MDSC 1404 – The Renal System: Purine nucleotide synthesis

Nucleotide Metabolism I & II: Purine Nucleotide Synthesis

Course Objectives

• Discuss purine and pyrimidine nucleotide metabolism.
• Understand the results of defects in the pathways involved, such as:
  • Lesch-Nyhan syndrome
  • Gout
  • Orotic aciduria

Nucleotides

• Common Purine Bases:
  • Adenine (Ade)
  • Guanine (Gua)
• Common Pyrimidine Bases:
  • Cytosine (Cyt)
  • Uracil (Ura)
  • Thymine (Thy)
• Nucleosides: Ribose/deoxyribose linked to a base.
• Nucleotides: Nucleoside phosphate (mono-, di-, tri-).

Biological Importance of Nucleotides

• Energy Sources
  • ATP: Most commonly used energy source.
  • GTP: Involved in protein synthesis and other metabolic processes.
  • UTP: For activating glucose and galactose.
  • CTP: In (phospho)lipid metabolism.
• Coenzymes: e.g., AMP is part of NAD+ and coenzyme A.
• Subunits of Nucleic Acids: DNA, RNA.

Nucleotide Synthesis

• Two Types of Pathways:
  • De novo synthesis: Starts with metabolic precursors (amino acids, ribose 5-phosphate, $CO<em>2$, $NH</em>3$).
  • Salvage pathways: Recycles free bases and nucleosides released by nucleic acid breakdown (turnover).
• Free bases are not intermediates in these pathways.
• Catalytic enzymes are large, multienzyme complexes.

Purines - De novo Synthesis

• Liver: Major site of purine synthesis (de novo and salvage).
• Two Parent Purine Nucleotides:
  • Adenosine 5'-monophosphate (AMP; adenylate).
  • Guanosine 5'-monophosphate (GMP; guanylate).
• Begins with the synthesis of 5-phosphoribosyl 1-pyrophosphate (PRPP).
• PRPP is also common to pyrimidine synthesis.

De novo Synthesis - Step 1

• Synthesis of PRPP from ribose-5-phosphate by the enzyme PRPP synthase, using ATP. The chemical reaction is as follows:
  • $ATP + Ribose-5-phosphate \rightarrow PRPP$

De novo Synthesis - Step 2

• Attachment of a nitrogen atom (from glutamine) to PRPP, forming highly unstable 5-phosphoribosylamine.
• Glutamine is converted to glutamate by glutamyl amidotransferase.

De novo Synthesis - Step 3

• Glycinamide R-5-P is formed by the addition of 3 atoms from glycine, via phosphoribosylglycinamide synthetase.

De novo Synthesis - Step 4

• The added glycine $NH_2$ group is then formylated by N10-formyltetrahydrofolate to form formylglycinamide R-5-P.

De novo Synthesis - Step 5

• Attachment of a second nitrogen atom (also from glutamine), forming formylglycinamidine R-5-P.

De novo Synthesis - Step 6

• Dehydration and ring closure leads to the formation of a 5-membered imidazole ring, resulting in 5-aminoimidazole ribonucleotide (AIR).

De novo Synthesis - Step 7

• Carboxylation by AIR carboxylase forms aminoimidazole carboxylate R-5-P (or AIR carboxylate).

De novo Synthesis - Steps 8 & 9

• Aspartate donates its amino group in two steps:
  • Aspartate attaches to AIR carboxylate via an amide bond.
  • The carbon skeleton is then eliminated as fumarate.

De novo Synthesis - Step 10

• The final carbon is contributed as a formyl group from N10-formyltetrahydrofolate.

De novo Synthesis - Step 11

• Second ring closure leads to the first “complete” intermediate, inosine monophosphate or inosinate (IMP).

De novo Synthesis - IMP Conversion

• IMP can be converted to either adenylate (AMP) or guanylate (GMP).
• IMP → AMP:
  • Insertion of $–NH_2$ from aspartate to form adenylosuccinate.
  • The enzyme involved is adenylosuccinate synthase, and GTP hydrolysis is used to provide the needed energy.
  • Removal of fumarate from the attached aspartate backbone by adenylosuccinate lyase → AMP.
• IMP → GMP:
  • Inosinate is first oxidized at C-2 by IMP dehydrogenase (uses $NAD^+$) to form xanthylate (XMP).
  • Next, $–NH_2$ is added to XMP from glutamine by XMP-glutamine amidotransferase → GMP.

De novo Synthesis - Nucleoside Monophosphates Conversion

• Nucleoside monophosphates (NMPs) are then converted to diphosphates (NDPs).
• Catalyzed by base-specific nucleoside monophosphate kinases.
• Usually, phosphate is obtained from ATP (abundant).
  • $AMP + ATP \rightleftharpoons 2 ADP$ (adenylate kinase)
  • $GMP + ATP \rightleftharpoons GDP + ADP$ (guanylate kinase).

De novo Synthesis - Nucleoside Diphosphates Conversion

• Finally, NDPs are reversibly converted to nucleotides (NTPs) by nucleoside diphosphate kinase.
• This enzyme has a broad specificity (indiscriminate with respect to base).
  • $GDP + ATP \rightleftharpoons GTP + ADP$

De novo Synthesis Regulation

• The rate of de novo synthesis depends on [PRPP], which is dependent on:
  • Rate of usage, degradation, and synthesis.
  • [ribose-5-P].
  • PRPP synthase.
• PRPP synthase:
  • A major regulatory control point.
  • Negative feedback by AMP, ADP, GMP, and GDP.

De novo Synthesis Regulation - Glutamine-PRPP amidotransferase

• Also regulated at the conversion of PRPP to 5-phospho-ribosylamine.
  • Catalyzed by glutamine-PRPP amidotransferase.
  • The enzyme is allosterically inhibited by AMP, GMP, and IMP.
• AMP and GMP regulate their conversion from IMP:
  • AMP feedback-inhibits adenylosuccinate synthase.
  • GMP feedback-inhibits IMP dehydrogenase.

De novo Synthesis Regulation - Cross-regulation

• Cross-regulation decreases the synthesis of one purine when the other is at low concentrations:
  • AMP synthesis (IMP → adenylosuccinate) requires GTP.
  • GMP synthesis (XMP → GMP) requires ATP.

Purines - Salvage Reactions

• Require less energy than de novo synthesis.
• Utilizes freed purines, nucleosides, and deoxynucleosides → purine mononucleotides.
• Recycles molecules from regular nucleic acid turnover.
• Basic mechanism – phosphoribosylation of a free purine using PRPP.

Salvage Reactions - Bases Involved

• Three bases involved in purine salvage reactions – adenine, guanine, and hypoxanthine (deaminated adenine).
• In irreversible reactions:
  • Adenine → AMP (adenine phosphoribosyltransferase, APRT).
  • Hypoxanthine → IMP (hypoxanthine-guanine phosphoribosyltransferase or HGPRT).
  • Guanine → GMP (also HGPRT).

Deoxynucleotides

• Building blocks of DNA are derived from the corresponding ribonucleotides (purine or pyrimidine).
• Direct reduction at the 2'-carbon atom of the D-ribose → 2'-deoxy derivative.
  • Reduction is at a nonactivated carbon (unusual).
  • Catalyzed by ribonucleotide reductase complex.
  • Requires thioredoxin, thioredoxin reductase, and NADPH.

Purine Nucleotide Degradation

• First purine nucleotides lose their phosphate via a 5'-nucleotidase:
  • Adenylate: AMP → adenosine.
  • Guanylate: GMP → guanosine.
• Next steps:
  • Deamination by base-specific deaminases.
  • Removal of ribose 5-phosphate by nucleosidase (a.k.a. purine nucleoside phosphorylase).

Purine Nucleotide Degradation - Guanosine & Adenosine

• For guanosine:
  • Sugar removal: guanosine → guanine (nucleosidase).
  • Deamination: guanine → xanthine (guanosine deaminase).
• For adenosine:
  • Deamination: adenosine → inosine (adenosine deaminase).
  • Sugar removal: inosine → hypoxanthine (nucleosidase).
  • Oxidation: hypoxanthine → xanthine (xanthine oxidase).

Purine Nucleotide Degradation - Xanthine Oxidase

• Xanthine oxidase then oxidizes xanthine.
  • It’s a flavoenzyme.
  • Prosthetic group - molybdenum atom and 4 Fe-S centers.
  • Molecular $O_2$ is the electron acceptor in the reaction.
  • The end product in primates, birds, and some animals is uric acid.
• Most mammals and other vertebrates convert uric acid → allantoin by urate oxidase.

Disorders of Purine Metabolism

• Gout:
  • Joints become inflamed, painful, and arthritic due to elevated concentrations of uric acid in the blood and tissues.
  • Leads to abnormal deposition of sodium urate crystals.
  • Kidneys are especially affected with deposits in tubules.
  • The disease occurs predominantly in males.

Gout - Causes

• Most cases are linked to underexcretion of urate (i.e., abnormalities in renal handling).
• Genetic defects in PRPP synthase are also a cause:
  • e.g., abnormally high Vmax or feedback-inhibition resistance.
  • The defective enzyme causes overproduction of purines.
  • Excess purines → excess urate.
  • Past solubility limit, urate crystallizes in tissue and joints (gouty arthritis).

Gout - Treatment

• Treatment involves diets low in nucleic acids (e.g., no liver) and drug therapy.
• Allopurinol is a drug that inhibits xanthine oxidase.
  • Xanthine oxidase converts allopurinol → oxypurinol.
  • Oxypurinol remains bound to the enzyme's active site.
  • Hypoxanthine and xanthine (intermediate products from AMP, GMP) remain.
  • These are more soluble than uric acid – less crystal deposits.

Lesch-Nyhan Syndrome

• A case of hyperuricemia (uric acid above the threshold).
• Inherited disorder with virtually complete deficiency of HGPRT.
  • Inability to salvage hypoxanthine or guanine.
  • Less PRPP usage, decreased IMP & GMP → increased de novo synthesis (energy consuming).
  • Without salvaging → increased degradation of purines → excessive uric acid and eventual gout-like damage.

Lesch-Nyhan Syndrome - Neurologic Symptoms

• Neurologic symptoms include mental retardation, involuntary movement, and self-mutilation.
• The brain is very dependent on salvage pathways:
  • It cannot perform de novo synthesis.
  • A possible reason for neurologic symptoms is inefficient nucleic acid recycling due to need for cell growth.

Other Enzyme Deficiencies - Hypouricemia

• Hypouricemia:
  • Uric acid is below the normal threshold value.
  • Xanthine oxidase deficiency → genetic defect (xanthinuria) or severe liver damage.
  • Can lead to increased hypoxanthine and xanthine excretion.
  • Xanthine kidney stones (xanthine lithiasis) and renal failure are possible as the condition progresses.
  • Note that drugs like allopurinol can cause this too!

Other Enzyme Deficiencies - Adenosine Deaminase (ADA) Deficiency

• Leads to severe combined immunodeficiency disease (SCID).
• Also known as Bubble Boy disease.

Other Enzyme Deficiencies - ADA Deficiency cont.

• T lymphocytes and B lymphocytes do not develop properly.
• Lack of ADA → 100-fold increase in cellular [dATP].
• High [dATP] inhibits ribonucleotide reductase → a general deficiency of other dNTPs in T cells.
• The cause in B cells remains unclear.

Other Enzyme Deficiencies - Purine Nucleoside Phosphorylase Deficiency

• Also associated with immune system impairment.
• Leads to dATP and dGTP accumulation that inhibit ribonucleotide reductase (as in ADA deficiency).
• Appears to affect only T cells (severe deficiency).
• B cells appear to develop normally.