Catalytic and Molecular Modifications of Succinate Dehydrogenase by Monovalent Inoganic Anions

Abstract

The enzymatic activity and the oxidation state of soluble, activated, substrate-reduced succinate dehydrogenase are modified by the presence of bromide. The anion inhibits the enzyme by two different mechanisms which depend on the ratio of bromide to succinate. At high ratios binding of two bromide ions is required and a competitive inhibition is observed: removal of succinate from the substrate binding site (Kd = 0.1 mM) leads to oxidation of the flavin. At lower ratios but with sufficient succinate to saturate a site with Kd = 1.52 mM, uncompetitive inhibition by a single bromide ion is observed. 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. 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. On the other hand, competitive inhibition of the succinate dehydrogenase of Mytilus californiae mantel by NO;, Br-, C1-and acetate, in decreasing order of potency, has been observed [7]. It appeared to us that investigation of this set of actions might provide valuable information.Of the monovalent anions, bromide has been studied by previous authors in more detail [l, 31. We focussed our attention on the enzyme having the catalytic site occupied by succinate, so as to obtain better evidence of other possible interactions of the anion with the molecule. This information was expected to complete what was already known on the effect in the absence of succinate and of other positive effectors, namely activation.In this paper the different forms of enzyme with respect to catalytic activity are indicated as follows : inactivated = combined with oxaloacetate; activated = associated with positive effectors; inactive = activated, but catalytically incompetent; active = activated and catalytically competent. MATERIALS A N D METHODSSolublc succinate dehydrogenase, prepared as described elsewhere as (he gel eluate [8], was precipitated with ammonium sulphate, redissolved in 50 mM Tris acetate, 5 mM succinate pH 7.5 and passed on a column (4 x 20 cm) of Sephadex G-25 fine equilibrated in the same buffer. The enzyme (4.1 nmol prot...