Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione S-transferase.
Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione S-transferase.
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DOI：
10.1016/s0021-9258(18)42831-1
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发表时间：
1992-03
期刊：
影响因子：
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通讯作者：
Suxing Liu;Pinghui Zhang;Xinhua Ji;William W. Johnson;G. Gilliland;Richard N. Armstrong
中科院分区：
文献类型：
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作者：
Suxing Liu;Pinghui Zhang;Xinhua Ji;William W. Johnson;G. Gilliland;Richard N. Armstrong
通过X射线晶体学和用苯丙氨酸对残基进行位点特异性取代以及评估突变酶的催化特性，研究了酪氨酸6的羟基在大鼠谷胱甘肽S-转移酶同工酶3-3的催化机制中的作用。这种特殊的酪氨酸残基在所有胞质酶的序列中都是保守的，并且在 mu 基因类同工酶 3-3 和 pi 基因类同工酶的晶体结构中被发现最接近于活性位点结合的谷胱甘肽 (GSH) 或谷胱甘肽磺酸盐的硫。同工酶3-3和GSH二元复合物的2.2-A结构表明Tyr6的羟基距离GSH的硫3.2-3.5 A，完全在氢键距离内。 Tyr6 羟基的去除对 GSH 的解离常数 (22 +/- 3 microM) 基本上没有影响。然而，当在 pH 6.5 下用 1-氯-2,4-二硝基苯 (CDNB) 或 4-苯基-3-丁烯-2-酮进行测定时，Y6F 突变体的周转数仅为天然酶的 1% 左右。二元酶-GSH复合物的UV差异光谱表明，Y6F突变体活性位点中GSH的主要电离态是中性硫醇（例如EY6F.GSH），这与硫醇基本上去质子化的天然酶（例如E.GS-）相反。分光光度滴定表明，E.GSH 复合物中硫醇的 pKa 为 6.9 +/- 0.3，而 EY6F.GSH 二元复合物中硫醇的 pKa 大于或等于 8。此外，kcat/KmCDNB 的 pH 依赖性表明，天然酶和 Y6F 突变体催化的反应依赖于 E.GSH 和 EY6F.GSH 复合物中的单电离，pKa 分别为 6.2 +/- 0.1 和 7.8 +/- 0.3。结果表明，Tyr6 和酶结合亲核试剂之间的氢键有助于降低二元酶-底物复合物中 GSH 的 pKa。
The role of the hydroxyl group of tyrosine 6 in the catalytic mechanism of isoenzyme 3-3 of rat glutathione S-transferase has been examined by x-ray crystallography and site-specific replacement of the residue with phenylalanine and evaluation of the catalytic properties of the mutant enzyme. This particuar tyrosine residue is conserved in the sequences of all of the cytosolic enzymes and is found, in crystal structures of both isoenzyme 3-3 from the mu-gene class and an isoenzyme from the pi-gene class, to be proximal to the sulfur of glutathione (GSH) or glutathione sulfonate bound at the active site. The 2.2-A structure of the binary complex of isoenzyme 3-3 and GSH indicates that the hydroxyl group of Tyr6 is located 3.2-3.5 A from the sulfur of GSH, well within hydrogen bonding distance. Removal of the hydroxyl group of Tyr6 has essentially no effect on the dissociation constant (22 +/- 3 microM) for GSH. Nevertheless the Y6F mutant exhibits a turnover number which is only about 1% that of the native enzyme when assayed at pH 6.5 with either 1-chloro-2,4-dinitrobenzene (CDNB) or 4-phenyl-3-buten-2-one. UV difference spectra of the binary enzyme-GSH complexes suggest that the predominant ionization state of GSH in the active site of the Y6F mutant is the neutral thiol (e.g. EY6F.GSH) which is in contrast to the native enzyme in which the thiol is substantially deprotonated (e.g. E.GS-). Spectrophotometric titration suggests that the pKa of the thiol is 6.9 +/- 0.3 in the E.GSH complex and greater than or equal to 8 in the EY6F.GSH binary complex. In addition, the pH dependence of kcat/KmCDNB reveals that the reactions catalyzed by the native enzyme and the Y6F mutant are dependent on a single ionization in the E.GSH and EY6F.GSH complexes with pKa = 6.2 +/- 0.1 and 7.8 +/- 0.3, respectively. The results suggest that the hydrogen bond between Tyr6 and the enzyme-bound nucleophile helps to lower the pKa of GSH in the binary enzyme-substrate complex.