Sekharudu C, Ramakrishnan B, Huang B, Jiang R T, Dupureur C M, Tsai M D, Sundaralingam M
Department of Chemistry, Ohio State University, Columbus 43210.
Protein Sci. 1992 Dec;1(12):1585-94. doi: 10.1002/pro.5560011206.
The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.
磷脂酶A2（PLA2）催化膜磷脂sn-2酯键的水解。该酶中高度保守的酪氨酸残基52和73与催化性天冬氨酸-99的羧基形成氢键。这些氢键最初被认为对界面识别和整个催化网络的稳定性至关重要。通过将酪氨酸-52和-73的酚羟基替换为苯丙氨酸来消除涉及它们的氢键，降低了稳定性，但并未显著影响该酶的催化活性。已测定双突变体Y52F/Y73F的X射线晶体结构，分辨率为1.93 Å，以研究该突变对结构的影响。晶体为三方晶系，空间群P3(1)21，晶胞参数a = b = 46.3 Å，c = 102.95 Å。强度数据在西门子面积探测器上收集，在总共27,203个反射中，8,024个反射是独立的，R(sym)为4.5%。使用XPLOR对所有独立反射进行结构精修，对于955个蛋白质原子、91个水分子和1个钙离子，最终R因子为18.6%。双突变体与野生型之间α碳原子的均方根偏差为0.56 Å。晶体结构表明，催化网络中失去了四个氢键；三个涉及酪氨酸，一个涉及脯氨酸-68。然而，催化三联体、组氨酸-48、天冬氨酸-99和催化水的氢键得以保留。活性位点没有额外的溶剂分子来取代缺失的羟基；相反，与野生型相比，酚羟基被氢原子取代使苯丙氨酸残基更靠近相邻残基；苯丙氨酸-52向催化二元组的组氨酸-48和天冬氨酸-99移动，苯丙氨酸-73向甲硫氨酸-8移动，两者均移动约0.5 Å。羟基留下的空隙的闭合增加了疏水相互作用，补偿了失去的氢键。三联体氢键的保留以及通过增加疏水相互作用实现的活性位点稳定可以解释为什么双突变体具有与野生型相似的活性。结果表明，催化三联体的天冬氨酰羧基可以单独发挥作用，无需两个酪氨酸残基的氢键提供额外支持。
