谷胱甘肽转移酶A1-1 c端区芳香族残基影响催化机制中速率决定步骤

Lisa O. Nilsson, Maryam Edalat, Pär L. Pettersson, Bengt Mannervik
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引用次数: 35

摘要

人谷胱甘肽转移酶A1-1 (GST A1-1)具有一个柔性的c端片段,当谷胱甘肽与小的亲电底物如1-氯-2,4-二硝基苯(CDNB)结合时,形成螺旋状(α9)关闭活性位点。在没有活性位点配体的情况下,c端段不固定在一个位置,在晶体结构中无法检测到。α9螺旋上的一个关键残基是Phe 220,它可以与酶结合的谷胱甘肽和第二底物相互作用,并可能引导反应物进入过渡态。结果表明,Phe 220突变为Ala和Thr会降低GST A1-1的催化效率。另一个残基Phe 222的突变导致活性进一步下降。反应介质中粘原的存在降低了野生型GST A1-1催化CDNB偶联的动力学参数Kcat和Kcat/Km,符合产物释放是底物饱和酶限速的观点。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的催化作用有关。与替代底物Δ5-androstene-3,17-二酮(AD)的异构化反应以类似的方式受到粘原的影响。突变对Kcat/Km的影响表明,与CDNB偶联反应一样,异构化反应的过渡态能被Phe 220降低。结果表明,动态c端段的Phe 220和Phe 222对取代反应和异构化反应的催化机制中的速率决定步骤都有影响。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism

Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (α9) 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 α9-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 Kcat and Kcat/Km 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 α9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Δ5-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 Kcat/Km. 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.

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