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-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 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 1H-NMR spectroscopic studies suggest the possible formation of a substrate carbanion when D-lactate is bound at the active site of D-LDH.
Sun
Z Y
ZY
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Dowd
S R
SR
Felix
C
C
Hyde
J S
JS
Ho
C
C
D015086:2,6-Dichloroindophenol; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D016227:Benzoquinones / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism; D019344:Lactic Acid; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013113:Spin Labels
D001425:Bacterial Outer Membrane Proteins; D016227:Benzoquinones; D007773:Lactates; D013113:Spin Labels; C025232:semiquinone radicals; D019344:Lactic Acid; D015086:2,6-Dichloroindophenol; D007770:L-Lactate Dehydrogenase