The Interaction of the Chemotherapeutic Drug Chlorambucil with Human Glutathione Transferase A1-1: Kinetic and Structural Analysis

Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/CBL-GSH complex was solved at 2.1 Å resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the C-terminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated.

谷胱甘肽转移酶（Glutathione transferases, GSTs）是一类通过催化谷胱甘肽（glutathione, GSH）对多种亲电性外源化合物（含多种化疗药物）的亲核进攻，参与细胞解毒过程的酶类。本研究采用动力学分析、蛋白质晶体学与分子动力学技术，探究了化疗药物苯丁酸氮芥（chlorambucil, CBL）与人源GSTA1-1（hGSTA1-1）的相互作用。在GSH存在的条件下，CBL可作为hGSTA1-1的高效底物。CBL与GSH之间催化反应的限速步骤具有粘度依赖性，动力学数据表明产物释放为该反应的限速环节。研究人员通过分子置换法，解析了分辨率为2.1 Å的hGSTA1-1/CBL-GSH复合物晶体结构。CBL结合于与GSH巯基相连的H位点，呈部分有序构象且暴露于溶剂环境，可与酶产生特异性相互作用。基于该晶体结构开展的分子动力学模拟显示，CBL基团具有较高的流动性，且加合物的存在可稳定酶的C端螺旋结构。在缺乏GSH的情况下，CBL可作为hGSTA1-1的烷基化不可逆抑制剂。CBL介导的酶失活过程遵循双相准一级饱和动力学规律，每摩尔二聚体酶可结合约1摩尔的CBL。结构分析表明，发生共价修饰的氨基酸残基为位于H位点入口处的Cys112。研究结果显示亚基间存在结构通讯：由于Cys112的修饰具有互斥性，提示该酶的两个活性位点大概率存在协同调控。