Preparation and 13C NMR characterization of [[epsilon-13C] methionine-192]-alpha-chymotrypsin. The demethylation of [S-[13C] methylmethionine-192]-alpha-chymotrypsin by an active site-directed thiol.

Met-192 forms part of the binding crevice of alpha-chymotrypsin. The aim of this investigation was to find a nucleophile that would displace a methyl group from the sulfonium cation of [S-[13C]methylmethionine-192]-alpha-chymotrypsin without disrupting the five disulfide bridges of the protein, thereby producing [[epsilon-13C]methionine-192]-alpha-chymotrypsin, an isotopically enriched version of the native enzyme desirable for 13C NMR studies. Treatment of [S-methylmethionine-192]-alpha-chymotrypsin with mercaptoethanol and dithiothreitol failed to produce the latter protein, as deduced from elution profiles of reaction mixtures chromatographed on affinity columns of immobilized lima bean trypsin inhibitor. In contrast, when [S-methylmethionine-192]-alpha-chymotrypsin was incubated in a 3.0 mM solution of the active site-directed reagent 2-mercaptoacetyl-4'-methoxyanilide at pH 8.6 and 5 degrees C for 48 h, affinity chromatograms indicated the presence of a protein corresponding to native alpha-chymotrypsin. Upon repeating the experiment with [S-[13C]methylmethionine-192]-alpha chymotrypsin, we isolated in 40% yield a protein which was identified as [[epsilon-13C]methionine-192]-alpha-chymotrypsin by a combination of 13C NMR and chemical criteria. This work represents the first active site-directed demethylation of an S-[13C]methylmethionine residue at the binding site of an enzyme.