Detection of an enzyme bound γ-glutamyl acyl ester of carbamyl phosphate synthetase ofEscherichia coli
- 14 December 1992
- journal article
- Published by Wiley in FEBS Letters
- Vol. 314 (2) , 135-138
- https://doi.org/10.1016/0014-5793(92)80959-k
Abstract
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% γ-glutamylhydroxamate, and 70% pyrrollidonecarboxylic acid by hydrolysis with hydroxylamine. These results provide direct evidence for a γ-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 K d of 7 ± 1 μM, indicating that Cys269 is essential for acyl ester formation but not for binding of glutamine.Keywords
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