Carbamoyl phosphate synthetase is an enzyme in pyrimidine biosynthesis that catalyzes the reaction below.

Glutamine + ATP + HCO3^- <=> Glutamate + Carbamoyl Phosphate + ADP

The enzyme is unusual in assimilating bicarbonate without using biotin as a cofactor.

Carbamoyl phosphate synthetase is the primary regulatory enzyme in pyrimidine biosynthesis of eukaryotes (Figure 11.35). In mammals, carbamoyl phosphate synthetase II is inhibited by the following pyrimidine-containing compounds: UDP, UTP, CTP, dUDP, and UDP-glucose.

By contrast, aspartate transcarbamoylase (ATCase) is the major control point in the pyrimidine pathway synthesis in bacteria.