MODIFICATION OF ALPHA-CHYMOTRYPSIN BY BROMOMETHYL NAPHTHYL KETONES AND BEHAVIOR OF REPORTER GROUPS IN THE ACTIVE-SITE

The reaction of alpha-chymotrypsin (ChT) with bromomethyl 1-naphthyl ketone (1-BNK) or with bromomethyl 2-naphthyl ketone (2-BNK) took place at the active site causing inactivation of the enzyme according to second-order kinetics. In addition to this finding, a comparison of the absorption spectrum of 2-BNK-modified ChT (2-BNK-ChT) with those of native ChT and the model dimethyl(2-naphthoylmethyl)sulfonium bromide (2-NSS) at low pH revealed that the alkylating agent, BNK, reacts with methionine-192 on ChT in a 1 : 1 stoichiometric ratio. Evidence for the existence of BNK-derived reporter groups at the enzyme active site came from the observation of (1) positive induced circular dichroism bands of these groups and of (2) pronounced absorption spectral changes of the 2-BNK-derived pendant on denaturation of the alkylated enzyme with guanidine hydrochloride at pH 7.0. From the pH dependence of the absorption spectra of the modified enzymes and the model sulfonium salts, we determined the pK(a) values of 1-BNK-ChT, 1-NSS, 2-BNK-ChT, and 2-NSS to be 6.6, 7.5, 5.2, and 8.4 at 25+/-1-degrees-C, respectively. The much larger difference in pK(a) between 2-BNK-ChT and 2-NSS (DELTApK(a)=3.2) compared with that between 1-BNK-ChT and 1-NSS (DELTApK(a)=0.9) was explained in terms of stabilization of the 2-BNK-derived ylide pendant due to a configurationally rigid hydrogen-bonding interaction between the ylide carbonyl oxygen atom with a negative charge and the serine hydroxyl group located near the substrate binding site of ChT. Supporting evidence for this interpretation was obtained from solvent effects on the absorption spectra of model compounds as well as pH effects on the remaining activity of the alkylated enzymes.