Effect of Pressure on the Pre-Steady-State Kinetics of the Hydrolysis of p-Nitrophenyl Pivalate Catalyzed by α-Chymotrypsin

Abstract

Abstract The pre-steady-state of the hydrolysis of p-nitrophenyl pivalate (pNPT) catalyzed by α-chymotrypsin (α-CHT) in Tris buffer solutions was measured up to 2.4 k bar at 25 °C. The reaction was initiated by substrate binding, and followed by acylation and deacylation processes. From the pressure dependence on the dissociation constants (Ks), the volume change ΔVKs was −14±1 cm3 mol−1. From the pressure dependence on the rate constants (kacyl and kdeacyl) of the acylation and deacylation processes, the activation volumes ΔVacyl\eweq and ΔVdeacyl\eweq were −24±1 and −2±1 cm3 mol−1, respectively. The reaction mechanisms of α-CHT are discussed in terms of the reaction and activation volumes.