Olson S T, Massey V
Biochemistry. 1979 Oct 16;18(21):4714-24. doi: 10.1021/bi00588a036.
A pyridine nucleotide independent D-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975 J. Bacteriol. 124, 1454--1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for D-lactate, and no inhibition is observed with L-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a gamma/2 of 0.224 and 0.442, respectively. Except for this atypically high oxygen reactivity, D-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by D-lactate. Two electrons per flavin were consumed in a dithionite titration, implyine with varying ratios of D-lactate and pyruvate, an Em7 of -0.219 +/- 0.007 V at 20 degrees C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivation results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation.
已从厌氧细菌埃氏巨球形菌中纯化出一种不依赖吡啶核苷酸的D-乳酸脱氢酶，达到了表观均一性。通过沉降平衡分析，该酶的分子量为105000，经十二烷基硫酸钠凝胶电泳测定，其亚基分子量为55000，因此可能是由相同亚基组成的二聚体。每摩尔蛋白质亚基含有约1摩尔FAD和1克原子的Zn2+，且黄素的荧光强度是游离FAD的1.7倍。早期的纯化方法[布罗克曼，H. L.，& 伍德，W. A.（1975年，《细菌学杂志》124卷，1454 - 1461页）]会导致该酶的锌大量流失，而锌是催化活性所必需的。新的纯化方法得到的酶量比之前分离的量多5倍以上。该酶对D-乳酸具有特异性，对L-乳酸无抑制作用。令人惊讶的是，该酶具有显著的氧化酶活性，这取决于离子强度。在离子强度γ/2分别为0.224和0.442时，Vmax值分别为190和530分钟-1。除了这种非典型的高氧反应性外，D-乳酸脱氢酶与其他黄素酶脱氢酶相似之处在于，黄素不与亚硫酸盐反应，色氨酸含量低，并且在光化学还原时形成中性蓝色半醌。该酶黄素可通过连二亚硫酸盐、在光照下通过草酸盐加催化量的5-脱氮黄素或通过D-乳酸进行还原。在连二亚硫酸盐滴定中，每分子黄素消耗两个电子，结合不同比例的D-乳酸和丙酮酸，在20℃下计算出黄素的Em7为-0.219±0.007V。该酶需要二硫苏糖醇来保持稳定性。当酶与亚化学计量水平的Cu2+一起孵育时会迅速失活。这种失活可被二硫苏糖醇逆转。有人提出该酶具有一对能够轻易形成二硫键的半胱氨酸残基。
