Abstract
Abstract The acetylenic hydroxy acid, 2-hydroxy-3-butynoate, is an irreversible inactivator of d-lactate dehydrogenase and d-lactate-dependent lactose, proline, and valinomycin-induced rubidium transport in isolated membrane vesicles from Escherichia coli. The compound serves as a substrate for d-lactate dehydrogenase which undergoes some 15 to 30 turnovers prior to inactivation. Similarly, a low activity, membrane-bound l-lactate dehydrogenase is also inactivated presumably by the l isomer of the acetylenic substrate. These effects are highly specific as evidenced by the following observations. 1. Inactivation of d-lactate dehydrogenase in membrane vesicles or in a solubilized, partially purified preparation of this enzyme exhibits similar properties. 2. Although d- and l-lactate oxidation by membrane vesicles and transport in the presence of these electron donors is inactivated by hydroxybutynoate, the oxidation of succinate and NADH, and succinate- and NADH-stimulated transport are not affected. Moreover, ascorbate-phenazine methosulfate-dependent transport is not inactivated by hydroxybutynoate. 3. 3-Butynoate inhibits neither d-lactate dehydrogenase activity nor d-lactate-dependent transport; and 2-hydroxy-3-butenoate (vinylglycolate) serves as a substrate for d-lactate dehydrogenase, and is thus an effective electron donor for transport. 4. Kinetics of inactivation of d-lactate dehydrogenase activity and d-lactate-dependent lactose, proline, and valinomycin-induced rubidium transport are virtually identical with respect to both inhibitor concentration and time. 5. Inactivation of d-lactate-dependent transport by 2-hydroxy-3-butynoate is blocked by d-lactate, but not by succinate and NADH. 6. α-Glycerol-P-dependent threonine transport in Staphylococcus aureus vesicles is not inactivated by hydroxy-butynoic acid. A possible mechanism of inhibition is discussed.
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