STOPPED-FLOW KINETIC AND BIOPHYSICAL STUDIES OF MEMBRANE-ASSOCIATED D-LACTATE DEHYDROGENASE OF ESCHERICHIA-COLI

The enzyme kinetics of the FAD-containing membrane-associated D-lactat e 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 c hange for the enzyme-bound FAD. The fast stage with a maximum rate of 400 s(-1) represents the rapid formation of the enzyme-substrate compl ex and the formation of the equilibrium between the oxidized and the r educed enzyme-substrate complexes. The slow stage, which occurs on the order of 0.36 s(-1), represents the slow release of the product, pyru vate, from the reduced enzyme. The formation of a D-LDH semiquinone ra dical was not observed during the oxidation of D-lactate by D-LDH at 2 5 degrees 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 H-1-NMR spectroscopic studies suggest the possible formation of a substrate c arbanion when D-lactate is bound at the active site of D-LDH.