Steady-state kinetic studies on D-lactate dehydrogenase from Megasphera elsdenii

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Abstract
Initial rate measurements were made of the oxidation of D-lactate and D-.alpha.-hydroxybutyrate by oxygen and potassium ferricyanide, catalyzed by D-lactate dehydrogenase (EC 1.1.1.27) from M. elsdenii. The detailed kinetic work indicates a ternary complex type mechanism, with a complex of keto acid and reduced enzyme reacting with the electron acceptor at pH 8. As the pH is lowered, the double-reciprocal plots become nonlinear, with a downward curvature. This seems to be due to negative interactions within the protein rather than to a complexity of the kinetic mechanism. The variation of initial rate parameters at pH 8 with temperature yields nonlinear Arrhenius plots with a greater activation energy above the break point than below. This type of behavior has been previously reported only for fumarase (Massey, 1953). Studies with deuterated D-lactate show only a small isotope effect on .vphi.0 and .vphi.1 (Km/Vmax for lactate) but a large effect on .vphi.2 (Km/Vmax for ferricyanide).