Steady-state carbon-13 nuclear magnetic resonance spectra of acyl-alpha-chymotrypsin.

When [13C]carbonyl-enriched p-nitrophenyl 5-n-propyl-2-furoate is incubated with alpha-chymotrypsin, a new peak appears in the 13C NMR spectrum. On the basis of its position and the fact that it is "chased" with unlabeled substrate, we conclude that this new signal is due to the acyl-enzyme intermediate. In spectra taken during steady-state turnover, the acyl-enzyme ester carbonyl 13C chemical shift displays a pH dependence that fits to a titration curve with an apparent pK of 7.1 (0.1). The apparent pK of the kcat vs. pH curve for enzyme-catalyzed hydrolysis of the same substrate under conditions differing only in reactant concentration is 7.0 (0.1). We have found no spectral evidence for a tetrahedral intermediate.