Tan K L, Chelvanayagam G, Parker M W, Board P G
Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra, Australia.
Biochem J. 1996 Oct 1;319 ( Pt 1)(Pt 1):315-21. doi: 10.1042/bj3190315.
The role of serine-11 in the catalytic mechanism of recombinant human GSTT2-2 was examined by site-directed mutagenesis. Amino acid sequence comparison of the Theta-class isoenzymes has identified a conserved serine residue in the N-terminal domain [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133-2143]. This conserved serine has been implicated in the activation of the enzyme-bound glutathione [Board, Coggan and Parker (1995) Biochem. J. 311, 247-250]. Mutating the equivalent serine (residue 11) of GSTT2-2 to Ala, Thr or Tyr abolished the catalytic properties of GSTT2-2 with cumene hydroperoxide and ethacrynic acid as second substrate. However, with l-menaphthyl sulphate (MSu) as the second substrate, the specific activity of the S11A mutant was doubled, while the S11T mutant retained half the wild-type activity and the S11Y mutant was inactive. The role of Ser-11 in catalysis seems to vary with different second substrates. In the substitution reaction with MSu, GSTT2-2 activity appears to depend on the size of the Ser-11 replacement rather than the presence of a side-chain hydroxy group. In addition, the reaction rate appears to be a function of pH, and there is no non-enzymic reaction even at high pH. We demonstrated that a reaction between MSu and an alternative thiol such as L-cysteine or 2-mercaptoethanol can take place in the presence of S-methylglutathione and GSTT2-2. We propose that the catalytic activity of GSTT2-2 with MSu is preceded by a conformational or charge modification to the enzyme upon the binding of glutathione or S-methylglutathione. This is followed by the binding of MSu and the subsequent removal of the sulphate group, giving rise to the carbonium ion of l-methylnaphthelene as the electrophile that reacts with the nucleophilic species. The reaction mechanism of GSTT2-2 with MSu may represent a novel function of GSTT2-2 as a glutathione-dependent sulphatase.
通过定点突变研究了丝氨酸-11在重组人GSTT2-2催化机制中的作用。θ类同工酶的氨基酸序列比较已确定在N端结构域有一个保守的丝氨酸残基[威尔西、博德、费尔和帕克（1995年）《欧洲分子生物学组织杂志》14卷，2133 - 2143页]。这个保守的丝氨酸与酶结合型谷胱甘肽的激活有关[博德、科根和帕克（1995年）《生物化学杂志》311卷，247 - 250页]。将GSTT2-2的等效丝氨酸（第11位残基）突变为丙氨酸、苏氨酸或酪氨酸，消除了GSTT2-2以氢过氧化异丙苯和依他尼酸作为第二底物时的催化特性。然而，以硫酸1-萘酯（MSu）作为第二底物时，S_{11}A突变体的比活性加倍，而S_{11}T突变体保留了野生型活性的一半，S_{11}Y突变体无活性。丝氨酸-11在催化中的作用似乎因不同的第二底物而异。在与MSu的取代反应中，GSTT2-2的活性似乎取决于丝氨酸-11取代基的大小，而不是侧链羟基的存在。此外，反应速率似乎是pH的函数，即使在高pH下也没有非酶促反应。我们证明了在S-甲基谷胱甘肽和GSTT2-2存在的情况下，MSu与另一种硫醇如L-半胱氨酸或2-巯基乙醇之间可以发生反应。我们提出，GSTT2-2与MSu的催化活性在谷胱甘肽或S-甲基谷胱甘肽结合后，酶会发生构象或电荷修饰。随后是MSu的结合以及随后硫酸根的去除，产生1-甲基萘的碳正离子作为与亲核物种反应的亲电试剂。GSTT2-2与MSu的反应机制可能代表了GSTT2-2作为一种谷胱甘肽依赖性硫酸酯酶的新功能。
