Li Dandan, Zhang Yunze, Song Haitao, Lu Liangqiu, Liu Deli, Yuan Yongze
Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan, Hubei, P. R. China.
Department of Biochemistry, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, Hubei, P.R. China.
PLoS One. 2017 Jan 13;12(1):e0169937. doi: 10.1371/journal.pone.0169937. eCollection 2017.
Aminoalcohols have been addressed as activating buffers for alkaline phosphatase. However, there is no record on the buffer activation regarding organophosphorus hydrolase (OPH). Here we reported the activating effects of aminoalcohols on OPH-catalyzed hydrolysis of diisopropylfluorophosphate (DFP), an analog molecule of G-type warfare agents. The kinetic parametors kcat, Vmax and kcat/Km in the OPH reaction were remarkably increased in the buffers (pH 8.0, 25°C) containing aminoalcohols with C2 between nitrogen (N) and oxygen (O) in their structures, including triethanolamine (TEA), diethanolamine, monoethanolamine, 1-amino-2-propanol, 2-amino-2-methyl-1-propanol, and triisopropanolamine. In contrast, much lower or no rate-enhancing effects were observed in the adding of amines, alcohols, amine/alcohol mixtures, or 3-amino-1-propanol (C3 between N and O). The 300 mM TEA further increased DFP-degrading activities of OPH mutants F132Y and L140Y, the previously reported OPH mutants with desirable activities towards DFP. However, the treatment of ethylenediaminetetraacetate (EDTA) markedly abolished the TEA-induced activation of OPH. The product fluoride effectively inhibited OPH-catalyzed hydrolysis of DFP by a linear mixed inhibition (inhibition constant Ki ~ 3.21 mM), which was partially released by TEA adding at initial or later reaction stage. The obtained results indicate the activation of OPH by aminoalcohol buffers could be attributed to the reduction of fluoride inhibition, which would be beneficial to the hydrolase-based detoxification of organophosphofluoridate.
氨基醇已被视为碱性磷酸酶的激活缓冲剂。然而，关于有机磷水解酶（OPH）的缓冲剂激活作用尚无相关记录。在此，我们报道了氨基醇对OPH催化水解二异丙基氟磷酸酯（DFP，一种G型战争毒剂类似物分子）的激活作用。在含有结构中氮（N）和氧（O）之间有C2的氨基醇的缓冲液（pH 8.0，25°C）中，包括三乙醇胺（TEA）、二乙醇胺、单乙醇胺、1-氨基-2-丙醇、2-氨基-2-甲基-1-丙醇和三异丙醇胺，OPH反应中的动力学参数kcat、Vmax和kcat/Km显著增加。相比之下，添加胺、醇、胺/醇混合物或3-氨基-1-丙醇（N和O之间有C3）时，观察到的速率增强作用要低得多或没有。300 mM的TEA进一步提高了OPH突变体F132Y和L140Y（先前报道的对DFP具有理想活性的OPH突变体）的DFP降解活性。然而，乙二胺四乙酸（EDTA）处理显著消除了TEA诱导的OPH激活。产物氟化物通过线性混合抑制（抑制常数Ki约为3.21 mM）有效抑制OPH催化的DFP水解，在初始或反应后期添加TEA可部分解除这种抑制。所得结果表明，氨基醇缓冲剂对OPH的激活作用可能归因于氟化物抑制作用的降低，这将有利于基于水解酶的有机磷氟化物解毒。
