Purine Synthesis

Overview of Nucleotide Synthesis and Degradation

• Most organisms synthesize nucleotides through two main pathways:
• De novo synthesis: Uses metabolic precursors.
• Salvage synthesis: Recycles purines and pyrimidines.
• Nitrogenous bases are not catabolized for energy; only the ribose ring undergoes catabolism.

Purine Synthesis

• N-1 atom comes from aspartic acid.
• N-3 and N-9 atoms come from glutamine.
• C-4, C-5, and N-7 atoms come from glycine.
• C-6 atom comes from CO2.
• C-2 and C-8 atoms come from THF (tetrahydrofolate, a one-carbon unit carrier).
• The purine ring is constructed on a ribose-5-phosphate platform.
• Inosine monophosphate (IMP) is the first purine derivative formed, as ribonucleotides not free bases.
• Ring system atoms are added successively to ribose-5-phosphate.
• R5P (ribose-5-phosphate) activation involves PPi transfer from ATP, to carbon-1 generating 5-phosphoribosyl-α-pyrophosphate (PRPP).
• PRPP is the limiting substrate.
• Step 2, the production of phosphoribosyl-β-amine, is the committed step in purine biosynthesis.
• GMP and AMP are synthesized from IMP in two steps.
• AMP synthesis requires 8 ATP equivalents, and GMP requires 6 ATP equivalents.
• First step involves activation of ribose-5-P by ATP-dependent PPi addition, forming 5-phosphoribosyl-1-pyrophosphate (PRPP).
• Glycine carboxyl condenses with amine of 5-phosphoribosyl-β-amine.
• Glycine carboxyl is activated by phosphorylation from ATP.
• Amine attacks the glycine carboxyl.
• The resulting product is glycinamide ribonucleotide (GAR).
• Formyl group of N10-formyl-THF is transferred to GAR's free amino group, producing α-N-formylglycinamide ribonucleotide.
• C-4 carbonyl forms a P-ester with phosphate from ATP.
• Active NH3 attacks C-4 to form formylglycinamidine ribonucleotide (FGAM).
• ATP activates the formyl group through phosphorylation, facilitating attack by N.
• The reaction leads to carboxyaminoimidazole ribonucleotide (CAIR) formation.
• Deprotonation of Asp-CH2 then leads to fumarate cleavage.
• Another one-carbon addition occurs using N-formyl-THF.
• The amino group attacks the formyl group closing the second ring.

Tetrahydrofolate

• Folic acid, a B vitamin in plants, fruits, yeast, and liver, named from folium (Latin for "leaf").
• Folates accept/donate one-carbon units for all carbon oxidation levels except CO2 (biotin is used).
• The active form of folate is tetrahydrofolate

Regulation of Purine Biosynthesis

• Regulation occurs through feedback inhibition and allosteric activation by α-PRPP.
• Step 1, feedback inhibition occurs by GDP and ADP.
• Step 2 feedback inhibition occurs by GMP, GDP, AMP, ADP, and ATP.
• Allosteric activation is achieved via α-PRPP.
• Fate of IMP is determined by GMP and AMP levels, leading to balanced nucleotide synthesis.
• GMP synthesis increases with rising ATP levels.
• AMP synthesis increases with rising GTP levels.
• GTP drives AMP biosynthesis, and ATP drives GMP biosynthesis.
• NMP is converted to NDP and then to NTP.
• Adenylate kinase facilitates AMP + ATP conversion to 2 ADP.
• Guanylate kinase facilitates GMP + ATP conversion to GDP + ADP.
• Nucleoside diphosphate kinase facilitates ATP + GDP conversion to ADP + GTP.

Purine Salvage

• Salvage pathways convert free nitrogenous bases to corresponding nucleotides.
• Phosphoribosyltransferase is involved.
• PPi drives the reaction forward through hydrolysis.
• APRT (Adenine phosphoribosyltransferase) exists.
• HGPRT (Hypoxanthine-guanine phosphoribosyltransferase).

Lesch-Nyhan Syndrome

• It is characterized by a buildup of uric acid.
• It causes hyperuricemia and hyperuricosuria.
• It is associated with severe gout and kidney problems.
• Neurological signs include poor muscle control and mental retardation.

Purine Degradation

• Nucleic acids are degraded by nucleases and phosphodiesterases into nucleotides.
• Further degradation results in ribose-1-phosphate and uric acid.
• Dietary nucleic acids are not readily incorporated into cellular nucleic acids.
• Most purines are from de novo synthetic pathways.
• Ingested bases are excreted as uric acid.

Xanthine Oxidase

• Xanthine oxidase is a mini electron-transport protein.
• Purine bases are degraded to xanthine, which is then oxidized to uric acid.
• Xanthine oxidase contains electron transfer prosthetic groups.
• The active site contains a molybdopterin complex with Mo (VI) and Mo (IV) oxidation states.
• The final electron acceptor is O2.
• It is found in large amounts in liver and milk.

Gout

• Gout stems from excess uric acid.
• Allopurinol, a hypoxanthine analog, is a xanthine oxidase inhibitor.
• Allopurinol binds tightly to xanthine oxidase, preventing the uric acid formation.
• Hypoxanthine and xanthine do not accumulate to harmful levels because they are more soluble.

Purine Nucleotide Cycle & Uric Acid Fate

• The purine nucleotide cycle is anaplerotic.
• It replenishes fumarate in the TCA cycle using aspartate.
• Significant in skeletal muscle deficient in typical anaplerotic enzymes.
• Fate of uric acid is oxidation and excretion.
• Gout is characterized by elevated uric acid levels.
• Symptoms include arthritis, pain, and joint inflammation.
• May result in kidney stones.

Adenosine Deaminase Deficiency

• SCID symptoms relate to diminished immune responses.
• 30% of SCID patients are deficient in adenosine deaminase.
• Deoxyadenosine is not deaminated to deoxyinosine due to this ABSENCE.

Pyrimidine Synthesis

• Pyrimidine ring is assembled before attachment to ribose-5-phosphate.
• Carbamoyl phosphate synthetase II is the committed step.
• Rxns 1-3 are on one polypeptide
• Rxns 5 & 6 are on one
• 2 ATP are required for orotate production from carbamoyl-Asp
• Three ATPs are required to convert dihydroorotate (DHO) to orotate.

UTP to CTP Conversion and Regulation

• UMP is converted to UTP and then to CTP.
• CTP is formed through amination of UTP by CTP synthetase.
• Feedback inhibition differs in bacteria and animals; indicates energy and purine sufficiency.

Pyrimidine Degradation

• Degradation occurs into β-alanine (C & U), β-aminoisobutyric acid (T), NH4+, and CO2.
• Pyrimidines in nucleosides can be reused, but free pyrimidines cannot be salvaged, only degraded.

Ribonucleotide Reductase (RNR)

• dNDPs are synthesized from NDPs.
• They are synthesized by the reduction at the 2’-OH position and not by synthesis from deoxyribose precursors.
• It has a heterotetramer (α2β2) structure, forming R1 and R2 subunits.
• NADPH donates electrons, which go to thioredoxin reductase, thioredoxin, and ribonucleotide reductase.
• Substrates for RNR are CDP, UDP, GDP, and ADP to form their deoxy counterparts

Control of Deoxyribonucleotide Biosynthesis

• RNR activity controls appropriate dNTP balance for DNA synthesis.
• ATP activates the enzyme.
• dATP inactivates the enzyme.
• In specificity site S, ATP favors CDP or UDP in catalytic site C, and dTTP favors GDP or ADP.

Thymine Nucleotides

• Both dUDP and dCDP are usable to make dUMP (the immediate precursor for dTMP synthesis).
• dCMP deaminase marks the second point of regulation in dNTP biosynthesis.
• The thymidylate synthase catalyzes the synthesis of dTMP from dUMP.
• Methylation occurs at the 5-position.
• N5, N10-methylene-THF acts as the methyl donor.
• 5,10-methylenetetrahydrofolate + dUMP = dihydrofolate + dTMP.
• Many cancers utilize inhibitors of TS to treat them