THIOLTRANSFERASE IN HUMAN RED-BLOOD-CELLS - KINETICS AND EQUILIBRIUM

Thioltransferase from human red blood cells (HRBC TTase), couled to GSSG reductase, catalyzed glutathione (GSH)-dependent reduction of prototype substrates hydroxyethyl disulfide (HEDS) and sodium S-sulfocysteine as well as of other homo- and heterodisulfides, including the protein mixed disulfide albumin-S-S-cysteine. Whereas apparent K(M) values for the substrates varied over more than a 20-fold range, the V(max) values agreed quite closely, usually within less than a factor of 2, suggesting that initial interaction of oxidized substrate with enzyme is not rate determining. HRBC TTase was inactivated by iodoacetamide (IAA), and this was prevented by pretreatment with disulfides. The pH dependence of IAA inactivation gave a remarkably low apparent pK(a) of 3.5, which was independent of ionic strength (0.05-2 M). At pH 6, one radiolabeled carboxyamidomethyl moiety was bound to the enzyme after treatment with [C-14]IAA. This unusual thiol reactivity suggests that the active-site cysteine moiety of the TTase may be involved in a hydrogen bond with a carboxylate moiety. In contrast, the pH dependence for GSH-dependent TTase catalysis of disulfide reduction displayed an inflection point near pH 8.0, also suggesting that the initial reaction of oxidized substrate with the active-site thiol is not involved in rate determination. Two substrate kinetic studies of HRBC TTase and rat liver TTase (e.g., [GSH] and [HEDS] varied independently) gave patterns of intersecting lines on double-reciprocal plots (1/upsilon vs 1/S), indicating a sequential mechanism for the TTase reactions, rather than a ping-pong mechanism. Thus, both GSH and the disulfide substrate appear to interact with the reduced enzyme before the products of disulfide reduction are released, rather than GSH acting simply as a reductant of the oxidized enzyme. When GSSG reductase was omitted, the TTase was shown to catalyze the approach to the same equilibrium position starting from either set of substrates (e.g., GSH + HEDS vs GSSG + beta-mercaptoethanol). Thus, TTase action is fully reversible, and the direction of catalysis and extent of reaction depend on the relative concentrations of the respective substrates and on the coupled action of GSSG reductase.