Nieslanik B S, Dietze E C, Atkins W M, Trong I L, Adman E
Department of Medicinal Chemistry, University of Washington, Seattle 98195-7610, USA.
Pac Symp Biocomput. 1999:554-65. doi: 10.1142/9789814447300_0055.
On the basis of available x-ray structures, A-class glutathione S-transferases (GSTs) contain at their C-termini a short alpha-helix that provides a 'lid' over the active site in the presence of the reaction products, glutathione-conjugates. However, in the ligand-free enzyme this helix is disordered and crystallographically invisible. An aromatic cluster including Phe-10, Phe-220, and the catalytic Tyr-9 within the C-terminal strand control the order of this helix. Here, preliminary x-ray crystallographic analyses of the wild type and F220Y rGSTA1-1 in the presence of GSH are described. Also, a transition state analysis is presented for ligand-dependent formation of the helix, based on variable temperature stopped-flow fluorescence. Together, the results suggest that the ligand-dependent ordering of the C-terminal strand occurs with a transition state that is highly desolvated, but with few intramolecular hydrogen bonds or electrostatic interactions. However, substitutions at Phe-220 modulate the activation parameters through interactions with the side chain of Tyr-9.
基于现有的X射线结构，A类谷胱甘肽S-转移酶（GSTs）在其C末端含有一个短的α-螺旋，在反应产物谷胱甘肽结合物存在时，该螺旋会在活性位点上方形成一个“盖子”。然而，在无配体的酶中，这个螺旋是无序的，在晶体学上不可见。C末端链内包括苯丙氨酸-10、苯丙氨酸-220和催化性酪氨酸-9的一个芳香族簇控制着这个螺旋的有序性。在此，描述了野生型和F220Y rGSTA1-1在谷胱甘肽存在下的初步X射线晶体学分析。此外，基于可变温度停流荧光，对配体依赖的螺旋形成进行了过渡态分析。综合来看，结果表明C末端链的配体依赖有序化发生时的过渡态是高度去溶剂化的，但分子内氢键或静电相互作用很少。然而，苯丙氨酸-220处的取代通过与酪氨酸-9的侧链相互作用来调节活化参数。
