Purine Synthesis

Nucleotide Biosynthesis Pathways

• Most organisms have two types of biosynthetic pathways for nucleotides:
  • De novo synthesis uses metabolic precursors to create nucleotide bases.
  • Salvage synthesis recycles purines and pyrimidines.
• Nitrogenous bases are not catabolized for energy; only the ribose ring is.

Purine Synthesis Sources

• Nine atoms of the purine ring come from:
  • N-1 from aspartic acid.
  • N-3, N-9 from glutamine.
  • C-4, C-5, N-7 from glycine.
  • C-6 from CO2.
  • C-2, C-8 from THF (one-carbon units).

Purine Synthesis Steps

• Inosine monophosphate (IMP) is the initially synthesized purine derivative.
• Atoms that comprise the purine ring system successively added to ribose-5-phosphate.
• Ribose-5-phosphate (R5P) activated by transfer of PPi from ATP, forming 5-phosphoribosyl-α-pyrophosphate (PRPP).
• PRPP Synthase is limiting substrate in purine biosynthesis.
• The step yielding phosphoribosyl-β-amine is the committed step.
• GMP and AMP are synthesized from IMP in two steps.
• Energy consumption:
  • AMP synthesis requires 8 ATP equivalents.
  • GMP synthesis requires 6 ATP equivalents.

De Novo Purine Synthesis

• The purine ring is built on a ribose-5-P platform.
• Ribose-5-P is activated by ATP-dependent PPi addition to form 5-phosphoribosyl-1-pyrophosphate (PRPP).
• PRPP is made by PRPP synthetase.
• It is the limiting substance for purine synthesis and a branch point in metabolism.
• Gln PRPP amidotransferase catalyzes the committed step.
• Glycine carboxyl condenses with the amine of 5-phosphoribosyl-β-amine in two steps
• Glycine carboxyl activated by phosphorylation from ATP
• The amine then attacks the glycine carboxyl, forming GAR.

Purine Biosynthesis Steps

• Step 4: The formyl group of N10-formyl-THF is transferred to the free amino group of GAR to form α-N-formylglycinamide ribonucleotide.
• Step 5: C-4 carbonyl forms a P-ester with phosphate from ATP, and active NH3 attacks C-4 to form an imine, forming formylglycinamidine ribonucleotide (FGAM).
• Step 6: Similar to step 5, ATP activates the formyl group by phosphorylation, facilitating the attack by N.
• Step 7: Carboxylation
• Step 8: The amino group of aspartate links to amino acid with the carboxyl group
• Step 9: Deprotonation: Deprotonation of Asp-CH2 leads to cleavage to form fumarate.
• Step 10: Another one-carbon addition is made from N-formyl-THF.
• Step 11: Amino group attacks the formyl group to close the second ring.

Tetrahydrofolate

• Folic acid, a B vitamin, is named from folium, Latin for "leaf".
• Folates accept and donate one-carbon units at all oxidation levels of carbon, except CO2.
• Tetrahydrofolate is the active form.

Folate Analogs

• De novo purine biosynthesis depends on N-formyl derivatives of THF at steps 4 and 10.
• Antagonists of folic acid metabolism indirectly inhibit purine formation, nucleic acid synthesis, cell growth, and cell development.

AMP and GMP Synthesis

• GTP provides energy for AMP synthesis.
• ATP provides energy for GMP synthesis.

Regulation of Purine Biosynthesis

• Feedback inhibition and allosteric activation of α-PRPP regulates purine biosynthesis
• Regulation occurs in 2 steps of pathway:
  • Step 1: Feedback inhibition by GDP and ADP.
  • Step 2: Feedback inhibition by GMP, GDP, AMP, ADP, and ATP.
  • Allosteric activation by α-PRPP.

Fate of IMP

• Fate of IMP is determined by GMP and AMP levels.
• Balanced synthesis of both nucleotides occurs, which occurs by:
  • GMP synthesis increasing as ATP increases.
  • AMP synthesis increasing as GTP increases.
• GTP drives AMP biosynthesis, and ATP drives GMP biosynthesis.

Kinases

• NMP → NDP → NTP
• Adenylate kinase: AMP + ATP ↔ 2 ADP
• Guanylate kinase: GMP + ATP ↔ GDP + ADP
• Nucleoside diphosphate kinase: ATP + GDP ↔ ADP + GTP

Purine Salvage

• Nucleic acids (especially RNA) are actively synthesized and degraded, resulting in free adenine, guanine, and hypoxanthine bases.
• Free nitrogenous bases are reconverted to corresponding nucleotides through salvage pathways.
• Phosphoribosyltransferase is involved with:
  • Base + PRPP ↔ NMP + PPi
• PPi hydrolysis drives reaction forward.
  • APRT: adenine phosphoribosyltransferase
  • HGPRT: hypoxanthine-guanine phosphoribosyltransferase

Lesch-Nyhan Syndrome

• HGPRT deficiency causes a build-up of uric acid.
• This can result in hyperuricemia and hyperuricosuria, associated with severe gout and kidney problems.
• Neurological signs consist of poor muscle control and moderate mental retardation.

Purine Degradation

• Nucleic acids degraded by nucleases & phosphodiesterases into nucleotides and further degraded into ribose-1-phosphate and uric acid.
• Dietary nucleic acid is incorporated into cellular nucleic acids.
• De novo synthetic pathways are primary sources for nucleic acid synthesis.
• Ingested bases are excreted as uric acid.
• Degradation to xanthine is the first step. Xanthine is then oxidized to uric acid.

Xanthine Oxidase

• Xanthine oxidase is a mini electron-transport protein.
• It contains e- transfer prosthetic groups and a molybdopterin complex in the active site.
  • The oxidation states include Mo (VI) & Mo (IV).
  • O2 is the final e- acceptor.
• Found in large amounts in liver and milk.

Gout

• Gout is caused by an excess of uric acid.
• Allopurinol, an analog of hypoxanthine, is a potent inhibitor of xanthine oxidase.
• Allopurinol binds tightly to xanthine oxidase, which prevents uric acid formation.
• Hypoxanthine and xanthine do not accumulate to harmful concentrations because they are more soluble and easily excreted.

Purine Nucleotide Cycle

• The purine nucleotide cycle is anaplerotic, replenishing fumarate in the TCA cycle using aspartate.
• This is important in skeletal muscle where many normal anaplerotic enzymes are missing.
• Fate of uric acid: oxidation and excretion
• Gout: a disease characterized by elevated levels of uric acid
• Arthritis: pain and joint inflammation
• Kidney stones: one of the types

Adenosine Deaminase Deficiency

• SCID is a group of related disorders involving diminished immune responses.
• Thirty percent of SCID patients lack the enzyme adenosine deaminase.
• In the absence of ADA, deoxyadenosine is not deaminated to deoxyinosine as normal.

Pyrimidine Synthesis

• Pyrimidine ring coupled to ribose-5-phosphate moiety after synthesis.
• Carbamoyl phosphate synthetase II catalyzes committed step.
  • Rxns 1-3 occur on one polypeptide
  • Rxns 5 & 6 occur on one
• UTP is intermediate

Regulation of Pyrimidine Biosynthesis

• UMP → UTP - Nucleotide Mono / diphosphate kinase
• CTP formed by amination of UTP by CTP synthetase.
• Feedback inhibition differs in bacteria and animals.
• Allosteric regulation also differs:
• ATCase
• CPS-II

Pyrimidine Degradation

• Degradation to β-alanine (C & U), β-aminoisobutyric acid (T), NH4+ & CO2

• In humans, pyrimidines in nucleosides can be used to resynthesize nucleotides. However, free pyrimidines cannot be salvaged, only degraded

Steps in degradation:

• Dephosphorylation

• Deamination

• Glycosidic bond cleavage

  • Uracil / (Thymine)

  • β-alanine / (β-aminoisobutyric acid)

  • Malanoyl CoA / (Methyl malanoyl CoA)

  • Succinyl CoA

Deoxyribonucleotide Synthesis

• Deoxyribonucleotides (dNDPs) synthesized from corresponding ribonucleotides (NDPs) by the reduction at 2’OH position (not de novo from deoxyribose precursors)
• Ribonucleotide Reductase (RNR)
• Heterotetramer (α2β2) form R1 & R2 subunits

Path of electrons (NADPH Thioredoxin reductase thioredoxin ribonucleotide reductase NDP) -Substrates for RNR are CDP, UDP, GDP, ADP; products= dCDP dUDP dGDP dADP

Ribonucleotide Reductase Structure

• Consists of three different nucleotide-binding sites, including active site.
• R2 contains dinuclear Fe(III) complex with a Tyr122 phenoxy radical.
• R1 contains 3 independent allosteric effector sites
• ATP levels regulate the overall rate of DNA synthesis and couples to cell's energy state.

Ribonucleotide Reductase Mechanism

• Free radical-mediated mechanism
• Tyr 122 radical is > 10 Å away from the substrate binding (active) site; another protein radical abstracts proton
• (1) Proton abstraction by RNR (2) & (3) Cleavage of C2’ - OH (4) Reduction of C2 (5) Proton abstraction by dNDP

Regulation of Deoxyribonucleotide Synthesis

• Ribonucleotide reductase activity controlled to make right amounts of dATP, dGTP, dCTP & dTTP for DNA synthesis

• (1) Overall activity site: ATP activates; dATP inactivates

• (2) Substrate-Specificity site: allosteric

• Energy status of cell is robust; [ATP] is high. Make DNA: 1 ATP occupies activity site A: ribonucleotide reductase ON 2 ATP in specificity site S favors CDP or UDP in catalytic site C[dCDP], [dUDP] ↑ 3 dCDP dUMPdTMPdTTP See next slide dUDP

4 dTTP occupies specificity site S, favoring GDP or ADP in catalytic site C GDPdGDPdGTP 5 dGTP occupies specificity site S, favoring ADP in catalytic site C[dADP]↑ 6 dATP replaces ATP in activity site A: ribonucleotide reductase OFF

Thymine Nucelotide Synthesis

• Both dUDP & dCDP can lead to the formation of dUMP (immediate precursor for dTMP synthesis.
• Free thymidine is not synthesized in the cell , and dCMP deaminase is a point of regulation in dNTP biosynthesis
• Thymidylate synthase catalyzes the synthesis of dTMP from dUMP. -Methylates dUMP at 5-position to make dTMP
• methyl donor is N5, N10-methylene-THF 5,10-methylenetetrahydrofolate + dUMP = dihydrofolate + dTMP

Thymidylate Synthase

• Plays a crucial role in the early stages of DNA biosynthesis.
• It is impacted by:
• DNA damage or deletion occur on a daily basis as a result of both endogenous and environmental factors. Such environmental factors include ultraviolet damage and cigarette smoke that contain a variety of carcinogens.
• The enzyme's reactions are necessary for cell growth
• Target for cancer treatment: TS's reaction can be impacted by chemotherapy. -The following cancers are impacted by issues with the TS: - colorectal/pancreatic/ovarian/gastric/breast cancers

Fluoro-Substituted Analogs

• Carbon-fluorine bonds are extremely rare in nature, and fluorine is not common in nature. Moreover, F is electronegative and relatively unreactive.
• Fluoro-substituted agents are potentially useful, and effect fluoro substitution effects thymidylate synthase.
  • The ternary complex is stable and prevents further enzyme turnover.