Nucleotide Synthesis & Degradation

Purine Synthesis yields Inosine Monophosphate (IMP) in an 11 step pathway:

• Activation of ribose-5-phosphate- product of pentose phosphate pathway, Ribose phosphate pyrophosphokinase activates the ribose by reacting it with ATP to form 5-Phosphoribosyl-alpha-pyrophosphate (PRPP)
• Acqusition of Purine atom N9- amidophosphoribosyl transferase catalyses the displacement of PRPP's pyrophosphate group by glutamine's amide nitrogen. The reaction occurs with inversion of the alpha configuration at C1 of PRPP forming Beta-5-Phosphoribosylamine.-Flux controlling step.
• Acquisition of purine atoms C4,C5 and N7. Glycine's carboxyl group forms an amide with the amino group of phosphoribosylamine yielding Glycinamide ribotide (GAR)- this reaction is reversible
• Acqusition of Purine atom C8- GAR's free alpha amino group is formylated to yield formylglycinamide ribotide (FGAR) this is catalysed by GAR transformylase. The formyl donor is N¹⁰ - formyl-THF.
• Acqusition of purine atom N3. The amide amino group of a second glutamine is transferred to the growing purine ring to form Formylglycinamidine ribotide (FGAM) this reaction is driven by the coupled hydrolysis of ATP to ADP and Pi.

• Formation of the purine imidazole ring- The ring is closed in an ATP requiring intramolecular condensation that yields 5-aminoimidazole ribotide (AIR). The aromatisation of the imidazole ring is facillitated by the tautomeric shift of the reactant from its imine to its enamine form.
• Acqusition of C6- Purine C6 is introduced as HCO3^-  in a reaction catalysed by AIR carboxylase that yields carboxyaminoimidazole ribotide (CAIR).
• Acqusition of N1. Contributed by aspartate in an amide-forming condensation reaction yielding 5-aminoimidazole-4-N-Succinylocarboxamide ribotide (SACAIR) this reaction is driven by the hydrolysis of ATP.
• Elimination of fumarate. SACAIR is cleaved with the release of fumarate yielding 5-aminoimidazole-4-carboxamide ribotide (AICAR)
• Acquisition of C2. The final purine ring atom is acquired through formylation by N¹⁰ -formyl-THF yielding 5-formaminoimidazole-4-carboxamide ribotide (FAICAR) This reaction is inhibited by sulfonamides.
• Cyclisation to form IMP- Ring closure to form IMP occurs through the elimmination of water. Unlike reaction 6 in the cyclisation that forms the imidazole ring this reaction does not require ATP hydrolysis.

Order of Formation-R5P,PRPP,PRA,GAR,FGAR,FGAM,AIR,CAIR,SAICAR,AICAR,FAICAR,IMP

IMP is rapidly converted into AMP in a 2 reaction pathway which replaces a 6-keto group with an amino group.

• Aspartate's amino group is linked to IMP in a reaction powered by the hydrolysis of GTP to GDP and Pi to yield adenylosuccinate.
• Adenylosuccinatelyase eliminates fumarate from adenylosccinate to form AMP.

GMP is also synthesised from IMP

• IMP is dehydrogenated via the reduction of NAD+ to form Xanthosine monophosphate XMP which is converted to GMP by the transfer of the glutamine amide nitrogen.
• This is driven by the hydrolysis of ATP to AMP + PPi

Adenylate kinase catalyses the phosphorylation of AMP to ADP

GDP + ATP ⇌ GTP + ADP

• The IMP pathway is regulated at the first two reactions, Ribose phosphate pyrophosphate kinase- the enzyme that catalyses reaction 1 of the pathway is inhibited by both ADP and GDP.

Amidophosphoribosyl transferase is allosterically stimulated by PRPP (feedforward activation)

• AMP and GMP are each competitive inhibitors of IMP in their synthesis. GTP powers the synthesis of AMP and ATP powers the synthesis of GMP therefore balancing production.
• Free purines are reconverted into their corresponding nucleotides through salavge pathways. These occur through 2 different enzymes.

Adenine phosphoribosyltransferase (APRT) mediates AMP formation using PRPP

Adenine + PRPP ⇌ AMP + PPi

Hypoxanthine-guanine phosphoribosltransferase (HGPRT) catalyses both Hypoxanthine and guanine.

Hypoxanthine + PRPP ⇌ IMP + PPi

Guanine + PRPP ⇌ GMP + PPi

Lesch-Nyhan Syndrome-HGPRT deficiency-sex linked defect- mental retardation-excessive uric acid production.

Atoms N1,C4,C5 and C6 of the pyrimidine ring are derived from aspartic acid, C2 arises from HCO3- and N3 is contributed by glutamine.

Uridine monophosphate (UMP) is synthesised in a six step reaction pathway:

• Synthesis of carbamoyl phosphate. From HCO3- and the amide nitrogen of glutamine by the cytosolic enzyme carbamoyl phosphate synthetase 2. This reaction consumes 2 ATP- one provides a phosphate group the other provides energy.
• Synthesis of Carbamoyl aspartate. Condensation of carbamoyl phosphate with aspartate to form carbamoyl aspartate is catalysed by aspartate transcarbamoylase ATCase.
• Ring Closure to form Dihydroorotate. Dihydroorotate is formed in an intramolecular condensation catalysed by dihydroortase.
• Dihydroortate is oxidised to orotate by dihydroorotate dehydrogenase.(Inhibition of this enzyme in T Lymphocytes blocks pyrimidine synthesis reducing arthritis)
• Orotate reacts with PRPP to yield Orotidine-5'-monophosphate (OMP) in a reaction catalysed by orotate phosphoribosyl transferase this enzyme also salvages other pyrimidine bases converting them to their corresponding nucleotides.
• Decarboxylation of OMP by OMP decarboxylase (ODCase) to form UMP.

UMP + ATP ⇌ UDP + ADP

UDP + ATP ⇌ UTP + ADP

CTP is formed by the amination of UTP by CTP synthetase. In animals the amino group is donated by glutamine , in bacteria it is supplied directly by ammonia.

In bacteria the pyrimidine biosynthetic pathway is regulated by reaction 2 (the ATCase reaction). In animals pyrimidne biosynethesis is controlled by the activity of carbamoyl phosphate synthetase II which is inhibited by UDP and UTP and actrivated by ATP and PRPP.

Ribonucleotide reductase converts ribonucleotides to deoxyribonucleotides by the reduction of their C2 position using a free radical mechanism. DNA contains 2'-deoxyribose residues rather than ribose and thymine rather than uracil.

Ribonucleotide reductase contains a binuclear Fe (III) prosthetic group, a tyrosyl radical and 3 redox active sulfhydryl groups.  It is regulated by allosteric effectors which ensure deoxynucleotides are syntheisised in the correct amounts needed for DNA synthesis.

Thymidylate synthase transfers a methyl group to dUMP to form thymine.Thymidylate (dTMP) is synthesised from dUMP by thymidylate synthase.

Tetrahydrofolate (THF) is regnerated in two reactions:

• DHF is reduced to THF by NADPH as catalysed by dihydrofolate reductase (DHFR)
• Serine hydroxymethyltransfertase transfers the hydroxymethyl group of serine to THF yielding N5,N10-methylene-THF and glycine.

Purines are broken down to uric acid which may be further catabolised for excretion.

Pyrimidines are converted to CoA derivatives for catabolism.

Nucleotides cannot pass through membranes are hydrolysed to nucleosides which are then directly absorbed by the intestinal mucosa or further degraded to ribose or free bases by nucleosidases and nucleoside posphorylases.

Nucleoside + H₂O ➔ Base + Ribose    Nucleoside + P_i ➔ Base + ribose-1-Phosphate

Purine Catabolism yields uric acid   

ribose-1-phosphate is the product of a reaction catalysed by purine nucleoside phosphorylase.The purine nucleotide cycle generates fumarate. The synthesis and degradation of AMP in the purine nucleotide cycle yield the citric acid cycle intermediate fumarate in muscles.Xanthine oxidase converts hypoxanthine to xanthine and then to uric acid .

In primates, birds, reptiles and insects the final prodcut of purine degradation is uric acid which is excreted other organisms degrade urate further.

Pyrimidines are broken down to Malonyl-CoA and Methylmalonyl-CoA

The end products of pyrimidine catabolism are Beta-alanine and Beta-aminoisobutyrate they are amino acids and metabolised as such. They are converted through transamination and activation reactions to malonyl-CoA- a precursor of fatty acid synthesis and methylmalonyl-CoA- converted to the citric acid cycle intermediate succinyl-CoA.-Catabolism of pyrimidine nucleotides contributes to the energy metabolism of the cell.