Volume profiles of the reaction mechanism of esterase: Evidence of a single-proton-transfer mechanism

Abstract

The pre-steady-state kinetics of hydrolysis of p-nitrophenyl trimethylacetate (pNPT), and the steady-state kinetic of hydrolysis of N-acetyl- L-tryptophanamide (ATA), N-acetyl- L-tryptophan p-chloroanilide (ATpCA), and succinyl- L-alanyl- L-alanine p-nitroanilide (SA 3pNA), catalyzed by α-chymotrypsin (α-CHT) have been studied in Tris buffer solution up to 2.4 kbar at 25°C. The reaction is initiated by the substrate binding, and then the acylation and deacylation occur. The volume changes ΔV Ks for the dissociation process of an enzyme-substrate complex, are (−14 ± 1) cm 3/mol for pNPT and (−3 ± 2) cm 3/mol for ATA. The activation volumes ΔV acyl ≠ for the acylation process are (−24 ±1) cm 3/mol for pNPT, (10±2) cm 3/mol for ATpCA, and (15±2) cm 3/mol for SA 3pNA, respectively. The activation volume ΔV deacyl ≠ of the deacylation is (−2±1) cm 3/mol. From the reaction and activation volumes, it os concluded that the catalytic reaction of α-CHT follows the single proton-transfer-mechanism.