Lien S, Gustafsson A, Andersson A K, Mannervik B
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden.
J Biol Chem. 2001 Sep 21;276(38):35599-605. doi: 10.1074/jbc.M103789200. Epub 2001 Jul 23.
A study of the kinetics of a heterodimeric variant of glutathione transferase (GST) A1-1 has led to the conclusion that, although the wild-type enzyme displays all-of-the-sites reactivity in nucleophilic aromatic substitution reactions, it demonstrates half-of-the-sites reactivity in addition reactions. The heterodimer, designed to be essentially catalytically inactive in one subunit due to a single point mutation (D101K), and the two parental homodimers were analyzed with seven different substrates, exemplifying three types of reactions catalyzed by glutathione transferases (nucleophilic aromatic substitution, addition, and double-bond isomerization reactions). Stopped-flow kinetic results suggested that the wild-type GST A1-1 behaved with half-of-the-sites reactivity in a nucleophilic aromatic substitution reaction, but steady-state kinetic analyses of the GST A1-D101K heterodimer revealed that this was presumably due to changes to the extinction coefficient of the enzyme-bound product. In contrast, steady-state kinetic analysis of the heterodimer with three different substrates of addition reactions provided evidence that the wild-type enzyme displayed half-of-the-sites reactivity in association with these reactions. The half-of-the-sites reactivity was shown not to be dependent on substrate size, the level of saturation of the enzyme with glutathione, or relative catalytic rate.
一项关于谷胱甘肽转移酶（GST）A1-1异二聚体变体动力学的研究得出结论：尽管野生型酶在亲核芳香取代反应中表现出全位点反应性，但在加成反应中表现出半位点反应性。该异二聚体因单点突变（D101K）而设计为在一个亚基中基本无催化活性，并且用七种不同底物对两个亲本同二聚体和该异二聚体进行了分析，这七种底物代表了谷胱甘肽转移酶催化的三种类型的反应（亲核芳香取代、加成和双键异构化反应）。停流动力学结果表明，野生型GST A1-1在亲核芳香取代反应中表现出半位点反应性，但对GST A1-D101K异二聚体的稳态动力学分析表明，这可能是由于酶结合产物的消光系数发生了变化。相比之下，对该异二聚体与三种不同加成反应底物的稳态动力学分析提供了证据，表明野生型酶在这些反应中表现出半位点反应性。半位点反应性显示不依赖于底物大小、酶与谷胱甘肽的饱和水平或相对催化速率。
