphoribosyltransferase) converts adenine → AMP.

What is salvage important for?

important in tissues that cannot perform de novo synthesis, like the brain

What does RNA/DNA turnover allow for?

Nucleotide → nucleoside → base, producing Adenine, Guanine, Hypoxanthine

What does diet allow for?

Digestion and absorption to produce Adenine, Guanine, Hypoxanthine

What does cell death allow for?

Nucleic acid degradation to produce Adenine, Guanine, Hypoxanthine

What are the steps of UMP synthesis?

1. via CPS II (carbamoyl phosphate synthetase II), Glutamine + CO₂ + 2 ATP → Carbamoyl phosphate

2. via Aspartate transcarbamoylase (ATCase)

Carbamoyl phosphate + Aspartate → Carbamoyl aspartate

3. Ring closure to form dihydroorotate

4. Oxidation to orotate (orotic acid) via Dihydroorotate dehydrogenase (only mitochondrial step)

5. Ribose attachment (orotate → OMP) via Orotate phosphoribosyltransferas, using PRPP to attach the ribose-phosphate backbone → OMP (orotidine

monophosphate)

6. Decarboxylation of OMP → UMP via OMP decarboxylase

What does CPI mean?

occurs in mitochondria and involved in urea cycle

What does CPII mean?

occurs in cytosol and involved in nucleotide synthesis

Describe CPSI urea cycle.

- occur in mitochondrial matrix

- N donor = Free NH₃ (ammonia)

- carbon source = CO₂ (as bicarbonate, HCO₃⁻)

- requires 2 ATP and activated by N- acetylglutamate (NAG)

- Couples to amino acid catabolism and ammonia disposal

- Product: Carbamoyl phosphate → enters urea cycle (via ornithine → citrulline)

Describe CPSII nucleotide synthesis.

- occur in Cytosol

- N donor = Glutamine (amide nitrogen)

- carbon source = CO₂ (as bicarbonate, HCO₃⁻)

- requires 2 ATP and inhibited by UTP, activated by PRPP

- Couples to nucleotide biosynthesis

- Product: Carbamoyl phosphate → enters pyrimidine synthesis (via aspartate → carbamoyl aspartate)

Describe UMP conversion to UTP.

• Enzymes: nucleoside monophosphate kinase (UMP →

UDP), then nucleoside diphosphate kinase (UDP → UTP)

• ATP donates the phosphate groups

Describe UTP conversion to CTP.

• Enzyme: CTP synthetase

• UTP + Glutamine + ATP → CTP + Glutamate + ADP + Pi

• A new amino group is added from glutamine to the C4

position of UTP

Describe regulation of pyrimidine biosynthesis in bacteria.

- ATCase (aspartate transcarbamoylase) is the key regulatory enzyme.

- Inhibited by CTP (end-product feedback).

- Activated by ATP — balances purine and pyrimidine levels.

Describe regulation of pyrimidine biosynthesis in animals.

- CPS II (Carbamoyl Phosphate Synthetase II) is the rate-limiting enzyme.

- Activated by PRPP and ATP (need to have balance A requires T)

- Inhibited by UTP (end-product)

- CTP synthetase is also feedback-inhibited by CTP

Describe regulation of pyrimidine biosynthesis overall.

1. Glutamine + CO₂ + ATP → Carbamoyl phosphate (via CPS II)

2. Carbamoyl phosphate + Aspartate → Carbamoyl aspartate (via ATCase)

3. Orotate + PRPP → OMP → UMP (via UMP synthase)

4. UTP → CTP (via CTP synthetase)

How costly is purine synthesis?

The purine ring (9 atoms) is built step by step directly on PRPP, requiring ~6 ATP equivalents and multiple amino acids (glycine, glutamine, aspartate, CO₂, and two formyl groups from folate)

- energy-sensitive and slow

Why is purine salvage optimal?

saves a lot of ATP, making it essential

How costly is pyrimidine synthesis?

The ring (6 atoms) is built separately from simple precursors — aspartate + carbamoyl phosphate — using only 2 ATP in carbamoyl phosphate synthesis. The ring is then attached to PRPP to form OMP → UMP, then phosphorylated to UTP > CTP

- low energy cost

What are Deoxyribonucleotides synthesized from?

- ribonucleotides (NDPs) by the enzyme ribonucleotide reductase (RNR).

- Key Reaction: NDP (ribonucleoside diphosphate) ⟶ dNDP (deoxyribonucleoside diphosphate)

Describe deoxyribonucleotide synthesis.

1. The 2'-OH group on the ribose sugar is replaced with a hydrogen (H), forming deoxyribose.

- requires electrons supplied by thioredoxin (small redox protein)

2. After dNDPs are formed, they can be converted to dNTPs (deoxyribonucleoside triphosphates) by nucleoside diphosphate kinases.

What reduces NDPs to dNDPs?

Ribonucleotide reductase

Describe the Catalytic Mechanism of Ribonucleotide

Reductase (RNR).

1. Initiation by radical formation: A thiyl radical is generated in the RNR enzyme (R1) & transferred to the active site (R1).

2. Hydrogen abstraction: The radical removes a hydrogen atom from the 3′-carbon of the ribose sugar of the NDP substrate.

3. Reduction of the 2′-OH group: The 2′-OH is converted to 2′-H via a thiol-mediated reduction. Two cysteine residues in the active site donate electrons to reduce

the 2′-OH to H.

4. Restoration of enzyme: The disulfide formed between cysteines must be reduced by thioredoxin or glutaredoxin, which in turn get reduced by thioredoxin reductase using NADPH

What is Thioredoxin?

a small redox protein that donates electrons to reduce the disulfide in ribonucleotide reductase, allowing the enzyme to continue its catalytic cycle

Describe the role of Thioredoxin in RNR Catalytic Cycle.

1. Reduced thioredoxin (-SH groups) reduces the disulfide bond in RNR → active enzyme restored.

2. Thioredoxin becomes oxidized (forms disulfide).

3. Thioredoxin reductase regenerates reduced thioredoxin using electrons from NADPH

Describe the conversion of dUMP to dTMP.

1. Dephosphorylation: dUDP → dUTP → dUMP (via

dUTPase)

2. Methylation: dUMP → dTMP (Thymidylate synthase)

3. Phosphorylation: dTMP → dTDP → dTTP

Describe the role of Thymidylate synthase (TS) in conversion of dUMP to dTMP.

catalyzes methylation of dUMP at the 5-position to form dTMP

1. The thiolate group of Cys 146 attacks C6 of dUMP to form a covalent adduct.

2. The methyl group donor is N⁵,N¹⁰-methylene tetrahydrofolate (methylene-THF), which is converted to dihydrofolate (DHF) in the process

What is the purpose of Dihydrofolate reductase (DHFR) in conversion of dUMP to dTMP.

regenerates THF from DHF using NADPH

What are the steps of Purine Catabolism?

1. Removal of phosphate group: AMP and GMP are dephosphorylated by nucleotidases to yield nucleosides (adenosine and guanosine).

2. Conversion to free bases: Adenosine is deaminated by adenosine deaminase to inosine. Inosine and guanosine are then cleaved by purine nucleoside phosphorylase to yield hypoxanthine and guanine, respectively.

3. Conversion to xanthine: Guanine is deaminated to xanthine by guanine deaminase. Hypoxanthine is oxidized to xanthine by xanthine oxidase.

4. Formation of uric acid: Xanthine oxidase further oxidizes xanthine to uric acid, which is excreted in humans

What is the final product of purine degradation in humans and primates?

Uric acid excreted in urine; highlevels can lead to gout

What is the final product of purine degradation in most mammals?

Allantoin via uricase conversion of uric acid

What is the final product of purine degradation in amphibians and most fish?

Allantoic acid or Urea

What is the final product of purine degradation in marine invertebrates and bacteria?

Ammonia (NH₃)

Describe gout.

- Cause: Excess uric acid (hyperuricemia) → crystallizes as monosodium urate in joints.

- Symptoms: Painful joint inflammation (often in the big toe).

- Treatment: Allopurinol (xanthine oxidase inhibitor; be recycled into nucleotides, or be excreted safely in urine), Low-purine diet, hydration, uricosuric drugs

What are the general steps of pyrimidine catabolism?

1. Dephosphorylation and deamination: CMP, UMP, dCMP, dUMP are dephosphorylated to nucleosides and then deaminated

- Cytidine is deaminated to uridine.

- Thymidine and uridine are processed similarly.

2. Cleavage to free bases: Nucleosides are cleaved by pyrimidine nucleoside phosphorylase to uracil and thymine.

3. Ring opening and reduction: Uracil is reduced to β-alanine. Thymine is reduced to β-aminoisobutyrate.

4. End products:

- β-alanine → into CoA biosynthesis (major)

- β-aminoisobutyrate → into methylmalonyl-CoA → succinyl-CoA (converted to succinate to OAA)