Hall M D, Levitt D G, Banaszak L J
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
J Mol Biol. 1992 Aug 5;226(3):867-82. doi: 10.1016/0022-2836(92)90637-y.
The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A. Molecular replacement, using the partially refined structure of porcine mitochondrial malate dehydrogenase as a probe, provided initial phases. The structure of this prokaryotic enzyme is closely homologous with the mitochondrial enzyme but somewhat less similar to cytosolic malate dehydrogenase from eukaryotes. However, all three enzymes are dimeric and form the subunit-subunit interface through similar surface regions. A citrate ion, found in the active site, helps define the residues involved in substrate binding and catalysis. Two arginine residues, R81 and R153, interacting with the citrate are believed to confer substrate specificity. The hydroxyl of the citrate is hydrogen-bonded to a histidine, H177, and similar interactions could be assigned to a bound malate or oxaloacetate. Histidine 177 is also hydrogen-bonded to an aspartate, D150, to form a classic His.Asp pair. Studies of the active site cavity indicate that the bound citrate would occupy part of the site needed for the coenzyme. In a model building study, the cofactor, NAD, was placed into the coenzyme site which exists when the citrate was converted to malate and crystallographic water molecules removed. This hypothetical model of a ternary complex was energy minimized for comparison with the structure of the binary complex of porcine cytosolic malate dehydrogenase. Many residues involved in cofactor binding in the minimized E. coli malate dehydrogenase structure are homologous to coenzyme binding residues in cytosolic malate dehydrogenase. In the energy minimized structure of the ternary complex, the C-4 atom of NAD is in van der Waals' contact with the C-3 atom of the malate. A catalytic cycle involves hydride transfer between these two atoms.
已确定来自大肠杆菌的苹果酸脱氢酶的晶体结构，对于8.0至1.87埃的X射线数据，所得的R因子为0.187。以猪线粒体苹果酸脱氢酶的部分精制结构为探针进行分子置换，提供了初始相位。这种原核酶的结构与线粒体酶密切同源，但与真核生物的胞质苹果酸脱氢酶的相似性略低。然而，所有这三种酶都是二聚体，并通过相似的表面区域形成亚基-亚基界面。在活性位点发现的一个柠檬酸离子有助于确定参与底物结合和催化的残基。与柠檬酸相互作用的两个精氨酸残基R81和R153被认为赋予底物特异性。柠檬酸的羟基与组氨酸H177形成氢键，类似的相互作用也可归因于结合的苹果酸或草酰乙酸。组氨酸177也与天冬氨酸D150形成氢键，形成经典的His.Asp对。对活性位点腔的研究表明，结合的柠檬酸将占据辅酶所需位点的一部分。在一项模型构建研究中，将辅因子NAD放入当柠檬酸转化为苹果酸且去除结晶水分子时存在的辅酶位点。对这种三元复合物的假设模型进行能量最小化处理，以便与猪胞质苹果酸脱氢酶的二元复合物结构进行比较。在能量最小化的大肠杆菌苹果酸脱氢酶结构中，许多参与辅因子结合的残基与胞质苹果酸脱氢酶中的辅酶结合残基同源。在三元复合物的能量最小化结构中，NAD的C-4原子与苹果酸的C-3原子处于范德华接触。催化循环涉及这两个原子之间的氢化物转移。
