Regulation of mitochondrial succinate dehydrogenase by substrate type activators

Abstract

The activation of succinate dehydrogenase in submitochondrial particles was measured in the presence of oxaloacetate (a negative modulator) and substrate type activators (succinate, malonate, and fumarate). Quantitative analysis of equilibrium and kinetic experiments led to the following model for activation OAA Act E,,OAA a E,OAA E, E,Act The only stable forms of the enzyme are the nonactive complex with oxaloacetate (ENAOAA) and the active complex with the activator (EAAct). The rate-limiting step in activation is the spontaneous conformation change from the nonactive to the active complex with oxaloacetate (EAOAA). This reaction is shown to precede both the dissociation of oxaloacetate and the binding of activator. The only stable form of active enzyme is O x a l o a c e t a t e has been known for many years to be a powerful inhibitor of succinate dehydrogenase (EC 1.3.99.1) (Das, 1937). Wojtczak et al. (1969) were the first to consider this inhibition as deactivation. In the recent years, the role of oxaloacetate as a deactivator has been studied extensively. Oxaloacetate and the enzyme form a very tight complex, which can be dissociated either by activation (Ackrell et al., 1974; or by denaturation. It was suggested that oxaloacetate is covalently linked as a thiohemiacetal (Vinogradov et al., 1972) with a SH group at the active site of the flavoprotein subunit of the enzyme (Kenney et al., 1976).The reaction of this SH group with MalNEt] is blocked by either activators or oxaloacetate (Kenney et al., 1976;Vinogradov et al., 1976). The dissociation constants of succinate and malonate, as calculated from their effect on rate of alkylation, are similar to the respective K , or K , values (Vinogradov et al., 1976). Yet, these studies measured the effect of only one modulator, activator or oxaloacetate, on the reactivity of this SH group, but not their simultaneous interaction with the proposed common active site. In order to understand the mechanism of this step, we looked for a quantitative correlation between the active fraction of the enzyme and the modulators' concentrations. I Abbreviations used: Act, activator; OAA, oxaloacetate; EA and ENA, active and nonactive forms of the enzyme, respectively; S D H , succinate dehydrogenase; ETPH, phosphorylating submitochondrial particles; PMS, phenazine methosulfate; DCPIP, dichlorophenol indophenol; MalNEt, N-ethylmaleimide; Mes, 2-(N-morpholino)ethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane.the active 1:l complex with the activator (EAAct). The free active enzyme (EA) is unstable in the sense that both activators and oxaloacetate rapidty react with it forming tight complexes. The reaction between oxaloacetate and the free active enzyme is extremely fast (k = 1.24 X IO6 M-' s-1 at 30 "C and pH 7.4). The role of activator is to stabilize EA in a complex which cannot react directly with oxaloacetate. Because of the low concentration of EA, the rate-limiting step in deactivation is the bimolecular reaction between EA and OAA. The kinetic and thermodynamic...

Regulation of dehydrogenases/one‐electron transferases by modification of flavin redox potentials

“…As shown by Gutman et al [3,4,34], oxaloacetate preferentially binds to the oxidized flavoenzyme, supposedly at a regulatory site, and decreases the midpoint potential of the (Fox/Fh,) couple value by 200 mV. The consequence is that the substrate, succinate, is no longer able to reduce the flavin.…”

The circular dichroism and optical absorbancy of the histidyl flavin during active—non‐active transition of soluble succinate dehydrogenase

“…As predicted by the values of dissociation constants from the regulatory site [4] excess succinate , or fumarate, added to the dehydrogenase either immediately after oxaloacetate or when the spectral modifications were completed, did not change the effect of oxaloacetate. Nevertheless dithionite restored the spectrum of the fully reduced enzyme.…”

“…Though the changes can not be subjected to quantitative analysis, they certainly indicate a major modification in the immediate vicinity of the flavin and of the iron-sulfur centers. Considering the fact that while modulating the enzyme's activity, oxaloacetate interacts with a site different from that where the substrate is oxidized [4,8], the changes in environment of flavin should be attributed to interaction of protein with the flavin moiety. The fact that addition of oxaloacetate induces oxidation of the flavin (flg.1) and shifts the redox potential of the flavin [8] suggests that these protein-flavin interactions are responsible for the alteration of the prosthetic group's properties.…”

“…The stable form of the non-active enzyme is a complex with oxaloacetate, in a 1: 1 ratio to &win, and the unchanged modulator is dislodged only by denaturation of the protein or by any type of activation [l-4]. In [4] it was proposed that the substrate binding site and the regulatory site are not the same and oxaloacetate does not form a thiohemiacetal linkage with an SH group essential for enzymic activity as suggested [5,6] but it induces a change in conformation of the enzyme.…”

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.…”