Robinson Anna, Huttley Gavin A, Booth Hilary S, Board Philip G
John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, ACT 2601, Australia.
Biochem J. 2004 May 1;379(Pt 3):541-52. doi: 10.1042/BJ20031656.
The Kappa class of GSTs (glutathione transferases) comprises soluble enzymes originally isolated from the mitochondrial matrix of rats. We have characterized a Kappa class cDNA from human breast. The cDNA is derived from a single gene comprising eight exons and seven introns located on chromosome 7q34-35. Recombinant hGSTK1-1 was expressed in Escherichia coli as a homodimer (subunit molecular mass approximately 25.5 kDa). Significant glutathione-conjugating activity was found only with the model substrate CDNB (1-chloro-2,4-ditnitrobenzene). Hyperbolic kinetics were obtained for GSH (parameters: K(m)app, 3.3+/-0.95 mM; V(max)app, 21.4+/-1.8 micromol/min per mg of enzyme), while sigmoidal kinetics were obtained for CDNB (parameters: S0.5app, 1.5+/-1.0 mM; V(max)app, 40.3+/-0.3 micromol/min per mg of enzyme; Hill coefficient, 1.3), reflecting low affinities for both substrates. Sequence analyses, homology modelling and secondary structure predictions show that hGSTK1 has (a) most similarity to bacterial HCCA (2-hydroxychromene-2-carboxylate) isomerases and (b) a predicted C-terminal domain structure that is almost identical to that of bacterial disulphide-bond-forming DsbA oxidoreductase (root mean square deviation 0.5-0.6 A). The structures of hGSTK1 and HCCA isomerase are predicted to possess a thioredoxin fold with a polyhelical domain (alpha(x)) embedded between the beta-strands (betaalphabetaalpha(x)betabetaalpha, where the underlined elements represent the N and C motifs of the thioredoxin fold), as occurs in the bacterial disulphide-bond-forming oxidoreductases. This is in contrast with the cytosolic GSTs, where the helical domain occurs exclusively at the C-terminus (betaalphabetaalphabetabetaalphaalpha(x)). Although hGSTK1-1 catalyses some typical GST reactions, we propose that it is structurally distinct from other classes of cytosolic GSTs. The present study suggests that the Kappa class may have arisen in prokaryotes well before the divergence of the cytosolic GSTs.
谷胱甘肽S-转移酶（GSTs）的κ类包含最初从大鼠线粒体基质中分离出的可溶性酶。我们已对来自人类乳腺的κ类cDNA进行了表征。该cDNA源自一个单基因，该基因由位于7号染色体q34 - 35上的8个外显子和7个内含子组成。重组人GSTK1 - 1在大肠杆菌中表达为同型二聚体（亚基分子量约为25.5 kDa）。仅发现其对模型底物1 - 氯 - 2,4 - 二硝基苯（CDNB）具有显著的谷胱甘肽结合活性。对于谷胱甘肽（GSH）呈现双曲线动力学（参数：表观米氏常数K(m)app，3.3±0.95 mM；最大表观反应速度V(max)app，每毫克酶21.4±1.8微摩尔/分钟），而对于CDNB呈现S形动力学（参数：半最大反应速度S0.5app，1.5±1.0 mM；V(max)app，每毫克酶40.3±0.3微摩尔/分钟；希尔系数，1.3），这反映了对两种底物的低亲和力。序列分析、同源建模和二级结构预测表明，人GSTK1与细菌的2 - 羟基色烯 - 2 - 羧酸盐（HCCA）异构酶最为相似，并且其预测的C末端结构域与细菌的形成二硫键的DsbA氧化还原酶几乎相同（均方根偏差为0.5 - 0.6 Å）。预测人GSTK1和HCCA异构酶的结构具有硫氧还蛋白折叠，在β链之间嵌入一个多螺旋结构域（α(x)）（β - α - β - α(x) - β - β - α，其中下划线部分代表硫氧还蛋白折叠的N和C基序），这与细菌形成二硫键的氧化还原酶中的情况相同。这与胞质GSTs不同，在胞质GSTs中，螺旋结构域仅出现在C末端（β - α - β - α - α - β - β - α(x)）。尽管人GSTK1 - 1催化一些典型的GST反应，但我们认为它在结构上与其他类别的胞质GSTs不同。本研究表明，κ类可能在胞质GSTs分化之前就已在原核生物中出现。
