Electronic effects in the acylation of alpha-chymotrypsin by substituted N-benzoylimidazoles.

Rate constants for the acylation of alpha-chymotrypsin by a series of acyl-substituted N-benzoylimidazoles have been determined by proflavin displacement from the active site. The second-order acylation rate constants k2/Km are large [e.g., that for N-(m-nitrobenzoyl)imidazole is 1.7 X 10(4) M-1 s-1 at pH 7.5], even though Km must be quite large (plots of k vs. k/[S]0 have infinite slopes). The values of k2/Km are nearly independent of pH in the range 5.0-9.0 when the substituent group is electron donating. Electron-withdrawing substituents produce an increase in k2/Km with increasing pH until a maximum is reached near pH 7. This is also the case in acylation by the N-[p-(dimethylamino)benzoyl]-N'-methylimidazolium ion (pKapp = 6.5). While the reaction of the N'-methylated derivative is via a positively charged species at all pH values, the unmethylated compounds react through both the neutral species and the conjugate acids, with the observed pH dependence depending on the relative values of the rate constants. The limiting value of k2/Km for the N-[p-(dimethylamino)benzoyl]-N'-methylimidazolium ion is 2.1 times less in D2O than in H2O. Thus, His-57 must be participating in the acylation reaction as a general base. The limiting values of k2/Km for the corresponding N'-methylated and unmethylated derivatives differ by a factor of only 150, which is similar to the difference in the second-order rate constants for nonenzymatic OH- -catalyzed hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)