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

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