Kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase

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Abstract
The kinetic mechanism of E. coli carbamoyl-phosphate synthetase was determined at pH 7.5, 25.degree. C, With NH3 as the N source, the initial velocity and product inhibition patterns are consistent with the ordered addition of MgATP, HCO3- and NH3. Phosphate is then released and the 2nd MgATP adds to the enzyme, which is followed by the ordered release of MgADP, carbamoyl phosphate and MgADP. With glutamine as the NH3 donor, the patterns are consistent with a 2-site mechanism in which glutamine binds randomly to the small MW subunit producing glutamate and NH3. Glutamate is released and the NH3 is transferred to the larger subunit. Carbamoyl-phosphate synthetase also required a free divalent cation for full activity.