Alkaline phosphatases of Neurospora crassa. Part III. Effects of pH and mechanism of action

Abstract

The effects of pH on the activity of a highly purified constitutive alkaline phosphatase from Neurospora crassa were studied. The pH optimum for the reaction was a linear function of the logarithm of the substrate concentration, as is typical for alkaline phosphatases. Other pH effects were complex and Vmax or pKm versus pH plots differed for the two substrates used and from similar plots prepared with the repressible enzyme from this or other sources. The Km for p-nitrophenyl phosphate increased 200-fold over the pH range 7.0–8.7. Substrate inhibition was more pronounced at low than high pH. Lineweaver-Burk plots were non-linear at high substrate concentration but linear at low substrate concentration. Substrate inhibition by glucose 6-phosphate was not observed.The data are consistent with the hypothesis that hydroxyl ion is a second substrate for the enzyme and is an activator at high and an inhibitor at low p-nitrophenyl phosphate and glucose 6-phosphate concentrations. A Bi-Bi mechanism with random substrate addition, dead-end substrate, and product inhibition, having ordered release of products is discussed and is consistent with kinetic and pH studies. A two-site model for the active center of the enzyme is presented to account for alternate product inhibition data.