Bruns C M, Hubatsch I, Ridderström M, Mannervik B, Tainer J A
Department of Molecular Biology MB4, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA, 92037, USA.
J Mol Biol. 1999 May 7;288(3):427-39. doi: 10.1006/jmbi.1999.2697.
The oxidation of lipids and cell membranes generates cytotoxic compounds implicated in the etiology of aging, cancer, atherosclerosis, neurodegenerative diseases, and other illnesses. Glutathione transferase (GST) A4-4 is a key component in the defense against the products of this oxidative stress because, unlike other Alpha class GSTs, GST A4-4 shows high catalytic activity with lipid peroxidation products such as 4-hydroxynon-2-enal (HNE). The crystal structure of human apo GST A4-4 unexpectedly possesses an ordered C-terminal alpha-helix, despite the absence of any ligand. The structure of human GST A4-4 in complex with the inhibitor S-(2-iodobenzyl) glutathione reveals key features of the electrophilic substrate-binding pocket which confer specificity toward HNE. Three structural modules form the binding site for electrophilic substrates and thereby govern substrate selectivity: the beta1-alpha1 loop, the end of the alpha4 helix, and the C-terminal alpha9 helix. A few residue changes in GST A4-4 result in alpha9 taking over a predominant role in ligand specificity from the N-terminal loop region important for GST A1-1. Thus, the C-terminal helix alpha9 in GST A4-4 provides pre-existing ligand complementarity rather than acting as a flexible cap as observed in other GST structures. Hydrophobic residues in the alpha9 helix, differing from those in the closely related GST A1-1, delineate a hydrophobic specificity canyon for the binding of lipid peroxidation products. The role of residue Tyr212 as a key catalytic residue, suggested by the crystal structure of the inhibitor complex, is confirmed by mutagenesis results. Tyr212 is positioned to interact with the aldehyde group of the substrate and polarize it for reaction. Tyr212 also coopts part of the binding cleft ordinarily formed by the N-terminal substrate recognition region in the homologous enzyme GST A1-1 to reveal an evolutionary swapping of function between different recognition elements. A structural model of catalysis is presented based on these results.
脂质和细胞膜的氧化会产生与衰老、癌症、动脉粥样硬化、神经退行性疾病及其他疾病病因相关的细胞毒性化合物。谷胱甘肽转移酶（GST）A4-4是抵御这种氧化应激产物的关键成分，因为与其他α类GST不同，GST A4-4对脂质过氧化产物如4-羟基壬-2-烯醛（HNE）具有高催化活性。人脱辅基GST A4-4的晶体结构出人意料地具有一个有序的C端α螺旋，尽管没有任何配体。人GST A4-4与抑制剂S-(2-碘苄基)谷胱甘肽复合物的结构揭示了亲电底物结合口袋的关键特征，这些特征赋予了对HNE的特异性。三个结构模块形成亲电底物的结合位点，从而决定底物选择性：β1-α1环、α4螺旋末端和C端α9螺旋。GST A4-4中的一些残基变化导致α9在配体特异性方面从对GST A1-1重要的N端环区域取代了主要作用。因此，GST A4-4中的C端螺旋α9提供了预先存在的配体互补性，而不是像在其他GST结构中观察到的那样作为一个灵活的帽。α9螺旋中的疏水残基与密切相关的GST A1-1中的不同，勾勒出一个用于脂质过氧化产物结合的疏水特异性峡谷。抑制剂复合物晶体结构所暗示的残基Tyr212作为关键催化残基的作用，通过诱变结果得到了证实。Tyr212的位置使其能够与底物的醛基相互作用并使其极化以进行反应。Tyr212还占用了同源酶GST A1-1中通常由N端底物识别区域形成的部分结合裂隙，以揭示不同识别元件之间功能的进化交换。基于这些结果提出了一个催化结构模型。
