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

Abstract

The activation of mitochondrial succinate dehydrogenase by various activators is a result of dissociation of oxaloacetate tightly bound to the nonactive enzyme. But, quantitative correlation between the effector concentrations and the active fraction of the enzyme was not at hand. In this study we measured the level of active succinate dehydrogenase equilibrated with a wide range of opposing effectors: oxaloacetate (1-500 muM) and activator (0.02-1.5 M NaBr). The results are compatible with a model assuming two stable forms of the enzyme: a nonactive enzyme-oxaloacetate complex and an active enzyme free of oxaloacetate. The active form is stabilized by binding two 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. kex at infinite Br- concentration is pH dependent but ka is not. The two reactions, activation and exchange, also differ in their activation energy bein 32 and 21.5 kcal/mol, respectively. It is concluded that, in the course of activation, Br- interacts at two distinct steps. First to produce 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. It is suggested that the equilibrium between the two stable forms of the enzyme can be reached by two 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.

Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart

“…Untreated heart mitochondrial membranes showed considerable lag phase during continuous assay of succinate oxidase reaction ( Fig. 4 A, trace 1), confirming previous observations with purified enzyme or SMP [4] , [6] , [7] , [38] . This phenomenon indicated slow (half-time of around 20 min) dissociation of OAA from the active centre of SDH during the time of the assay.…”

Activation of succinate dehydrogenase from adultFasciola hepatica(Trematoda)

“…It has long been known that the succinate dehydrogenase of mammals undergoes a relatively fast transformation from a low activity form to a high activity form when incubated with substrate, a process usually referred to as activation (Thorn, 1962). The active form of mammalian succinate dehydrogenase is deactivated by oxaloacetate, which is reversibly bound to the enzyme (Ackrell, Kearney & Mayr, 1974;Gutman, 1976). In addition to activation by succinate, the enzyme can be activated by incubation with compounds which bind to the active site (fumarate, malonate) and also by incubation with a variety of anions, nucleotides and reducing agents (for review see Singer, Kearney & Gutman, 1972).…”

Catalytic and Molecular Modifications of Succinate Dehydrogenase by Monovalent Inoganic Anions

“…Like the substrate or competitive inhibitors, at high concentration and neutral pH they bind and stabilize the molecules of activated enzyme originating in the dissociation equilibrium of the bound oxaloacetate [3,4]. On the other hand, when they act at low pH, binding of the positive effector precedes and facilitates dissociation of oxaloacetate, the same sequence of events observed in reductive activation [5,6l.…”

“…Mechanisms, as well as the possible physiological significance of the novel type of regulation of succinate dehydrogenase, are discussed.Inorganic monovalent anions have been described as positive modulators of succinate dehydrogenase [l -31 and it has been ascertained that activation by bromide releases bound oxaloacetate, as is generally the case in activation [2].The action of the anions seems to involve two different mechanisms. Like the substrate or competitive inhibitors, at high concentration and neutral pH they bind and stabilize the molecules of activated enzyme originating in the dissociation equilibrium of the bound oxaloacetate [3,4]. On the other hand, when they act at low pH, binding of the positive effector precedes and facilitates dissociation of oxaloacetate, the same sequence of events observed in reductive activation [5,6l.…”

“…The action of the anions seems to involve two different mechanisms. Like the substrate or competitive inhibitors, at high concentration and neutral pH they bind and stabilize the molecules of activated enzyme originating in the dissociation equilibrium of the bound oxaloacetate [3,4]. On the other hand, when they act at low pH, binding of the positive effector precedes and facilitates dissociation of oxaloacetate, the same sequence of events observed in reductive activation [5,6l. In the activation by monovalent anions of the oxaloacetatetreated enzyme no inhibitory effects have been reported, whereas such effects are found for dicarboxylic acids, for example ; therefore inorganic anions apparently do not interfere with the catalytic site.…”