Nilsson Lisa O, Edalat Maryam, Pettersson Pär L, Mannervik Bengt
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden
Biochim Biophys Acta. 2002 Jul 29;1598(1-2):199-205. doi: 10.1016/s0167-4838(02)00362-x.
Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k(cat) and k(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.
人谷胱甘肽转移酶A1-1（GST A1-1）具有一个灵活的C末端片段，该片段形成一个螺旋（α9），在谷胱甘肽和小的亲电底物如1-氯-2,4-二硝基苯（CDNB）结合时封闭活性位点。在没有活性位点配体的情况下，C末端片段不固定在一个位置，并且在晶体结构中不可检测。α9螺旋中的一个关键残基是苯丙氨酸220，它可以与酶结合的谷胱甘肽和第二种底物相互作用，并可能引导反应物进入过渡态。将苯丙氨酸220突变为丙氨酸和苏氨酸会降低GST A1-1的催化效率。另一个残基苯丙氨酸222的突变导致活性进一步降低。反应介质中存在增粘剂会降低野生型GST A1-1催化CDNB共轭反应的动力学参数k(cat)和k(cat)/K(m)，这与产物释放是底物饱和酶的限速步骤这一观点一致。这些突变导致两个动力学参数的粘度依赖性降低，表明α9螺旋的运动与野生型GST A1-1中的催化作用相关。与替代底物Δ(5)-雄烯-3,17-二酮（AD）的异构化反应受到增粘剂的类似影响。如突变对k(cat)/K(m)的影响所示，异构化反应的过渡态能量与CDNB共轭反应一样，被苯丙氨酸220降低。结果表明，动态C末端片段中的苯丙氨酸220和苯丙氨酸222影响取代反应和异构化反应催化机制中的限速步骤。
