Temperature and pH effects on immobilized lactate dehydrogenase kinetics.

Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing, and kinetic measurements made. The results were interpreted on the basis of the Kobayashi-Laidler treatment of immobilized enzymes in flow systems, various tests being applied to determine the degree of diffusion control. It was established in various ways that the degree of diffusion control increases with (a) decrease in flow rate, (b) decrease in substrate concentration, and (c) decrease in temperature. A number of quantitative relationships, predicted by the theory, were obeyed by the results, for example: (a) Km(app) varies linearly with vf-1/3, where vf is the flow rate, (b) the logarithm of the product concentration at the exit varies linearly with the logarithm of the flow rate, and (c) absolute calculations of product concentrations are in reasonable agreement with experiment. A value of 5 kcal . mol-1 is estimated for the activation energy of the diffusion processes, and of 1 kcal . mol-1 for the chemical processes. When the pH is varied the rates pass through a flat maximum, the pH dependence being less than with the free enzyme.