First-sphere and second-sphere electrostatic effects in the active site of a class mu gluthathione transferase.
First-sphere and second-sphere electrostatic effects in the active site of a class mu gluthathione transferase.
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mu 类谷胱甘肽转移酶活性位点的第一球体和第二球体静电效应。
DOI：
10.1021/bi960189k
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Armstrong,RN
中科院分区：
文献类型：
--
作者：
Xiao,G;Liu,S;Ji,X;Johnson,WW;Chen,J;Parsons,JF;Stevens,WJ;Gilliland,GL;Armstrong,RN
结合在大鼠 mu 类谷胱甘肽转移酶 M1-1 活性位点上的谷胱甘肽 (GSH) 硫醇的激活涉及氢键网络，其中包括 Y6 的羟基与 GSH 的硫之间的直接（第一球体）相互作用以及涉及 L12 的主链酰胺 N-H 与 Y6 和 Y6 的羟基之间的氢键的第二球体相互作用。 T13 的羟基和 Y6 的 π 电子云之间存在表面氢键（即 T13-OH- - -π-Y6-OH- - --SG）。这些氢键的功能已通过位点特异性诱变和 X 射线晶体学的结合进行了检查。 Y6 的羟基具有约10的正常pKa，即使它与溶剂隔离并且处于很大程度上疏水的环境中。二元Y6F·GSH复合物中GSH的表观pKa增加了1.6个对数单位，并且酶结合的亲核试剂的反应性降低。 Y6L突变体的催化性能与Y6F相同，表明芳环和硫之间的弱极性边相互作用对催化没有影响。与 GSH 复合的 Y6F 突变体的精细三维结构显示，除了硫配位环境的变化外，蛋白质没有发生重大结构扰动。去除 T13V 和 T13A 突变体中 T13 羟基之间的表面氢键的第二球影响，使酶结合 GSH 的 pKa 提高约 0.7 pKa 单位。这些突变体的晶体结构表明，活性位点的结构变化很小，表明 E·GSH 的 pKa 变化是由于表面氢键的存在或不存在造成的。 T13S 突变体在天然酶中具有与 T13 完全不同的侧链氢键几何结构，并且催化特性与 T13A 和 T13V 突变体相似，与表面氢键的缺失一致。 T13 的 γ-甲基对于强化表面氢键几何形状和防止羟基形成更有利的常规氢键至关重要。
The activation of the thiol of glutathione (GSH) bound in the active site of the class mu glutathione transferase M1-1 from rat involves a hydrogen-bonding network that includes a direct (first-sphere) interaction between the hydroxyl group of Y6 and the sulfur of GSH and second-sphere interactions involving a hydrogen bond between the main-chain amide N-H of L12 and the hydroxyl group of Y6 and an on-face hydrogen bond between the hydroxyl group of T13 and the π-electron cloud of Y6 (i.e., T13-OH- - -π-Y6-OH- - --SG). The functions of these hydrogen bonds have been examined with a combination of site-specific mutagenesis and X-ray crystallography. The hydroxyl group of Y6 has a normal pKaof about 10 even though it is shielded from solvent and is in a largely hydrophobic environment. The apparent pKaof GSH in the binary Y6F·GSH complex is increased by 1.6 log units, and the reactivity of the enzyme-bound nucleophile is reduced. The catalytic properties of the Y6L mutant are identical to those of Y6F, suggesting that the weakly polar on-edge interaction between the aromatic ring and sulfur has no influence on catalysis. The refined three-dimensional structure of the Y6F mutant in complex with GSH shows no major structural perturbation of the protein other than a change in the coordination environment of the sulfur. Removal of the second-sphere influence of the on-face hydrogen bond between the hydroxyl group of T13 as in the T13V and T13A mutants elevates the pKaof enzyme-bound GSH by about 0.7 pKaunits. Crystal structures of these mutants show that structural changes in the active site are minor and suggest that the changes in pKaof E·GSH are due to the presence or absence of the on-face hydrogen bond. The T13S mutant has a completely different side-chain hydrogen-bonding geometry than T13 in the native enzyme and catalytic properties similar to the T13A and T13V mutants consistent with the absence of an on-face hydrogen bond. The γ-methyl group of T13 is essential in enforcing the on-face hydrogen bond geometry and preventing the hydroxyl group from forming more favorable conventional hydrogen bonds.