Detection of an enzyme bound gamma-glutamyl acyl ester of carbamyl phosphate synthetase of Escherichia coli.

E. coli carbamyl phosphate synthetase binds 0.2-0.4 mol equivalents of glutamine in an acid resistant form. The bound material is quantitatively released as glutamate by weak base hydrolysis and as a mixture of 12% glutamate, 10% gamma-glutamylhydroxamate, and 70% pyrrollidonecarboxylic acid by hydrolysis with hydroxylamine. These results provide direct evidence for a gamma-glutamyl acyl ester on the enzyme. The absence of the acyl ester in a mutant carbamyl phosphate synthetase with a Cys269-->Ser substitution in the glutaminase subunit further suggests that the covalent intermediate is a thioester of Cys269. Under equilibrium conditions, the Cys269Ser mutant enzyme binds glutamine with a Kd of 7 +/- 1 microM, indicating that Cys269 is essential for acyl ester formation but not for binding of glutamine.