Sultone-Assisted Inactivation of α-Chymotrypsin by Hydroxylamine

Abstract

Sultone and phenylmethanesulfonyl fluoride (PMSF) form sulfonyl-enzymes with serine proteases in a similar fashion. However, the sultone-derived sulfonyl-enzyme is readily hydrolyzed to regenerate active enzyme, while that derived from PMSF is not. Thus, sultone is regarded as a substrate rather than an inhibitor of serine proteases. It has been assumed that the sulfonyl-enzyme derived from sultone is an analog of an acyl-enzyme in a normal reaction. Therefore, this sulfonyl-enzyme is presumably hydrolyzed by a mechanism similar to that of deacylation in the normal enzymatic reaction. We found that in the presence of a nucleophile such as hydroxylamine, sultone induced inactivation of α-chymotrypsin in a time-dependent manner. This inactivation was not seen when α-chymotrypsin was incubated with sultone or hydroxylamine alone. The inactivation presumably results from attack of hydroxylamine at the β-carbon of serine in the sulfonyl-enzyme. If so, the sulfonyl-enzyme is probably hydrolyzed by a mechanism similar to that of tosylate solvolysis rather than the previously assumed mechanism of acyl-enzyme hydrolysis. 1H-NMR and kinetic approaches were used to determine the p K a value (≅6.7) of the active-site histidine in the sulfonyl-enzyme complex. This information together with previously reported pH dependence of sulfonyl-enzyme formation suggests that the o-hydroxyl group on the aromatic ring of the sulfonyl-chymotrypsin is in the phenol form rather than the phenolate form. This o-hydroxyl group probably functions as a general acid to facilitate hydrolysis of the sulfonyl-enzyme. In the absence of this o-hydroxyl group, such as in the case of PMSF-derived sulfonyl-enzyme, hydrolysis of the sulfonyl-enzyme will be significantly slower due to the lack of the general acid catalysis.