Ion Pair Formation in Pig Heart Lipoamide Dehydrogenase

In this paper, we present rapid reaction kinetic data which provides additional evidence for the participation of a base in the active center of lipoamide dehydrogenase. We propose that this base deprotonates dihydrolipoamide, the substrate, activating it for nucleophilic attack on the enzyme active center disulfide. This mechanism for substrate activation predicts pH-independent rates of reduction of the enzyme by dihydrolipoamide at pH values above the pK of the base on oxidized enzyme. These rates (extrapolated to infinite dihydrolipoamide concentration) are found to be constant from pH 5.5 to pH 8.1. We have shown previously that although reduction of lipoamide dehydrogenase by dihydrolipoamide requires transfer of 2 protons as well as 2 electrons in the pH range from 5.5 to 7.6, the pH dependence of the spectrum of 2-electron-reduced enzyme indicates that only 1 of these protons is present in the reduced active center disulfide (Matthews, R. G., and Williams, C. H., Jr. (1976) J. Biol. Chem. 251, 3956-3964). These results were consistent with the formation of a thiollbase ion pair on reduction of the enzyme active center disulfide. In this paper, we present evidence that one of the oxidation-reduction protons dissociates with a pK of 7.9 on 2-electron-reduced enzyme, and that at pH values above this pK, the rate of reoxidation of 2electron-reduced enzyme by lipoamide (extrapolated to infinite lipoamide concentration) decreases. This decrease would be expected since microscopic reversibility implies that 2-electron-reduced enzyme must transfer both oxidation-reduction protons to lipoamide. Between pH 5.5 and pH 7.0, the rate of reoxidation of enzyme by lipoamide shows a plateau. The rate of alkylation of one of the active center thiols by iodoacetamide shows a similar pH profile, and suggests analogies with papain and other enzymes thought to contain a thiolatelimidazolium ion pair at the active center. We conclude that the base involved in ion pair formation in lipoamide dehydrogenase is required to be in its protonated form for enzymatic reduction of lipoamide and also for maximal reactivity of 2-electron-reduced enzyme with iodoacetamide.