The Activation of Alkaline Phosphatase by Effector Molecules: A Combined Kinetic and Hydrodynamic Study

Abstract

The results of our kinetic and hydrodynamic measurements indicate that alkaline phosphatase, from calf intestine and Escherichia coli, is in its active form a dimeric enzyme. The monomer has a very slight activity compared with the dimeric form of the enzyme. Tris and N‐dimethylethanolamine act as effectors on the enzyme activity and shift the monomer ⇌ dimer enzyme equilibrium toward the dimeric active species form, while L‐phenylalanine, a known non‐competetive inhibitor of alkaline phosphatase, inhibits the enzyme activity and prevents the formation of the dimeric enzyme. The effects of Tris and N‐dimethylethanolamine concentrations on the rate of hydrolysis of p‐nitrophenyl phosphate catalyzed by alkaline phosphatase (calf intestinal and E. coli) was studied at pH 9.0. A non‐linear 20‐fold increase of enzymatic activity was observed with increasing buffer concentrations (0.005–0.500 M) and a 40‐fold increase compared to that measured in 0.015 M NaHCO3, pH 9.0, under otherwise identical experimental conditions. Parallel to the kinetic measurements, three independent hydrodynamic measurements were made. First, active‐enzyme sedimentation was used to measure s20, w values at the same enzyme‐protein concentration (3 ng/ml) at which the kinetic experiments were performed; s20, w values obtained in 0.015 M NaHCO3, in 0.500 M Tris/Cl and in 0.500 M Tris/Cl containing 0.010 M L‐phenylalanine were: 5.57 S 6.46 S and 5.17 S, respectively. Second, s20, w values were obtained by boundary sedimentation velocity with the following results: 5.3 ± 0.3 S in 0.15 M NaHCO3, 5.3 ± 0.3 S in 0.015 M Tris/Cl and 6.3 ± 0.3 S in 0.500 M Tris/Cl. Third, molecular weights were determined by equilibrium sedimentation; the values obtained were 84500 in 0.015 M NaHCO3 and 157000 in 0.500 M Tris/Cl. Copyright © 1980, Wiley Blackwell. All rights reserved