Sun Z Y, Dowd S R, Felix C, Hyde J S, Ho C
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Biochim Biophys Acta. 1995 Oct 25;1252(2):269-77. doi: 10.1016/0167-4838(95)00120-j.
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.
利用停流光谱法研究了大肠杆菌中含黄素腺嘌呤二核苷酸（FAD）的膜相关D-乳酸脱氢酶（D-LDH）的酶动力学。通过酶结合FAD的吸光度变化观察到，底物D-乳酸对D-LDH的还原呈现出两阶段行为。最大速率为400 s-1的快速阶段代表酶-底物复合物的快速形成以及氧化型和还原型酶-底物复合物之间平衡的形成。以0.36 s-1左右的速率发生的缓慢阶段代表产物丙酮酸从还原型酶中的缓慢释放。在25℃下D-LDH氧化D-乳酸的过程中未观察到D-LDH半醌自由基的形成。然而，在随后从还原型酶到单电子受体氮氧化物自旋标记的电子转移过程中，通过光学光谱和电子顺磁共振光谱观察并鉴定出一种酶中间体为阴离子半醌。1H-NMR光谱研究结果表明，当D-乳酸结合在D-LDH的活性位点时，可能会形成底物碳负离子。
