Phillips M F, Mantle T J
Department of Biochemistry, Trinity College, Dublin, Ireland.
Biochem J. 1993 Aug 15;294 ( Pt 1)(Pt 1):57-62. doi: 10.1042/bj2940057.
Mouse liver glutathione S-transferase YfYf (Pi class) reacts with [14C]ethacrynic acid to form a covalent adduct with a stoichiometry of 1 mol per mol of subunit. Proteolytic digestion of the enzyme-[14C]ethacrynic acid adduct with V8 protease produced an 11 kDa fragment containing radioactivity. Sequencing revealed this to be an N-terminal peptide (minus the first 15 residues, terminating at Glu-112) which contains only one cysteine residue (Cys-47). This is tentatively identified as the site of ethacrynic attachment. Kinetic studies reveal that glutathione S-conjugates protect against inactivation by ethacrynic acid, but the level of protection is not consistent with their potency as product inhibitors. A model is proposed in which glutathione S-conjugates and ethacrynic acid compete for the free enzyme, and a second molecule of ethacrynic acid reacts covalently with the enzyme-ethacrynic acid complex. The native protein contains one thiol reactive with 5,5'-dithiobis-(2-nitrobenzoic acid) at neutral pH. The resultant mixed disulphide, like the ethacrynic acid adduct, is inactive, but treatment with cyanide (which incorporates on a mol for mol basis) restores activity to 35% of that of the native enzyme.
小鼠肝脏谷胱甘肽S-转移酶YfYf（Pi类）与[14C]依他尼酸反应，形成化学计量比为每摩尔亚基1摩尔的共价加合物。用V8蛋白酶对酶-[14C]依他尼酸加合物进行蛋白水解消化，产生了一个含有放射性的11 kDa片段。测序显示这是一个N端肽（减去前15个残基，终止于Glu-112），其中仅含有一个半胱氨酸残基（Cys-47）。初步确定这是依他尼酸附着的位点。动力学研究表明，谷胱甘肽S-共轭物可防止依他尼酸导致的失活，但保护水平与其作为产物抑制剂的效力不一致。提出了一个模型，其中谷胱甘肽S-共轭物和依他尼酸竞争游离酶，并且第二个依他尼酸分子与酶-依他尼酸复合物发生共价反应。天然蛋白在中性pH下含有一个可与5,5'-二硫代双（2-硝基苯甲酸）反应的硫醇。所得的混合二硫化物与依他尼酸加合物一样无活性，但用氰化物处理（按摩尔比加入）可使活性恢复至天然酶活性的35%。
