Biomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Sciences, Chung-Ang University, 84, Huksuk-Ro, Dongjak-Gu, Seoul 06974, Republic of Korea.
Biomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Sciences, Chung-Ang University, 84, Huksuk-Ro, Dongjak-Gu, Seoul 06974, Republic of Korea.
Pestic Biochem Physiol. 2023 Aug;194:105463. doi: 10.1016/j.pestbp.2023.105463. Epub 2023 May 11.
Plant glutathione S-transferase (GST, EC 2.5.1.18) is an enzyme that detoxifies various electrophilic compounds including herbicides and organic pollutants by catalyzing the formation of conjugates with reduced glutathione (GSH). Although the structure and function of the GST subunits in rice, an important food in Asia, are not well understood, they are crucial for herbicide development. To investigate the role of active site residues in rice Phi-class GSTF3 (OsGSTF3), evolutionarily conserved serine residues were replaced with alanine using site-directed mutagenesis to obtain the mutants S13A, S38A, S69A, and S169A. These four mutants were expressed in Escherichia coli and purified to electrophoretic homogeneity using immobilized GSH affinity chromatography. Mutation of Ser13 to Ala resulted in substantial reductions in specific activities and k/K values for the GSH-[1-chloro-2,4-dinitrobenzene (CDNB)] conjugation reaction. In contrast, mutations of Ser38, Ser69, and Ser169 to Ala had little effect on the activities and kinetic parameters. Additionally, the mutation of Ser13 to Ala significantly affected the K and I values of S-hexylglutathione and S-(2,4-dinitrophenyl)glutathione, which compete with GSH and the product of GSH-CDNB conjugation, respectively. A pH-log (k/K) plot was used to estimate the pK value of GSH in the enzyme-GSH complex of the wild-type enzyme, which was approximately 6.9. However, the pK value of GSH in the enzyme-GSH complex of the S13A mutant was approximately 8.7, which was about 1.8 pK units higher than that of the wild-type enzyme. OsGSTF3 was also crystallized for crystallographic study, and the structure analyses revealed that Ser13 is located in the active site and that its side chain is in close proximity to the thiol group of glutathione bound in the enzyme. Based on these substitution effects on kinetic parameters, the dependence of kinetic parameters on the pH and 3-dimensional structure, it was suggested that Ser13 in rice OsGSTF3 is the residue responsible for catalytic activity by lowering the pK of GSH in the enzyme-GSH complex and enhancing the nucleophilicity of the GSH thiol in the active site.
植物谷胱甘肽 S-转移酶（GST，EC 2.5.1.18）是一种酶，可通过催化与还原型谷胱甘肽（GSH）形成缀合物来解毒各种亲电化合物，包括除草剂和有机污染物。尽管亚洲重要粮食作物水稻中 GST 亚基的结构和功能尚未得到很好的理解，但它们对于除草剂的开发至关重要。为了研究活性位点残基在水稻 Phi 类 GSTF3（OsGSTF3）中的作用，使用定点突变将进化上保守的丝氨酸残基突变为丙氨酸，获得了 S13A、S38A、S69A 和 S169A 突变体。这四个突变体在大肠杆菌中表达，并通过固定化 GSH 亲和层析电泳纯化为电泳纯。将 Ser13 突变为 Ala 导致 GSH-[1-氯-2,4-二硝基苯（CDNB）]缀合反应的比活性和 k/K 值显著降低。相比之下，将 Ser38、Ser69 和 Ser169 突变为 Ala 对活性和动力学参数几乎没有影响。此外，Ser13 突变为 Ala 显著影响 S-己基谷胱甘肽和 S-（2,4-二硝基苯基）谷胱甘肽的 K 和 I 值，它们分别与 GSH 和 GSH-CDNB 缀合物的产物竞争。pH-log（k/K）图用于估计野生型酶中酶-GSH 复合物中 GSH 的 pK 值，约为 6.9。然而，S13A 突变体中酶-GSH 复合物中 GSH 的 pK 值约为 8.7，比野生型酶高约 1.8 pK 单位。OsGSTF3 也进行了结晶用于晶体学研究，结构分析表明 Ser13 位于活性位点，其侧链与结合在酶中的谷胱甘肽的巯基基团接近。基于这些对动力学参数的取代效应、动力学参数对 pH 和三维结构的依赖性，表明水稻 OsGSTF3 中的 Ser13 通过降低酶-GSH 复合物中 GSH 的 pK 值并增强活性位点中 GSH 巯基的亲核性来负责催化活性。
