THE EFFECTS OF pH AND TEMPERATURE ON THE KINETICS OF THE PHOSPHORYLASE REACTION

Abstract

Arsenate was used in place of phosphate in the phosphorylase a reaction because kinetic measurements were thereby simplified. The effect of an increase in pH from 5.5 to 8.0 was a decrease in the Km of arsenate by a factor of about 3, both in the presence and absence of 5[image]-AMP. Owing to this change in Km, the pH optimum was dependent on the substrate concentration. The effect of addition of 5[image]-AMP was a decrease in the Km of arsenate by a factor of about 5 at all pH values. At intermediate pH values the Vmax was nearly the same with and without 5[image]-AMP, but became lower without 5[image]-AMP at the extremes of the pH range. Large changes in the Km for arsenate and for glycogen were produced by an increase in temperature from 12[degree] to 38[degree] at constant pH. For this reason the temperature optimum of the phosphorylase reaction was also dependent on the substrate concentration. In an Arrhenius plot of the reaction rates obtained at saturating substrate concentrations up to a temperature of 31[degree], the energy of activation was lower in the presence of 5[image]-AMP than in its absence. Between 31[degree] and 38[degree], a new slope was established with and without 5[image]-AMP. In this temperature range there was likewise a change in the kinetics of the system such that the rate of the reaction occurred up to 48[degree]. At 50[degree] the rate was still 80 per cent of maximum. In the absence of 5[image]-AMP the rate declined much more rapidly with increasing temperature. These observations suggest that phosphorylase can undergo reversible changes in its quarternary structure, presumably as the result of altered subunit interaction.