Chambers Carolyn, Luo Chunyuan, Tong Min, Yang Yerie, Saxena Ashima
Walter Reed Army Institute of Research, Division of Biochemistry, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA; US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910-7500, USA.
Walter Reed Army Institute of Research, Division of Biochemistry, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA.
Toxicol In Vitro. 2015 Mar;29(2):408-14. doi: 10.1016/j.tiv.2014.11.001. Epub 2014 Nov 13.
In this study, we employed site-directed mutagenesis to understand the role of amino acids in the gorge in oxime-induced reactivation of nerve agent-inhibited human (Hu) acetylcholinesterase (AChE). The organophosphorus (OP) nerve agents studied included GA (tabun), GB (sarin), GF (cyclosarin), VX, and VR. The kinetics of reactivation were examined using both the mono-pyridinium oxime 2-PAM and bis-pyridinium oximes MMB4, HI-6, and HLö-7. The second-order reactivation rate constants were used to compare reactivation of nerve agent-inhibited wild-type (WT) and mutant enzymes. Residues including Y72, Y124 and W286 were found to play important roles in reactivation by bis-pyridinium, but not by mono-pyridinium oximes. Residue Y124 also was found to play a key role in reactivation by HI-6 and HLö-7, while E202 was important for reactivation by all oximes. Residue substitutions of F295 by Leu and Y337 by Ala showed enhanced reactivation by bis-pyridinium oximes MMB4, HI-6, and HLö-7, possibly by providing more accessibility of the OP moiety associated at the active-site serine to the oxime. These results are similar to those observed previously with bovine AChE and demonstrate that there is significant similarity between human and bovine AChEs with regard to oxime reactivation.
在本研究中，我们采用定点诱变来了解肟诱导神经毒剂抑制的人（Hu）乙酰胆碱酯酶（AChE）复活过程中氨基酸在峡部的作用。所研究的有机磷（OP）神经毒剂包括GA（塔崩）、GB（沙林）、GF（环沙林）、VX和VR。使用单吡啶肟2-PAM和双吡啶肟MMB4、HI-6及HLö-7检测复活动力学。用二级复活速率常数比较神经毒剂抑制的野生型（WT）和突变型酶的复活情况。发现包括Y72、Y124和W286在内的残基在双吡啶肟复活过程中起重要作用，但在单吡啶肟复活过程中不起作用。还发现残基Y124在HI-6和HLö-7复活过程中起关键作用，而E202对所有肟的复活都很重要。用亮氨酸取代F295以及用丙氨酸取代Y337的残基替换显示，双吡啶肟MMB4、HI-6和HLö-7的复活增强，这可能是因为使与活性位点丝氨酸结合的OP部分更容易与肟接触。这些结果与之前在牛AChE中观察到的结果相似，表明在肟复活方面，人和牛AChE之间存在显著相似性。
