Allosteric properties of glutaraldehyde-modified glycogen phosphorylase b. Selective desensitization of homotropic cooperativity.

The kinetic properties of a glutaraldehyde-modified phosphorylase b have been examined to understand further the mechanism of allosteric transition of this enzyme. The sigmoidal response to AMP, which is observed with native glycogen phosphorylase b, cannot be demonstrated with the modified enzyme. This enzyme derivative also exhibits no homotropic cooperativity of glucose 1 -phosphate under conditions for which native phosphorylase b gives rise to sigmoidal saturation curves. The conditions include (a) use of inosine monophosphate instead of AMP as activator and (b) the presence of various inhibitors, glucose, glucose-6-P, or 2’,3’cyclic AMP. In contrast to homotropic cooperativity, the heterotropic interactions which are characteristic of native phosphorylase b can be demonstrated with the glutaraldehyde-modilied enzyme. The K, values of the substrates, glucose-l-P, phosphate, and glycogen depend on the concentration of AMP. For instance, when AMP concentration is raised from 0.0125 to 1 mM, a decrease in K, of glucose-l-P from 13.7 to 6.6 mM can be observed. When IMP is used instead of AMP, the K, of glucose-l-P for the modified enzyme has been found to be 0.067 M irrespective of the nucleotide concentration. This result supports the view that the major difference between AMP and IMP activation of phosphorylase b is that only AMP enhances the enzyme affinity toward the substrate. In addition to the interactions between the nucleotide activators and substrates, negative heterotropic interactions between the inhibitors, glucose, glucose-6-P, or cyclic AMP and the substrate glucose-l-P can also be demonstrated in the glutaraldehyde-modified phosphorylase b. Thus, only homotropic cooperativity in glycogen phosphorylase b is desensitized by glutaraldehyde modification. The selectivity in the desensitization indicates that homotropic and heterotropic interactions in this enzyme are not linked functions. This property of the enzyme cannot be explained on the basis of the con-