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.
Wakayama
M
M
Department of Applied Chemistry, Faculty of Engineering, Oita University, Japan.
Tsutsumi
T
T
Yada
H
H
Sakai
K
K
Moriguchi
M
M
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001665:Binding Sites; D002264:Carboxylic Acids / Q000494:pharmacology; D002384:Catalysis; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011549:Pseudomonas / Q000201:enzymology*
D002264:Carboxylic Acids; D004791:Enzyme Inhibitors; D006639:Histidine; D000581:Amidohydrolases; C070988:N-acyl-D-glutamate deacylase; D004047:Diethyl Pyrocarbonate