Johansson A S, Stenberg G, Widersten M, Mannervik B
Department of Biochemistry, Uppsala University, Uppsala, S-751 23, Sweden.
J Mol Biol. 1998 May 8;278(3):687-98. doi: 10.1006/jmbi.1998.1708.
Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the KM value, while the kcat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both kcat and KM values were altered by the substitution of amino acid 105. The constant for formation of the sigma-complex between 1,3, 5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both KM values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1.
人谷胱甘肽转移酶P1-1（GSTP1-1）在位于底物结合H位点的氨基酸残基105处具有多态性。为了阐明该残基的作用，对GSTP1-1/Ile105和GSTP1-1/Val105进行了广泛的表征。构建了残基105体积和疏水性改变的突变酶GSTP1-1/Ala105和GSTP1-1/Trp105，并将其纳入研究。使用一组亲电底物测定稳态动力学参数和比活性，目的是涵盖不同类型的反应机制。稳态动力学参数分析表明，105位氨基酸取代的影响高度依赖于所使用的底物。当使用1-氯-2,4-二硝基苯作为底物时，残基105侧链的变化似乎主要导致KM值的变化，而kcat值没有受到明显影响。对于其他底物，如7-氯-4-硝基苯并-2-恶唑-1,3-二唑和依他尼酸，kcat和KM值都因105位氨基酸的取代而改变。结果表明，1,3,5-三硝基苯与谷胱甘肽之间形成σ-复合物的常数取决于105位氨基酸的体积。还表明105位氨基酸的性质会影响酶在50℃时的热稳定性，表明该残基在酶的稳定性中起重要作用。根据半衰期判断，GSTP1-1/Ile105变体比Val105变体稳定约两到三倍。孵育缓冲液中谷胱甘肽的存在使酶的半衰期增加了三倍。因此，酶的热稳定性以及取决于底物的情况，KM值和周转数都受到GSTP1-1第105位取代的影响。
