Detection and accumulation of tetrahedral intermediates in elastase catalysis.
Tetrahedral intermediates in the reaction of elastase with specific di- and tripeptide p-nitroanilide substrates have been detected, accumulated, and stabilized at high pH by using subzero temperatures and fluid aqueous/organic cryosolvents. The tetrahedral adducts are characterized by spectra with lambda max of 359 +/- 2 nm, compared with thata of 380 nm for p-nitroaniline and 315-320 nm for the substrates. The maximal concentration of intermediate that could be accumulated varied with the different substrates from 40 to 100% of the active enzyme present. The pH dependence of the reactions indicated that formation of the tetrahedral intermediates was rate-limiting at low pH (pK* = 7.0 at -39 degrees C) and that collapse to the acylenzymes was rate-determining at high pH. When corrected for the effect of temperature and cosolvent, the rate of intermediate formation was in good agreement with that measured at 25 degrees C in aqueous solution by stopped-flow techniques.
D000818:Animals; D004285:Dogs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009842:Oligopeptides; D010196:Pancreatic Elastase / Q000378:metabolism*; D011487:Protein Conformation; D013053:Spectrophotometry
D009842:Oligopeptides; D010196:Pancreatic Elastase