Relationship between Structural Change and Allosteric Transitions of Glycogen Phosphorylase a

The activation and dissociation of tetrameric phosphorylase a to a dimer upon prior incubation with glucose and AMP has been studied in relation to the allosteric transitions of this enzyme. The dependence of both the initial rate and extent of activation upon glucose concentration are characteristic of homotropic interaction having Hill coefficients of 2.9 and 1.5, respectively. The initial rate as well as the extent of phosphorylase a activation is also dependent upon AMP concentration. The maximum extent of AMP activation of the enzyme is much lower than that of glucose activation. In the presence of 0.005 M to 0.1 M glucose, AMP causes inactivation rather than activation. The final enzyme activity obtained at saturating AMP is dependent upon the glucose concentration. At 0.2 M glucose, AMP has no effect on the enzyme activity. The reversal of glucose dissociation by AMP shows a similar dependence upon glucose concentration. Both glucose and AMP affect the rate of inactivation of phosphorylase a in the presence of trypsin or p-hydroxymercuribenzoate (HMB). Although 0.2 M glucose enhances the rate of inactivation of phosphorylase a by trypsin, this effect can be completely reversed by AMP. Glucose at 0.2 M retards the initial rate of phosphorylase a inactivation by HMB. Although AMP by itself provides slight protection of the enzyme against HMB inactivation, it enhances the inactivation rate when 0.2 M glucose is present. These results support the view that enzyme dissociation is a result of conformational changes of the enzyme subunits. The effect of modifier concentration on phosphorylase a activation may be explained by the model proposed by Monod, Wyman, and Changeux if three conformational states are postulated for the enzyme. This model, however, cannot readily account for the effect of modifiers on the rate of enzyme inactivation by HMB and trypsin. The present results suggest that the allosteric transitions of phosphorylase a may be best explained by a direct application of Koshland's “induced fit” theory.