van Iersel M L, Ploemen J P, Lo Bello M, Federici G, van Bladeren P J
Department of Toxicology, Wageningen Agricultural University, The Netherlands.
Chem Biol Interact. 1997 Dec 12;108(1-2):67-78. doi: 10.1016/s0009-2797(97)00096-3.
In the present study the irreversible inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by alpha, beta-unsaturated aldehydes and ketones was studied. When GSTP1-1 was incubated with a 50-fold molar excess of the aldehydes acrolein (ACR) and 4-hydroxy-2-nonenal (HNE) and the ketones curcumin (CUR) and ethacrynic acid (EA) at 22 degrees C, all of them inactivated GSTP1-1. The remaining activity after 4 h of incubation in all cases was lower than 10%. The aldehydes crotonaldehyde (CRA), cinnamaldehyde (CA) and trans-2-hexenal were found to inhibit GSTP1-1 only at a 5000-fold molar excess and even then, for example, for CA a higher remaining activity of 17% was observed. The same inhibition experiments were conducted with 3 mutants of GSTP1-1: the C47S and C101S mutants and the double mutant C47S/C101S. Remaining activity for C47S varied between +/- 40% for CRA, CA, CUR and HEX and +/- 80% for ACR, EA and HNE. For C101S it varied between 0 and 9% and for the double mutant C47S/C101S, activity after 4 h of incubation was variable. Again it varied between +/- 40% for CRA, CA, CUR and HEX and +/- 80% for ACR, EA and HNE. EA is known to react almost exclusively with cysteine 47. When [14C]EA was incubated with the GSTP1-1, modified by the alpha, beta-unsaturated carbonyl compounds, no [14C]EA was incorporated in the enzyme, indicating that in all cases this cysteine residue was one of the major targets. Since Michael addition with these reagents is known to be reversible, the results of incubation of the inactivated enzymes with an excess of glutathione (GSH) were determined. For all compounds, a restoration of the catalytic activity was observed. The results indicate that alpha, beta-unsaturated carbonyl derivatives inhibit GSTP1-1 irreversibly mainly by binding to cysteine residues of GSTP1-1, especially Cys-47, This means that some of these compounds (e.g. CUR) might modify GST activity in vivo when GSH concentrations are low by covalent binding to the enzyme.
在本研究中，对α,β-不饱和醛和酮对人谷胱甘肽S-转移酶P1-1（GSTP1-1）的不可逆抑制作用进行了研究。当GSTP1-1在22℃下与50倍摩尔过量的醛丙烯醛（ACR）、4-羟基-2-壬烯醛（HNE）以及酮姜黄素（CUR）和依他尼酸（EA）一起孵育时，它们均使GSTP1-1失活。在所有情况下，孵育4小时后的剩余活性均低于10%。发现醛巴豆醛（CRA）、肉桂醛（CA）和反式-2-己烯醛仅在5000倍摩尔过量时才抑制GSTP1-1，即便如此，例如对于CA，仍观察到较高的17%的剩余活性。对GSTP1-1的3个突变体进行了相同的抑制实验：C47S和C101S突变体以及双突变体C47S/C101S。C47S对于CRA、CA、CUR和HEX的剩余活性在±40%之间变化，对于ACR、EA和HNE在±80%之间变化。对于C101S，其在0%至9%之间变化，对于双突变体C47S/C101S，孵育4小时后的活性是可变的。同样，对于CRA、CA、CUR和HEX，其在±40%之间变化，对于ACR、EA和HNE在±80%之间变化。已知EA几乎只与半胱氨酸47反应。当将[14C]EA与经α,β-不饱和羰基化合物修饰的GSTP1-1一起孵育时，没有[14C]EA掺入该酶中，这表明在所有情况下，该半胱氨酸残基都是主要靶点之一。由于已知与这些试剂的迈克尔加成是可逆的，因此测定了将失活酶与过量谷胱甘肽（GSH）一起孵育的结果。对于所有化合物，均观察到催化活性的恢复。结果表明，α,β-不饱和羰基衍生物主要通过与GSTP1-1的半胱氨酸残基，尤其是Cys-47结合，不可逆地抑制GSTP1-1。这意味着当谷胱甘肽浓度较低时，这些化合物中的一些（例如CUR）可能通过与该酶共价结合在体内改变GST活性。
