Gleason W B, Fu Z, Birktoft J, Banaszak L
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455.
Biochemistry. 1994 Mar 1;33(8):2078-88. doi: 10.1021/bi00174a014.
The crystal structure of mitochondrial malate dehydrogenase from porcine heart contains four identical subunits in the asymmetric unit of a monoclinic cell. Although the molecule functions as a dimer in solution, it exists as a tetramer with 222 point symmetry in the crystal. The crystallographic refinement was facilitated in the early stages by using weak symmetry restraints and molecular dynamics. The R-factor including X-ray data to 1.83-A resolution was 21.1%. The final root mean square deviation from canonical values is 0.015 A for bond lengths and 3.2 degrees for bond angles. The resulting model of the tetramer includes independent coordinates for each of the four subunits allowing an internal check on the accuracy of the model. The crystalline mitochondrial malate dehydrogenase tetramer has been analyzed to determine the surface areas lost at different subunit-subunit interfaces. The results show that the interface with the largest surface area is the same one found in cytosolic malate dehydrogenase. Each of the subunits contains a bound citrate molecule in the active site permitting the elaboration of a model for substrate binding which agrees with that found for the crystalline enzyme from Escherichia coli. The environment of the N-terminal region of the crystallographic model has been studied because the functional protein is produced from a precursor. This precursor form has an additional 24 residues which are involved in mitochondrial targeting and, possibly, translocation. The crystallographic model of mitochondrial malate dehydrogenase has been compared with its cytosolic counterpart from porcine heart and two prokaryotic enzymes. Small but significant differences have been found in the polar versus nonpolar accessible surface areas between the mitochondrial and cytosolic enzymes. Using least squares methods, four different malate dehydrogenases have been superimposed and their consensus structure has been determined. An amino acid sequence alignment based on the crystallographic structures describes all the conserved positions. The consensus active site of these dicarboxylic acid dehydrogenases is derived from the least squares comparison.
猪心线粒体苹果酸脱氢酶的晶体结构在单斜晶胞的不对称单元中包含四个相同的亚基。尽管该分子在溶液中以二聚体形式发挥功能，但在晶体中它以具有222点对称性的四聚体形式存在。在早期阶段，通过使用弱对称性限制和分子动力学促进了晶体学精修。包括1.83埃分辨率的X射线数据在内的R因子为21.1%。与标准值的最终均方根偏差对于键长为0.015埃，对于键角为3.2度。所得的四聚体模型包括四个亚基各自的独立坐标，从而可以对模型的准确性进行内部检查。已对结晶的线粒体苹果酸脱氢酶四聚体进行分析，以确定在不同亚基-亚基界面处损失的表面积。结果表明，表面积最大的界面与胞质苹果酸脱氢酶中的相同。每个亚基在活性位点含有一个结合的柠檬酸分子，从而可以构建一个底物结合模型，该模型与大肠杆菌结晶酶的模型一致。由于功能性蛋白质由前体产生，因此对晶体学模型的N端区域环境进行了研究。这种前体形式有另外24个残基，它们参与线粒体靶向，可能还参与转运。已将线粒体苹果酸脱氢酶的晶体学模型与其猪心胞质对应物以及两种原核酶进行了比较。在线粒体和胞质酶之间的极性与非极性可及表面积方面发现了微小但显著的差异。使用最小二乘法，将四种不同的苹果酸脱氢酶进行了叠加，并确定了它们的共有结构。基于晶体学结构的氨基酸序列比对描述了所有保守位置。这些二羧酸脱氢酶的共有活性位点源自最小二乘法比较。
