Kropp Timothy J, Richardson Rudy J
Toxicology Program, Department of Environmental Health Sciences, The University of Michigan, Ann Arbor, Michigan 48109-2029, USA.
Chem Res Toxicol. 2007 Mar;20(3):504-10. doi: 10.1021/tx600310y. Epub 2007 Feb 27.
Elucidating mechanisms of aging of esterases inhibited by organophosphorus (OP) compounds is important for understanding toxicity and developing biomarkers of exposure to these agents. Aging has classically been thought to involve net loss of a single side group from the OP moiety of phosphylated esterases, rendering the enzyme refractory to reactivation. However, recent evidence has shown that acetylcholinesterase (AChE) and the catalytic domain of human neuropathy target esterase (NEST) undergo aging by alternative mechanisms following their inhibition with N,N'-diisopropylphosphorodiamidofluoridate (mipafox, MIP). This study was performed to determine whether MIP-inhibited butyrylcholinesterase (BChE) ages conventionally, by net loss of a single side group, or by an alternate route, e.g., reversible deprotonation or displacement of both isopropylamine groups, as recently observed for MIP-inhibited NEST and AChE, respectively. Diisopropylphosphorofluoridate (DFP), the phosphate analogue of the phosphoroamidate MIP, was used for comparison. Kinetic values for MIP against BChE were as follows: ki = (1.28 +/- 0.053) x 10(6) M-1 min-1; k3 = 0.004,15 +/- 0.000,27 min-1; k4 = 0.008,49 +/- 0.000,99 min-1. Kinetic values for DFP against BChE were as follows: ki = (1.83 +/- 0.18) x 10(6) M-1 min-1; k3 = 0.004,88 +/- 0.000,24 min-1; k4 = 0.0121 +/- 0.0028 min-1. Mass spectrometric studies revealed a mass shift of 123.4 +/- 0.7 Da for the active-site peptide peak of aged DFP-inhibited BChE, corresponding to a monoisopropylphosphate adduct. Similarly, the analogous mass shift for aged MIP-inhibited BChE was 122.4 +/- 0.7 Da, corresponding to a monoisopropylphosphoroamido adduct. Therefore, we conclude that the MIP-BChE conjugate ages by loss of a single isopropylamine group, in contrast to MIP-inhibited AChE or NEST.
阐明有机磷（OP）化合物抑制的酯酶的老化机制，对于理解这些药剂的毒性和开发暴露生物标志物非常重要。传统上认为老化涉及磷酸化酯酶的OP部分单个侧基的净损失，使酶难以再活化。然而，最近的证据表明，乙酰胆碱酯酶（AChE）和人类神经病靶标酯酶（NEST）的催化结构域在用N，N'-二异丙基磷酰二氨基氟化物（丙胺氟磷，MIP）抑制后通过替代机制发生老化。进行这项研究是为了确定MIP抑制的丁酰胆碱酯酶（BChE）是按传统方式老化，即单个侧基的净损失，还是通过替代途径老化，例如可逆去质子化或两个异丙胺基团的置换，这分别是最近在MIP抑制的NEST和AChE中观察到的。磷酸氨基酯MIP的磷酸盐类似物二异丙基磷酰氟（DFP）用于比较。MIP对BChE的动力学值如下：ki =（1.28±0.053）×10（6）M-1 min-1；k3 = 0.004,15±0.000,27 min-1；k4 = 0.008,49±(0.000,99) min-1。DFP对BChE的动力学值如下：ki =（1.83±0.18）×10（6）M-1 min-1；k3 = 0.004,88±0.000,24 min-1；k4 = 0.0121±0.0028 min-1。质谱研究显示，老化的DFP抑制的BChE活性位点肽峰的质量位移为123.4±0.7 Da，对应于单异丙基磷酸加合物。同样，老化的MIP抑制的BChE的类似质量位移为122.4±0.7 Da，对应于单异丙基磷酰胺加合物。因此，我们得出结论，与MIP抑制的AChE或NEST相反，MIP - BChE共轭物通过单个异丙胺基团的损失而老化。
