Irreversible inhibition of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius
Irreversible inhibition of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius
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DOI：
10.1007/s00792-008-0179-1
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发表时间：
2008-09-01
期刊：
影响因子：
2.9
通讯作者：
Manco, Giuseppe
中科院分区：
文献类型：
--
作者：
Febbraio, Ferdinando;D'Andrea, Sandro Esposito;Manco, Giuseppe
近年来，不可逆抑制的动力学研究因其与基础科学兴趣问题的相关性及其实际重要性而受到越来越多的关注。我们的研究致力于表征众所周知的乙酰胆碱酯酶不可逆抑制剂对嗜热真细菌酸热脂环酸芽孢杆菌的羧酸酯酶（EST2）的影响。特别是，磺酰抑制剂和有机磷杀虫剂磷酸二乙基对硝基苯酯（对氧磷）已得到研究。根据伪一级动力学，EST2 与磺酰抑制剂的孵育导致时间依赖性失活。另一方面，对氧磷引起的 EST2 失活过程是添加抑制剂后立即完成的抑制反应，不能描述为伪一级动力学，而最好认为是高亲和力抑制。对氧磷失活的表观速率常数值通过监测抑制剂存在下的酶/底物反应来确定，并与磺酰抑制剂的表观速率常数值进行比较。还研究了竞争性抑制剂对 EST2 不可逆抑制的保护作用，以及羟胺和 2-PAM 对复合酶/不可逆抑制剂的重新激活。考虑到所使用的不可逆抑制剂能够区分两个不同的结合位点，根据最近描述的 EST2 双底物结合模式讨论了数据。
Kinetic studies of irreversible inhibition in recent years have received growing attention owing to their relevance to problems of basic scientific interest as well as to their practical importance. Our studies have been devoted to the characterization of the effects that well-known acetylcholinesterase irreversible inhibitors exert on a carboxylesterase (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius. In particular, sulfonyl inhibitors and the organophosphorous insecticide diethyl-p-nitrophenyl phosphate (paraoxon) have been studied. The incubation of EST2 with sulfonyl inhibitors resulted in a time-dependent inactivation according to a pseudo-first-order kinetics. On the other hand, the EST2 inactivation process elicited by paraoxon, being the inhibition reaction completed immediately after the inhibitor addition, cannot be described as a pseudo-first-order kinetics but is better considered as a high affinity inhibition. The values of apparent rate constants for paraoxon inactivation were determined by monitoring the enzyme/substrate reaction in the presence of the inhibitor, and were compared with those of the sulfonyl inhibitors. The protective effect afforded by a competitive inhibitor on the EST2 irreversible inhibition, and the reactivation of a complex enzyme/irreversible-inhibitor by hydroxylamine and 2-PAM, were also investigated. The data have been discussed in the light of the recently described dual substrate binding mode of EST2, considering that the irreversible inhibitors employed were able to discriminate between the two different binding sites.