NATURALLY-OCCURRING HUMAN GLUTATHIONE-S-TRANSFERASE GSTP1-1 ISOFORMS WITH ISOLEUCINE AND VALINE IN POSITION-104 DIFFER IN ENZYMATIC-PROPERTIES
NATURALLY-OCCURRING HUMAN GLUTATHIONE-S-TRANSFERASE GSTP1-1 ISOFORMS WITH ISOLEUCINE AND VALINE IN POSITION-104 DIFFER IN ENZYMATIC-PROPERTIES
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DOI：
10.1111/j.1432-1033.1994.00893.x
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发表时间：
1994-09-15
期刊：
影响因子：
--
通讯作者：
AWASTHI, YC
中科院分区：
文献类型：
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作者：
ZIMNIAK, P;NANDURI, B;AWASTHI, YC
谷胱甘肽S - 转移酶p1 - 1异构体在单个氨基酸残基（异亮氨酸104或缬氨酸104）上存在差异，先前已在人胎盘中被鉴定出来[Ahmad, H., Wilson, D. E., Fritz, R. R., Singh, S. V., Medh, R. D., Nagle, G. T., Awasthi, Y. C. 和Kurosky, A. (1990)《生物化学与生物物理学报》278, 398 - 408]。在本报告中，对这两种蛋白质的酶学性质进行了比较。[I104]谷胱甘肽S - 转移酶P1 - 1已从其在大肠杆菌中的cDNA表达出来，并通过亲和层析纯化至均一；该cDNA已被突变，将异亮氨酸104替换为缬氨酸104，并且[V104]谷胱甘肽S - 转移酶P1 - 1按照[I104]谷胱甘肽S - 转移酶P1 - 1所述进行表达和分离。这两种酶在其比活性以及对亲电底物的亲和力方面存在差异（[I - 104]谷胱甘肽S - 转移酶p1 - 1和[V - 104]谷胱甘肽S - 转移酶P1 - 1对1 - 氯 - 2,4 - 二硝基苯的K - M值分别为0.8 mM和3.0 mM），但它们对谷胱甘肽的亲和力相同。此外，这两种酶可通过其热稳定性加以区分，在45℃时的半衰期分别为19分钟和51分钟。底物的存在对热变性的抗性有不同的调节作用。这些数据结合分子模拟表明，104位的残基有助于确定疏水性底物结合位点的几何形状，并且还可能通过与直接参与底物结合的残基相互作用来影响活性。
Glutathione S-transferase p1-1 isoforms, differing in a single amino acid residue (Ile104 or Val104), have been previously identified in human placenta [Ahmad, H., Wilson, D. E., Fritz, R. R., Singh, S. V., Medh, R. D., Nagle, G. T., Awasthi, Y. C. and Kurosky, A. (1990) Arch, Biochem. Biophys. 278, 398-408]. In the present report, the enzymic properties of these two proteins are compared. [Il04]glutathione S-transferase P1-1 has been expressed from its cDNA in Escherichia coli and purified to homogeneity by affinity, chromatography; the cDNA has been mutated to replace Ile104 by Val104, and [V104]glutathione S-transferase P1-1 was expressed and isolated as described for [I104]glutathione S-transferase P1-1. The two enzymes differed in their specific activity and affinity for electrophilic substrates (K-M values for 1-chloro-2,4-dinitrobenzene were 0.8 mM and 3.0 mM for [I-104]glutathione S-transferase p1-1 and [V-104]glutathione S-transferase P1-1, respectively), but were identical in their affinity for glutathione. In addition, the two enzymes were distinguishable by their heat stability, with half-lives at 45 degrees C of 19 min and 51 min, respectively. The resistance to heat denaturation was differentially modulated by the presence of substrates. These data, in conjunction with molecular modeling, indicate that the residue in position 104 helps to define the geometry of the hydrophobic substrate-binding site, and may also influence activity by interacting with residues directly involved in substrate binding.