Details of the acyl-enzyme intermediate and the oxyanion hole in serine protease catalysis.

Raman, absorbance, and kinetic measurements were used to determine how the serine protease active site feature known as the oxyanion hole interacts with an acyl-enzyme intermediate. The substrate, p-(dimethylamino)benzoylimidazolide (DAB-Im), was synthesized and used to prepare DAB-acyl-enzymes of wild-type (WT) and N155G subtilisin-BPN' (the N155G mutant lacks a fully functioning oxyanion hole), alpha-chymotrypsin (CHT), and bovine trypsin (TRY). DAB-acyl-enzyme deacylation rate constants, k3, were found to span a 720-fold range at pH 7.8 (DAB-WT > DAB-TRY > DAB-N155G > DAB-CHT). DAB-N155G was found to deacylate 80-fold slower than DAB-WT, indicating a 2.6 kcal/mol loss of transition-state binding energy due to this mutation. Absorbance spectra revealed strongly red-shifted absorbance lambda max values for all of the DAB-acyl-enzymes. The red shift was found to be 2.0 nm less in DAB-N155G, indicating that the oxyanion hole is partially responsible for this electronic perturbation of the DAB chromophore at the active site. Raman difference spectra of the DAB-acyl-enzymes measured at pH 5.0 and 8.6, with 18O-labeling of the carbonyl, show that the molecular motions most perturbed by the active site are three associated with the scissile acyl bond. Most interesting is the carbonyl stretching vibration, v(C = O), whose motion extends into the hydrolytic reaction coordinate. Comparison of the v(C = O) of DAB-WT and DAB-N155G reveals that the oxyanion hole does indeed form a hydrogen-bonding interaction with the carbonyl oxygen, the strength of which increases at pH 8.6. Interestingly, the DAB-TRY carbonyl forms very strong hydrogen bonds, even at pH 5.0, but DAB-CHT does not, even at pH 8.6. The low-frequency (1661 cm-1) v(C = O)'s of pH 5.0 DAB-TRY and pH 8.6 DAB-WT are proposed to correspond to a tetrahedrally distorted carbonyl center like that observed in the crystal structure of guanidinobenzoyl-TRY (Mangel et al., 1990). The strength of hydrogen bonding between the DAB-acyl-enzyme's carbonyl and the oxyanion hole, as gauged by the v(C = O) frequency, was found to correlate positively with an increased deacylation rate. This correlation, as well as calculated acyl-enzyme carbonyl bond lengths, which indicate a 0.015-A lengthening due to the oxyanion hole interaction, was found to be in good agreement with previously published resonance Raman data of alpha, beta-unsaturated arylacryloyl-acyl-enzymes (Tonge & Carey, 1990b, 1992).