Synthesis of Pyrimidine Nucleotides Dr. Shumaila Asim Lecture # 5

De-novo synthesis of Pyrimidines (Uracil, Thymine & Cytosine) Biosynthesis of pyrimidines is simple than that of purines. Unlike purine synthesis, pyrimidines are synthesized as bases and latter it is added to ribose sugar, i.e., the ring is completed before being it is linked to ribose-5- phosphate.

Synthesis of Pyrimidine Nucleotides Precursors Amino acids (Aspartate, Glutamine) CO2 (from HCO3-) Ribose-5-phosphate (HMP shunt & nucleosides) Enzymes Coenzymes and Cofactors NAD+, NADPH, Mg++ ATPs Energy PRPP formation

Sources of Atoms of Pyrimidine Nucleus N1, C6, C5 and C4 are derived from aspartate N3 is derived from glutamine C2 is derived from HCO3- (bicarbonate)

Step-1: Synthesis of carbamoyl phosphate: With the hydrolysis of two ATP molecules, bicarbonate and amide nitrogen of glutamine combine to form carbamoyl phosphate in the presence of enzyme carbamoylphosphate synthetase II. Step-2: Synthesis of carbamoyl aspartate: Carbamoyl phosphate reacts with aspartate to yield carbamoyl aspartate catalyzed by the enzyme aspartate transcarbamoylase (ATCase). Step-3: Ring closure & dihydroorotate formation: By the elimination (condensation reaction) of one molecule of water, the carbamoyl aspartate is converted to a ring compound – dihydroorotate catalyzed by dihydroorotase enzyme.

Step-4: Oxidation of dihydroorotate: Dihydroorotate is dehydrogenated to form orotate with the enzyme dihydroorotate dehydrogenase. Step-5: Acquisition of the ribose phosphate moiety: Orotate reacts with PRPP to produce orotidine-5′- monophosphate (OMP) with the enzyme orotate phosphoribosyl transferase. The anomeric form of pyrimidine nucleotides is fixed in in the β- configuration. Step-6: Decarboxylation to form UMP:OMP undergoes decarboxylation with assistance of enzyme OMP decarboxylase (ODCase) to form uridine monophosphate (UMP).

Synthesis of CTP CTP is synthesized by the amination of UTP by the enzyme CTP synthase. In animals amino group is donated by glutamine whereas in bacteria, the amino group is donated directly by ammonia

Synthesis of Thymine (5-methyluracil) as deoxyTTP: Thymine, which is present in DNA and not in RNA, is a methylated uracil residue. Thymine in the cell is synthesized as dTTP from dUMP by methylation in four steps. Step-1: dUTP is hydrolyzed to dUMP and PPi by the enzyme dUTP diphosphohydrolase (dUTPase) Step-2: dUMP is then methylated to form dTMP Step-3 & 4: dTMP is then phosphorylated with ATP in two rounds to form dTTP

Regulatory Control of Pyrimidine Synthesis Regulation at carbamoyl phosphate synthetase II UDP and UTP inhibit enzyme; ATP and PRPP activate it UMP and CMP competitively inhibit OMP Decarboxylase *Purine synthesis inhibited by ADP and GDP at ribose phosphate pyrophosphokinase step, controlling level of PRPP  also regulates pyrimidines

Synthesis Of Pyrimidine Nucleotides By Salvage Pathway

salvage of Uracil The salvage of pyrimidine bases has less clinical significance than that of the purines, owing to the solubility of the by-products of pyrimidine catabolism. Uracil can be salvaged to form UMP through the action of uridine phosphorylase and uridine kinase, as indicated: uracil + ribose-1-phosphate <——> uridine + Pi uridine + ATP ——> UMP + ADP

salvage of Deoxyuridine Deoxyuridine is also a substrate for uridine phosphorylase. Formation of dTMP, by salvage of dTMP requires thymine phosphorylase and the previously encountered thymidine kinase: thymine + deoxyribose-1-phosphate <——> thymidine + Pi thymidine + ATP ——> dTMP + ADP

salvage of deoxycytidine The salvage of deoxycytidine is catalyzed by deoxycytidine kinase: deoxycytidine + ATP <——> dCMP + ADP Deoxyadenosine and deoxyguanosine are also substrates for deoxycytidine kinase, although the Km for these substrates is much higher than for deoxycytidine.

salvage of thymidine The salvage pathway to dTTP synthesis involves the enzyme thymidine kinase which can use either thymidine or deoxyuridine as substrate: thymidine + ATP <——> TMP + ADP deoxyuridine + ATP <——> dUMP + ADP The activity of thymidine kinase (one of the various deoxyribonucleotide kinases) is unique in that it fluctuates with the cell cycle, rising to peak activity during the phase of DNA synthesis; it is inhibited by dTTP.

Synthesis of deoxyribonucleotides

Summary of pyrimidine biosynthesis UMP