Carbamoyl phosphate synthetase (glutamine-hydrolysing) (EC 6.3.5.5) is an enzyme that catalyzes the reactions that produce carbamoyl phosphate in the cytosol (as opposed to type I, which functions in the mitochondria). Its systemic name is hydrogen-carbonate:L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating).^{[1][2][3][4][5][6][7][8]}

In pyrimidine biosynthesis, it serves as the rate-limiting enzyme and catalyzes the following reaction:

2 ATP + L-glutamine + HCO₃⁻ + H₂O ${\displaystyle \rightleftharpoons }$ 2 ADP + phosphate + L-glutamate + carbamoyl phosphate (overall reaction)

(1a) L-glutamine + H₂O ${\displaystyle \rightleftharpoons }$ L-glutamate + NH₃

(1b) 2 ATP + HCO₃⁻ + NH₃ ${\displaystyle \rightleftharpoons }$ 2 ADP + phosphate + carbamoyl phosphate

It is activated by ATP and PRPP^[9] and it is inhibited by UTP (Uridine triphosphate)^[10] Neither CPSI nor CPSII require biotin as a coenzyme, as seen with most carboxylation reactions.

It is one of the three enzyme functions coded by the CAD gene. It is classified under EC 6.3.5.5.

• Carbamoyl phosphate synthetase I
• Carbamoyl phosphate synthetase III