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.