The Kinetics of the α-Chymotrypsin-catalyzed Hydrolysis of p-Nitrophenyl Acetate in Organic Solvent-Water Mixtures

The α-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate has been studied at pH 7 and 25° in isopropyl alcoholwater and dioxane-water mixtures. It was found that the initial reaction in which the enzyme is acylated follows second order kinetics rather than Michaelis-Menten kinetics as previously reported. The second order rate constant for this reaction is markedly decreased by addition of either isopropyl alcohol or dioxane in a manner which can be formally attributed to either competitive or noncompetitive inhibition. The rate of the steady state reaction increases with substrate concentration in a manner which can be formally described in terms of substrate activation. After correction for this effect, the rate at substrate saturation is increased by organic solvents (by a factor of 3.5 in 20% (v/v) dioxane); the apparent Michaelis-Menten constant is also greatly increased (by a factor of 150 in 20% (v/v) dioxane). The primary amine tris(hydroxymethyl)aminomethane, which was used as buffer, is shown to have a strong inhibitory effect on the pre-steady state reaction, and to be very much more effective than water as an acyl acceptor. Although several of these effects can be expressed in reasonably quantitative terms, it is not possible to offer convincing explanations for them. Organic solvents such as dioxane may produce more or less extensive changes in the enzyme, perhaps conformational in character, which drastically alter its kinetic properties; they may also exert an influence by disrupting the water structure in the vicinity of the active site.