Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100, Pavia, Italy.
Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136, Bologna, Italy.
Biochimie. 2022 Aug;199:23-35. doi: 10.1016/j.biochi.2022.03.008. Epub 2022 Apr 7.
Among the functions exerted by eukaryotic lactate dehydrogenases, it is of importance the generation of lactate in muscles subjected to fatigue or to limited oxygen availability, with both these conditions triggering a decrease of cellular pH. However, the mutual dependence between lactate dehydrogenase (LDH) catalytic action and lactic acidosis is far from being fully understood. Here we show that the tetrameric LDH from rabbit skeletal muscle undergoes allosteric transitions as a function of pH, i.e. the enzyme obeys Michaelis-Menten kinetics at neutral or slightly alkaline pH values, and features sigmoidal kinetics at pH 6.5 or lower. Remarkably, we also report that a significant dissociation of tetrameric rabbit LDH occurs under acidic conditions, with pyruvate/NAD or citrate counteracting this effect. Moreover, citrate strongly activates rabbit LDH, inducing the enzyme to feature Michaelis-Menten kinetics. Further, using primary rabbit skeletal muscle cells we tested the generation of lactate as a function of pH, and we detected a parallel decrease of cytosolic pH and secretion of lactate. Overall, our observations indicate that lactic acidosis is antagonized by LDH dissociation, the occurrence of which is regulated by citrate and by allosteric transitions of the enzyme induced by pyruvate.
在真核生物乳酸脱氢酶发挥的众多功能中，其在肌肉产生乳酸中起着重要作用，这些肌肉会经历疲劳或氧气供应有限的情况，这两种情况都会导致细胞 pH 值下降。然而，乳酸脱氢酶 (LDH) 催化作用和酸中毒之间的相互依赖关系远未得到充分理解。在这里，我们表明兔骨骼肌中的四聚体 LDH 会根据 pH 值发生变构转变，即该酶在中性或略碱性 pH 值下遵循米氏-门捷列夫动力学，而在 pH 值为 6.5 或更低时呈现出 S 形动力学。值得注意的是，我们还报告说，在酸性条件下，四聚体兔 LDH 会发生显著的解离，而丙酮酸/NAD 或柠檬酸盐会拮抗这种效应。此外，柠檬酸盐强烈激活兔 LDH，诱导酶呈现米氏-门捷列夫动力学。此外，我们使用原代兔骨骼肌细胞测试了 pH 值对乳酸生成的影响，我们检测到胞质 pH 值的平行下降和乳酸的分泌。总体而言，我们的观察结果表明，酸中毒可通过 LDH 解离拮抗，其发生受柠檬酸盐和由丙酮酸诱导的酶变构转变调节。
