1. Synthesis of Pyrimidine Nucleotides
Dr. Shumaila Asim
Lecture # 3

2. 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.

3. 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

5. 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.

7. 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.

9. 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.

10. 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

11. 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

13. 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

14. 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

15. 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

17. 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