Department of Pharmacology, University of California, San Diego, La Jolla, California, 92093.
Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California, 92093.
Protein Sci. 2018 Feb;27(2):463-471. doi: 10.1002/pro.3338. Epub 2017 Nov 21.
Malate dehydrogenase (MDH) and citrate synthase (CS) are two pacemaking enzymes involved in the tricarboxylic acid (TCA) cycle. Oxaloacetate (OAA) molecules are the intermediate substrates that are transferred from the MDH to CS to carry out sequential catalysis. It is known that, to achieve a high flux of intermediate transport and reduce the probability of substrate leaking, a MDH-CS metabolon forms to enhance the OAA substrate channeling. In this study, we aim to understand the OAA channeling within possible MDH-CS metabolons that have different structural orientations in their complexes. Three MDH-CS metabolons from native bovine, wild-type porcine, and recombinant sources, published in recent work, were selected to calculate OAA transfer efficiency by Brownian dynamics (BD) simulations and to study, through electrostatic potential calculations, a possible role of charges that drive the substrate channeling. Our results show that an electrostatic channel is formed in the metabolons of native bovine and recombinant porcine enzymes, which guides the oppositely charged OAA molecules passing through the channel and enhances the transfer efficiency. However, the channeling probability in a suggested wild-type porcine metabolon conformation is reduced due to an extended diffusion length between the MDH and CS active sites, implying that the corresponding arrangements of MDH and CS result in the decrease of electrostatic steering between substrates and protein surface and then reduce the substrate transfer efficiency from one active site to another.
苹果酸脱氢酶（MDH）和柠檬酸合酶（CS）是参与三羧酸（TCA）循环的两种起搏酶。草酰乙酸（OAA）分子是从 MDH 转移到 CS 以进行连续催化的中间底物。已知，为了实现中间产物转运的高通量并降低底物泄漏的可能性，形成 MDH-CS 代谢物以增强 OAA 底物的通道化。在这项研究中，我们旨在了解不同结构取向的复合物中可能的 MDH-CS 代谢物中的 OAA 通道化。选择了最近发表的三种来自天然牛、野生型猪和重组来源的 MDH-CS 代谢物，通过布朗动力学（BD）模拟计算 OAA 转移效率，并通过静电势能计算研究驱动底物通道化的电荷的可能作用。我们的结果表明，在天然牛和重组猪酶的代谢物中形成了静电通道，该通道引导带相反电荷的 OAA 分子穿过通道并提高转移效率。然而，由于 MDH 和 CS 活性位点之间的扩散长度延长，建议的野生型猪代谢物构象中的通道化概率降低，这意味着 MDH 和 CS 的相应排列导致底物和蛋白质表面之间的静电引导减少，从而降低从一个活性位点到另一个活性位点的底物转移效率。
