Dependence on pH of the activity of pig liver esterase

Abstract

The dependence on pH of the kinetic parameters for the hydrolysis of phenyl acetate catalyzed by pig liver carboxylesterase was examined for purified high-isoelectric point and low-isoelectric point fractions of enzyme that were separated by isoelectric focusing. The values of k cat are half-maximal at pH 4.3 and 5.1 for the high- and low-isoelectric point forms, respectively, and show a shallow dependence on pH with a value of n = 0.5. The absence of a change in the pH dependence of k cat for the high-isoelectric point enzyme in the presence of high concentrations of methanol, which reacts with the acetyl-enzyme intermediate to give methyl acetate, provides evidence that the pH dependence is not caused by a change in rate-determining step. This means that if an imidazole group is involved in catalysis its p K must be perturbed downward by 2–3 units. The pH dependence of k cat K m is biphasic with apparent p K values for dissociations of the free enzyme near 7 and 4 for both the high- and low-isoelectric point enzymes. Inhibition by a second molecule of substrate and by methanol are strongest for high-pH forms of the enzyme.