人胎盘谷胱甘肽S-转移酶中巯基的研究

Studys on Sulfhydryl Group of Human Placenta Glutathione S-transferase

用巯基试剂5.5'-二硫双(2-硝基苯甲酸)(DTNB)测得人胎盘谷胱甘肽S-转移酶(GST-π)的总巯基数为每亚基2个,均为表面巯基,,其中一个与DTNB反应快,被修饰后可导致酶活力全部丧失。另一巯基与DTNB反应较慢,可能与酶活力无关。用在12℃测定剩余巯基和Stallcup-Koshland作图法求得DTNB修饰快反应和慢反应巯基的速度常数分别为44056和162min^(-1)(mol/L)^(-1)。底物谷胱甘肽的衍生物S-正辛烷谷胱甘肽(S-o-GSH)能保护GST-π能保护的快反应巯基免受DTNB的修饰,使反应速度常数随着S-o-GSH浓度的增高而降低。S-o-GSH也能保护酶被N-乙基马来酰亚胺(NEMI)修饰失活,但不能保护慢反应巯基被DTNB修饰。另一底物2,4-二硝基氯苯(CDNB)对NEMI的修饰失活没有保护作用。上述结果提示快反应巯基参与GST-π和谷胱甘肽的结合,是组成活性中心的重要基因。

Two sulfhydryl(SH)groups per subunit of placenta glutathione S-transferase (GST-π) could be detected by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), both being located on the surface of the enzyme. One SH group reacted quickly with DTNB, and the enzyme was entirely inactivated after this SH group was modified. The another SH group reacted with DTNB relativelyslowly, and possibly not related to enzyme activity. With the method of determinating the residual SH group at 12℃ and the Stallcup-Koshland plot, the rate constants of DTNB with the fast and slow-reacting SH-group were determined to be 44056 and 162 min-1 (mol/L)-1 respectively. A derivative of the substrate GSH, S-n-octylglutathione (S-o-GSH) , protected the fast-reacting SH-group of GST-π from modification by DTNB, and the rate constant decreased with increasing concentration of S-o-GSH. S-o-GSH also protected the enzyme from modification by NEMI, but it did not protect the slow-reacting SH-group from modification by DTNB. Another substrate, l-chloro-2,4-dinitrobenzene (CDNB) showed no effect on GST-π modification and inactivation by NEMI. All the above mentioned results suggested that the fast-reacting SH-group participated in the binding of GSH to GST-π as an important group in the active center.