Chemical modification of salicylate hydroxylase

Salicylate hydroxylase from Pseudomonas cepacia was found to be inactivated by phenyl glyoxal, a reagent specific for arginyl residues. The reaction was found to display pseudo-first order kinetics but was not retarded by the addition of any of the substrates before initiating the inactivation. This suggests that the modification does not take place at the enzyme active site. Salicylate hydroxylase can also be effectively inactivated by di ethyl pyrocarbonate, a reagent highly reactive with histidyl residues. The enzyme inactivation was retarded by the addition of salicylate but not NADH, suggesting the modification was limited to the salicylate substrate site. However, the inactivation of enzyme with an excess amount of di ethyl pyrocarbonate did not display pseudo-first order kinetics, due to the appreciable autodecay of the reagent in an aqueous medium. A new method is described for the determination of both the pseudo-first order rate constant and the second order rate constant for the enzyme inactivation by a reagent such as di ethyl pyrocarbonate, which undergoes exponential decays. The validity of this method has been demonstrated by the successful application to the analysis of results of salicylate hydroxylase and bacterial luciferase inactivations by diethyl pyrocarbonate. The effects of temperature, pH and concentration of phosphate buffer on the stability of this reagent have been examined.