Stopped-flow kinetic and biophysical studies of membrane-associated d-lactate dehydrogenase of Escherichia coli

Abstract

The enzyme kinetics of the FAD-containing membrane-associated d-lactate dehydrogenase ( d-LDH) of Escherichia coli have been investigated by stopped-flow spectroscopy. The reduction of d-LDH by the substrate, d-lactate, exhibits a two-stage behavior as observed by the absorbance change for the enzyme-bound FAD. The fast stage with a maximum rate of 400 s −1 represents the rapid formation of the enzyme-substrate complex and the formation of the equilibrium between the oxidized and the reduced enzyme-substrate complexes. The slow stage, which occurs on the order of 0.36 s −1, represents the slow release of the product, pyruvate, from the reduced enzyme. The formation of a d-LDH semiquinone radical was not observed during the oxidation of d-lactate by d-LDH at 25°C. However, during the subsequent electron transfer from the reduced enzyme to a nitroxide spin-label, a one-electron acceptor, an enzyme intermediate has been observed and identified by both optical and EPR spectroscopies as an anionic semiquinone. Results from 1 H-NMR spectroscopic studies suggest the possible formation of a substrate carbanion when d-lactate is bound at the active site of d-LDH.