Synthesis of Pyrimidine Nucleotides Dr. Shumaila Asim Lecture # 3

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

De novo synthesis of pyrimidine nucleotides - Overview De novo synthesis of pyrimidine starts with simple organic materials, contains 7 reactions, and produces the first pyrimidine base orotate and then UMP, UDP, UTP and CTP.

1: Formation of carbamoyl phosphate Starting materials for pyrimidine biosynthesis includes bicarbonate, amine from glutamine, and phosphate from ATP to form carbamoyl phosphate. The reaction is catalyzed by the cytosolic enzyme carbamoyl phosphate synthetase II (CPS II) and is the committed step in the mammalian pathway.

2 and 3: Formation of carbamoyl aspartate and dihyroorotate Carbamoyl phosphate condenses with aspartate to form carbamoyl aspartate. Then, dihydroorotate, a closed 6-membered ring structure is formed by the loss of H2O.

4 to 6: Formation of UMP, UDP, and UTP NAD+ NADH+/H+ PRPP Ppi orotidine mono-phosphate (OMP) dihydroorotate Orotic acid CO2 ATP ADP ATP ADP OMP UMP UDP UTP Dihydroorotate is oxidized to orotic acid, which in turn combined with PRPP to form orotidine mono-phosphate (OMP). OMP is decarboxylated to form uridine monophosphate (UMP) UDP is formed from UMP by phosphorylation, catalyzed by a specific nucleoside monophosphate kinase, UMP kinase UDP is converted to UTP by another phosphorylation step catalyzed by nucleoside diphosphate kinase, an enzyme with broad specificity for nucleoside diphosphates

7. Formation of CTP UTP CTP CTP is formed from UTP An amino group from glutamine replaces the keto group in uridine triphosphate (UTP) to form cytidine triphosphate (CTP). Note that: UMP does not form CMP, nor does UDP form CDP. UMP and UDP have to be converted to UTP first. glutamine glutamate UTP CTP ATP ADP + Pi

Synthesis of UTP from UMP UMP is converted to UTP in two step kinase reaction with 2 molecules of ATP        UMP  +  ATP   ⇌   UDP  +  ADP UDP  +  ATP    ⇌  UTP  +  ADP

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

Formation of deoxyTMP dTMP dTTP N5,N10-methylene-tetrahydrofolic Acid dTDP dTTP dUMP dUDP dCMP dCDP N5,N10-methylene-tetrahydrofolic Acid ATP ADP dTMP synthetase UDP

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