Characterization of the activity and folding of the glutathione transferase from Escherichia coli and the roles of residues Cys(10) and His(106).
Characterization of the activity and folding of the glutathione transferase from Escherichia coli and the roles of residues Cys(10) and His(106).
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DOI：
10.1042/bj20071702
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发表时间：
2009
期刊：
影响因子：
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通讯作者：
Xin-Yu Wang;Zai-Rong Zhang;S. Perrett
中科院分区：
文献类型：
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作者：
Xin-Yu Wang;Zai-Rong Zhang;S. Perrett
GSTs（谷胱甘肽转移酶）是一类重要的酶参与细胞解毒。gst存在于所有种类的生物体中，与对药物、杀虫剂、除草剂和抗生素的耐药性有关。因此，真核生物GSTs的活性、结构和折叠，特别是GSTs的活性、结构和折叠，已被广泛研究。从大肠杆菌中提取的GST的晶体结构早在10年前就有报道，认为Cys（10）和His（106）是潜在的催化残基。然而，这些残基在催化中的作用尚未得到进一步的研究，也没有描述蛋白质的折叠特性。在本研究中，我们研究了残基Cys（10）和His（106）对EGST活性和稳定性的贡献。我们发现EGST显示出一个复杂的平衡展开剖面，涉及至少两个部分折叠的中间体，其中一个是二聚体。残基Cys（10）和His（106）的突变导致蛋白质的稳定，并影响酶催化的表观稳态动力学参数。结果表明His（106）的咪唑环在酶的催化机制中起重要作用，而Cys（10）则参与了底物谷胱甘肽的结合。Cys（10）位点的工程设计可以提高EGST的稳定性和GST活性。然而，除了GST活性，我们发现EGST还具有硫醇：二硫氧化物还原酶活性，其中残基Cys（10）起着至关重要的作用。此外，色氨酸猝灭实验表明，在Cys（10）的游离巯基和谷胱甘肽底物之间形成了混合二硫。
GSTs (glutathione transferases) are an important class of enzymes involved in cellular detoxification. GSTs are found in all classes of organisms and are implicated in resistance towards drugs, pesticides, herbicides and antibiotics. The activity, structure and folding, particularly of eukaryotic GSTs, have therefore been widely studied. The crystal structure of EGST (GST from Escherichia coli) was reported around 10 years ago and it suggested Cys(10) and His(106) as potential catalytic residues. However, the role of these residues in catalysis has not been further investigated, nor have the folding properties of the protein been described. In the present study we investigated the contributions of residues Cys(10) and His(106) to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving a population of at least two partially folded intermediates, one of which is dimeric. Mutation of residues Cys(10) and His(106) leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His(106) plays an important role in the catalytic mechanism of the enzyme, whereas Cys(10) is involved in binding of the substrate, glutathione. Engineering of the Cys(10) site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol:disulfide oxidoreductase activity, for which the residue Cys(10) plays an essential role. Further, tryptophan quenching experiments indicate that a mixed disulfide is formed between the free thiol group of Cys(10) and the substrate, glutathione.