The reaction of tris (hydroxymethyl) aminomethane with calf intestinal alkaline phosphatase

The effect of tris (hydroxymethyl) aminomethane concentrations on the rate of calf intestinal alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate was studied, in the pH range 8-10, where no transphosphorylation reaction could be detected. Kinetic analysis of the results permitted description of the effect of Tris concentrations T on the rate of enzyme catalyzed hydrolysis (V) by the following equation: (see article). The rate-accelerating effect of Tris concentrations can be ascribed to two different mechanisms: At moderate Tris concentrations (0.01-0.20 M) the enzyme forms a reversible addition complex with a Tris molecule. This complex has an enhanced catalytic activity. We suggest that the binding of Tris to the enzyme could potentiate a second active site of the enzyme, due to its ionization effect upon an acidic group of the enzyme of pK = 8.9. The modest linear rate accelerating effect of Tris at high concentrations (0.20-0.60 M) could be ascribed to the change of the dielectric constant of the medium, the degree of solvation of the protein, or change in the tertiary structure of the enzyme.