Allosteric Properties of Phosphorylase b: III. INACTIVATION BY CYANATE AND BINDING STUDIES

Abstract

Abstract 1. The rate of inactivation of phosphorylase b by isocyanate is affected by various ligands which bind to the enzyme. 2. AMP, the activator of the enzyme, decreases the rate of inactivation in proportion to the amount added. 3. The substrates, Pi and glucose-1-P, and the competitive inhibitor, UDP-glucose, by themselves have no effect on the rate of inactivation by isocyanate but in the presence of AMP they provide further protection. 4. The allosteric inhibitors, ATP, ADP, and glucose-6-P, do not affect the rate of inactivation but they antagonize the protection offered by AMP. 5. The interrelationships among these ligands are analogous to those previously seen in kinetic studies. They have been interpreted on the basis of the proposed model for allosteric transitions between two conformational states. This interpretation assumes that the enzyme in these different conformations is inactivated at different rates by isocyanate. 6. Glycogen caused only a slight effect on the rate of inactivation by isocyanate and its possible role in allosteric transitions is difficult to assess at this time. 7. The protection against isocyanate inactivation has been used to calculate an apparent dissociation constant of 2 x 10-4 m for AMP and the free enzyme. Similarly, the apparent dissociation constants for enzyme already associated with AMP were calculated to be 2.3 mm for UDP-glucose, 6 mm for glucose-1-P, and 11 mm for Pi. Comparisons with dissociation constants determined by other means have been presented and discussed. 8. Studies on the binding of AMP to phosphorylase b by the ultracentrifugal separation or equilibrium dialysis methods indicated positive homotropic cooperativity between the two binding sites. ATP inhibits the binding while glucose-1-P improves it.