Kolm R H, Sroga G E, Mannervik B
Department of Biochemistry, University of Uppsala, Sweden.
Biochem J. 1992 Jul 15;285 ( Pt 2)(Pt 2):537-40. doi: 10.1042/bj2850537.
The coding region of cDNA corresponding to human class Pi glutathione transferase P1-1 was amplified by the PCR, subcloned into an expression vector, pKHP1, expressed in Escherichia coli, and characterized. The physicochemical and catalytic properties of recombinant glutathione transferase P1-1 were indistinguishable from those of the enzyme previously isolated from human placenta. The active-site residue Tyr-8 of the wild-type enzyme was converted into Phe by means of oligonucleotide-directed mutagenesis. The mutant enzyme Y8F displayed a 300-fold decrease in specific activity, ascribable mainly to a lowered k(cat.) (or V) value. Kinetic parameters reflecting binding affinity, S0.5 (substrate concn. giving 1/2V) and I50 (concn. of inhibitor giving 50% remaining activity), were only moderately elevated in the mutant enzyme. These results indicate that Tyr-8 contributes primarily to catalysis as such, rather than to binding of the substrates. The dependence of k(cat.)/Km on pH shows an optimum at pH 7.0, defined by acidic and basic ionic dissociation constants with pKa1 = 6.7 and pKa2 = 7.3 respectively. The mutant enzyme Y8F does not display the basic limb of the k(cat.)/Km versus pH profile, but shows a monotonic increase of k(cat.)/Km with an apparent pKa1 of 7.1. The results indicate that the phenolic hydroxyl group of Tyr-8 in un-ionized form, but not the phenolate of Tyr-8, contributes to catalysis by glutathione transferase P1-1.
通过聚合酶链反应（PCR）扩增了与人类π类谷胱甘肽转移酶P1-1相对应的cDNA编码区，将其亚克隆到表达载体pKHP1中，在大肠杆菌中表达并进行了表征。重组谷胱甘肽转移酶P1-1的物理化学和催化特性与先前从人胎盘中分离出的酶无法区分。通过寡核苷酸定向诱变将野生型酶的活性位点残基Tyr-8转化为Phe。突变酶Y8F的比活性降低了300倍，这主要归因于k(cat.)（或V）值的降低。反映结合亲和力的动力学参数S0.5（产生1/2V的底物浓度）和I50（产生50%剩余活性的抑制剂浓度）在突变酶中仅适度升高。这些结果表明，Tyr-8主要对催化本身起作用，而不是对底物的结合起作用。k(cat.)/Km对pH的依赖性在pH 7.0处显示出最佳值，分别由酸性和碱性离子解离常数定义，pKa1 = 6.7和pKa2 = 7.3。突变酶Y8F没有显示出k(cat.)/Km与pH曲线的碱性部分，但显示出k(cat.)/Km随表观pKa1为7.1单调增加。结果表明，未电离形式的Tyr-8的酚羟基，而不是Tyr-8的酚盐，对谷胱甘肽转移酶P1-1的催化有贡献。
