Dissociative mechanism of thermal denaturation of rabbit skeletal muscle glycogen phosphorylase b

The thermal stability of rabbit skeletal muscle glycogen phosphorylase b wa s characterized using enzymological inactivation studies, differential scan ning calorimetry, and analytical ultracentrifugation. The results suggest t hat denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monom ers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibi tor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that t he cofactor of glycogen phosphorylase-pyridoxal 5'-phosphate-stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheate d at 53 degreesC occurs after cooling of the enzyme solution to 30 degreesC . The fact that the rate of reactivation decreases with dilution of the enz yme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phos phorylase b reactivation.