Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 105
Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 105
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DOI：
10.1006/jmbi.1998.1708
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发表时间：
1998-05-08
影响因子：
5.6
通讯作者：
Mannervik, B
中科院分区：
文献类型：
--
作者：
Johansson, AS;Stenberg, G;Mannervik, B
人谷胱甘肽转移酶P1-1(GSTP1-1)在第105位氨基酸残基上具有多态性，位于底物结合II位点。为了阐明该残基的作用，我们对GSTP1-1/Ile105和GSTP1-1/Val105进行了广泛的表征。构建了残基105的体积和疏水性改变的突变体酶GSTP1-1/Ala105和GSTP1-1/Trp105。使用一组亲电底物测定了稳态动力学参数和比活性，目的是覆盖不同类型的反应机理。对稳态动力学参数的分析表明，第105位氨基酸的取代对底物的影响很大。当以1-氯-2，4-二硝基苯为底物时，残基105侧链的改变似乎主要引起K-M_1值的变化，而k(CAT)值不受明显影响。对于其他底物，如7-氯-4-硝基-2-氧杂-1，3-二氮唑和乙丙氨酸，其K(CAT)和K-M值都被氨基酸105取代而改变。1，3，5-三硝基苯与谷胱甘肽形成Sigma络合物的常数与105位氨基酸的体积有关。第105位氨基酸的性质也影响了酶在50℃下的热稳定性，这表明该残基在酶的稳定中起着重要作用。从半衰期来看，GSTP1-1/Ile105变异体的稳定性大约是Val105变异体的两到三倍。在孵化缓冲液中，谷胱甘肽的存在使酶的半衰期增加了三倍。因此，GSTP1-1第105位的取代会影响酶的热稳定性，并且根据底物的不同，K-M值和周转次数都会受到影响。(C)1998年学术出版社有限公司。
Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding II-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 us ed. 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 K-M,value, while the k(cat) value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both k(cat) and K-M 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, tl-le thermal stability of the enzyme and depending on substrate, both K-M values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1. (C) 1998 Academic Press Limited.