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 in teracts with an acyl-enzyme intermediate. The substrate, p-(dimethylam ino)benzoylimidazolide (DAB-Im), was synthesized and used to prepare D AB-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 const ants, 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 s lower than DAB-WT, indicating a 2.6 kcal/mol loss of transition-state binding energy due to-this mutation. Absorbance spectra revealed stron gly 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, indic ating that the oxyanion hole is partially responsible for this electro nic perturbation of the DAB chromophore at the active site. Raman diff erence spectra of the DAB-acyl-enzymes measured at pH 5.0 and 8.6, wit h 180-labeling of the carbonyl, show that the molecular motions most p erturbed by the active site are three associated with the scissile acy l bond. Most interesting is the carbonyl stretching vibration, nu(C=0) , whose motion extends into the hydrolytic reaction coordinate. Compar ison of the nu(C=0) 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, bu t DAB-CHT does not, even at pH 8.6. The low-frequency (1661 cm-1) nu(C =0)'s of pH 5.0 DAB-TRY and pH 8.6 DAB-WT are proposed to correspond t o a tetrahedrally distorted carbonyl center like that observed in the crystal structure of guanidinobenzoyl-TRY (Mangel et al., 1990). The s trength of hydrogen bonding between the DAB-acyl-enzyme's carbonyl and the oxyanion hole, as gauged by the nu(C=0) frequency, was found to c orrelate positively with an increased deacylation rate. This correlati on, as well as calculated acyl-enzyme carbonyl bond lengths, which ind icate a 0.015-angstrom lengthening due to the oxyanion hole interactio n, was found to be in good agreement with previously published resonan ce Raman data of alpha,beta-unsaturated arylacryloyl-acylenzymes (Tong e & Carey, 1990b, 1992).