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.
Lusty
C J
CJ
Department of Molecular Genetics, Public Health Research Institute, New York, NY 10016.
D000143:Acids / Q000494:pharmacology; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D004952:Esters / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D006877:Hydroxamic Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry / Q000378:metabolism; D012694:Serine
D000143:Acids; D004952:Esters; D005971:Glutamates; D006877:Hydroxamic Acids; D005973:Glutamine; C050614:glutamate-gamma-hydroxamic acid; D012694:Serine; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D003545:Cysteine; D011761:Pyrrolidonecarboxylic Acid