Chemical modification of histidine residue of N-acyl-D-Glutamate amidohydrolase from Pseudomonas sp. 5f-1.

N-Acyl-D-glutamate amidohydrolase (D-AGase) from Pseudomonas sp. 5f-1 was inactivated by diethyl pyrocarbonate (DEP). The chemical modification by DEP showed a difference spectrum at 246 nm due to the N-carbethoxyhistidine residue. Removal of the carbethoxy group from inactivated enzyme with hydroxylamine restored enzyme activity. The inactivation by DEp proceeded with pseudo-first-order kinetics, and was protected in the presence of the substrate N-acetyl-D-glutamate (Glu), or the competitive inhibitor sodium alpha-ketoglutarate (alpha-KGA). These results suggest the presence of an essential histidine residue at or near of the active site of the enzyme.