The kinetics of hydrolysis of phenyl phosphate by alkaline phosphatases

Abstract

The hydrolysis of phenyl phosphate by purified alkaline phosphatase from calf intestinal mucosa was investigated at substrate concentration from 2.5 x 10-5 [image] to 7.5 x 10-2 [image] and at pH values from 8.25 to 10.1, the concentration of added magnesium chloride (enzyme activator) being maintained constant at 10-2 [image]. There is marked inhibition by high concentrations of substrate, especially at lower pH values. The optimum pH for hydrolysis varies considerably with substrate concentration. The optimum substrate concentration also varies with pH. At a fixed substrate concentration, the optimum pH is influenced by the nature of the metal activator. The existence of several equilibria involving enzyme, substrate and metal are postulated to explain these results. Evidence is presented which supports this hypothesis. The Dixon (1933) plot of pKm against pH reveals a discontinuity at about pH 9.2 (at 38[degree]). This is interpreted as indicating that a phenolic hydroxyl group of a tyrosine residue of the enzyme is directly involved in the formation of a rate-limiting enzyme complex. The turnover number of intestinal alkaline phosphatase is calculated to be about 394,000 moles of phenyl phosphate/minute/100,000 g of protein (236,000 moles/minutes/mole of enzyme) at 38[degree]. Results obtained with purified alkaline phosphatase from milk support earlier observations by Folley and Kay (1935) using alkaline phosphatase from mammary gland of the guinea pig. The behavior of the milk (mammary-gland) phosphatase suggests that the several equilibria recognized for the intestinal phosphatase system are also important for the milk phosphatase system.