Abad-Zapatero C, Griffith J P, Sussman J L, Rossmann M G
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
J Mol Biol. 1987 Dec 5;198(3):445-67. doi: 10.1016/0022-2836(87)90293-2.
The crystal structure of M4 apo-lactate dehydrogenase from the spiny dogfish (Squalus acanthius) was initially refined by a constrained-restrained, and subsequently restrained, least-squares technique. The final structure contained 286 water molecules and two sulfate ions per subunit and gave an R-factor of 0.202 for difraction data between 8.0 and 2.0 A resolution. The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.25 A. The elements of secondary structure of the refined protein have not changed from those described previously, except for the appearance of a one-and-a-half turn 3(10) helix immediately after beta J. There is also a short segment of 3(10) helix between beta C and beta D in the part of the chain that connects the two beta alpha beta alpha beta units of the six-stranded parallel sheet (residues Tyr83 to Ala87). Examination of the interactions among the different elements of secondary structure by means of a surface accessibility algorithm supports the four structural clusters in the subunit. The first of the two sulfate ions is in the active site and occupies a cavity near the essential His195. Its nearest protein ligands are Arg171, Asp168 and Asn140. The second sulfate ion is located near the P-axis subunit interface. It is liganded by His188 and Arg173. These two residues are conserved in bacterial lactate dehydrogenase and form part of the fructose 1,6-bisphosphate effector binding site. Two other data sets in which one (collected at pH 7.8) or both (collected at pH 6.0) sulfate ions were replaced by citrate ions were also analyzed. Five cycles of refinement with respect to the pH 6.0 data (25 to 2.8 A resolution) resulted in an R value of 0.191. Only water molecules occupy the subunit boundary anion binding site at pH 7.8. The amino acid sequence was found to be in poor agreement with (2Fobs-Fcalc) electron density maps for the peptide between residues 207 and 211. The original sequence WNALKE was replaced by NVASIK. The essential His195 is hydrogen bonded to Asp168 on one side and Asn140 on the other. The latter residue is part of a turn that contains the only cis peptide bond of the structure at Pro141. The "flexible loop" (residues 97 to 123), which folds down over the active center in ternary complexes of the enzyme with substrate and coenzyme, has a well-defined structure. Analysis of the environment of Tyr237 suggests how its chemical modification inhibits the enzyme.
来自棘鲨（Squalus acanthius）的M4脱辅基乳酸脱氢酶的晶体结构最初通过约束-限制，随后是限制的最小二乘法技术进行了优化。最终结构每个亚基包含286个水分子和两个硫酸根离子，对于8.0至2.0埃分辨率的衍射数据，R因子为0.202。原子坐标精度的上限估计为0.25埃。优化后的蛋白质二级结构元件与先前描述的没有变化，只是在βJ之后立即出现了一个1.5圈的3(10)螺旋。在连接六链平行片层的两个β-α-β-α-β单元的链段中，βC和βD之间也有一段短的3(10)螺旋（残基Tyr83至Ala87）。通过表面可及性算法检查二级结构不同元件之间的相互作用，支持了亚基中的四个结构簇。两个硫酸根离子中的第一个在活性位点，占据了必需的His195附近的一个腔。它最近的蛋白质配体是Arg171、Asp168和Asn140。第二个硫酸根离子位于P轴亚基界面附近。它由His188和Arg173配位。这两个残基在细菌乳酸脱氢酶中保守，并且是果糖1,6-二磷酸效应物结合位点的一部分。还分析了另外两个数据集，其中一个（在pH 7.8下收集）或两个（在pH 6.0下收集）硫酸根离子被柠檬酸根离子取代。对pH 6.0数据（25至2.8埃分辨率）进行五个循环的优化，得到R值为0.191。在pH 7.8时，只有水分子占据亚基边界阴离子结合位点。发现氨基酸序列与残基207和211之间肽段的(2Fobs - Fcalc)电子密度图不一致。原来的序列WNALKE被NVASIK取代。必需的His195一侧与Asp168形成氢键，另一侧与Asn140形成氢键。后一个残基是一个转角的一部分，该转角包含结构中唯一的脯氨酸Pro141处的顺式肽键。在酶与底物和辅酶的三元复合物中折叠在活性中心上方的“柔性环”（残基97至123）具有明确的结构。对Tyr237环境的分析表明了其化学修饰如何抑制酶。
