基于分子动力学模拟研究温度对乳酸脱氢酶活性的影响

Investigation on the Effect of Temperature on the Activity of Lactate Dehydrogenase Based on Molecular Dynamics Simulation

乳酸脱氢酶是糖无氧酵解及糖异生的重要酶系之一,它能够催化丙酮酸形成乳酸,在食品发酵工业中具有很高的应用价值,但该酶易受高温影响,导致乳酸制品产量下降。为了研究不同温度对乳酸脱氢酶的构象及活性的影响,采用分子动力学模拟的方法,针对4种不同温度条件下(37、55、70和85℃)的乳酸脱氢酶分别进行了80 ns的计算模拟,分析了构象变化及酶活性中心的差异。研究发现,在37和55℃条件下,乳酸脱氢酶比较稳定;当温度升高至70和85℃,乳酸脱氢酶的均方根误差、均方根波动、回旋半径值和溶剂可及表面积显著增加,而85℃时的蛋白二级结构已发生较大改变,这表明高温会导致蛋白质构象不稳定。对比37和85℃条件下该酶的底物丙酮酸的结合能力,发现高温会导致丙酮酸的结合位点残基之间的距离增大,进而破坏底物分子结合的微环境。因此,乳酸脱氢酶在温度超过70℃时发生变性,并随着温度的升高变性程度增加,进而导致酶活性丧失,不利于其在食品发酵等方面的应用。本研究在原子水平上分析了4种不同温度对乳酸脱氢酶的影响,揭示了其酶活性及构象变化的关键信息,为乳酸制品在发酵过程中选择合适温度提供了理论支撑。

Lactate dehydrogenase(LDH) is one of the important enzymes for anaerobic glycolysis and gluconeogenesis. It could catalyze pyruvate to form lactic acid, which had high application value in food fermentation industry. However, LDH was easily affected by high temperature, which leaded to the decline of lactic acid production. To study the effects of different temperature on the conformation and activity of LDH, molecular dynamics simulations at four different temperatures(37, 55, 70 and 85 ℃) were performed for 80 ns, respectively. The overall conformational changed and the differences of enzyme active centers were analyzed. The results showed that LDH was relatively stable at 37 and 55 ℃, and the root mean square error, root mean square fluctuation, radius gyration and solvent accessible surface area of LDH increased significantly at 70 and 85 ℃. Moreover, the secondary structure of protein had changed greatly at 85 ℃, which indicated that high temperature would lead to protein conformational instability. Comparing the binding ability of pyruvate at 37 and 85 ℃, which was the substrate of the enzyme, we found that high temperature would increase the distance between the residues of pyruvate binding sites, and then destroy the microenvironment of substrate molecular binding.Therefore, the denaturation of LDH occurred when the temperature exceeded 70 ℃, and the denaturation degree was positively correlated with the increase of temperature. This would lead to the loss of its enzyme activity, which was not conducive to the application value in food industry. In this study, the effects of four different temperatures on LDH were analyzed at atomic level, and the key information of enzyme activity and conformation changes were revealed, which provided theoretical support for the selection of appropriate temperature in the fermentation process of lactic acid products.