Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate.
Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.
Kim
Y S
YS
Department of Biochemistry, College of Science, Yonsei University, Seoul, Korea.
Park
C
C
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000084:Acetate Kinase / Q000037:antagonists & inhibitors*; D000150:Acinetobacter / Q000201:enzymology*; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005561:Formates / Q000494:pharmacology*; D006639:Histidine / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010658:Phenylglyoxal / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*
D005561:Formates; D006639:Histidine; D002731:4-Chloromercuribenzenesulfonate; D004228:Dithionitrobenzoic Acid; D010770:Phosphotransferases; D000084:Acetate Kinase; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal; D005033:Ethylmaleimide