Detection of an enzyme bound γ‐glutamyl acyl ester of carbamyl phosphate synthetase of Escherichia coli

[摘要]

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.

[学科分类] 生物化学/生物物理

[关键词] Carbamyl phosphate synthetase;Thioester intermediate;Glutamine amidotransferase;Glutamine hydrolysis;HEPES;4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;CAD;the multifunctional pyrimidine-specific enzyme comprising glutamine-dependent carbamyl phosphate synthetase;aspartate transcarbamylase and dihydroorotase.