The effect of opposing effectors on activation level of succinate dehydrogenase: equilibrium and kinetic studies

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Abstract
The activation of beef heart mitochondrial succinate dehydrogenase by various activators is a result of dissociation of oxaloacetate tightly bound to the nonactive enzyme. Quantitative correlation between the effector concentrations and the active fraction of the enzyme was not at hand. The level of active succinate dehydrogenase equilibrated with a wide range of opposing effectors: oxaloacetate (1-500 .mu.M) and activator (0.02-1.5 M NaBr) was measured. The results are compatible with a model assuming 2 stable forms of the enzyme: a nonactive enzyme-oxaloacetate complex and an active enzyme free of oxaloacetate. The active form is stabilized by binding 2 Br- and one H+. The rate of activation (ka) and exchange between enzyme bound and free oxaloacetate k(ex) were measured. Both ka and kex are hyperbolically dependent on Br- concentration but differ in magnitude and pH dependence. The constant kex at infinite Br- concentration is pH dependent, but ka is not. The 2 reactions, activation and exchange, also differ in their activation energy being 32 and 21.5 kcal/mol, respectively. In the course of activation Br- interacts at 2 distinct steps. It first produces a ternary, nonactive [enzyme-oxaloacetate-Br-] complex. From this complex, oxaloacetate dissociates and the oxaloacetate-free enzyme assumes its active form. Finally, the active enzyme is stabilized by binding another Br-. The rate-limiting step in deactivation is binding of oxaloacetate to active enzyme. The complex formed undergoes a very rapid transformation to the stable nonactive form. This pathway, under certain conditions, can reverse its direction and contribute to the overall rate of activation. The equilibrium between the 2 stable forms of the enzyme can be reached by 2 parallel pathways, each contributing independently to the observed rate of activation, while the final equilibrium is determined by the free energy between the products and the reactants.