Enzymic Detoxification of Organophosphate Nerve Agents

描述（由申请人提供）： 本提案中所述研究的长期目标是设计和构建一个强大的催化系统，该系统可用于有机磷神经毒剂的去污，破坏和检测。神经毒剂沙林（GB），梭曼（GD）和VX是有史以来报道的毒性和致命性最强的神经毒剂。由于这些化合物易于合成和传播，它们对文明社会的健康和福祉构成严重威胁。为了准备一个系统，可以解毒的有机磷神经毒剂，我们将操纵和增强野生型蛋白质的酶的力量，作为催化剂的识别和水解这些剧毒物质的周转。已经证明，嵌入在细菌磷酸三酯酶和α-氨酰基脯氨酸二肽酶的活性位点内的催化机制能够在扩散控制极限下水解某些有机磷酸神经毒剂，例如对氧磷。这些酶的活性位点结构将通过合理和组合突变技术进行重新设计，以创建具有改变的催化特性的突变酶库。这些酶库将有效地评估高通量筛选方案，使用荧光和可见光谱与GB，GD和VX的手性类似物。限制性神经毒剂的催化活性将通过与阿伯丁试验场的DeFrank小组的直接合作进行优化。野生型和突变蛋白的结构分析将由威斯康星州的霍尔顿小组进行。

DESCRIPTION (provided by applicant): The long-term objective for the research described in this proposal is the design and construction of a robust catalytic system that can be exploited for the decontamination, destruction, and detection of organophosphate nerve agents. The nerve agents sarin (GB), soman (GD), and VX are among the most toxic and deadly nerve agents ever reported. Since these compounds are easy to synthesize and distribute, they represent a serious threat to the health and well being of civilized societies. In order to prepare a system that can detoxify organophosphate nerve agents, we will manipulate and enhance the enzymatic power of wild type proteins to serve as catalysts for the recognition and hydrolytic turnover of these highly toxic materials. It has been demonstrated that the catalytic machinery embedded within the active sites of the bacterial phosphotriesterase and alpha-prolidase are capable of hydrolyzing certain organophosphate nerve agents such as paraoxon at the diffusion controlled limit. The active site structures of these enzymes will be reengineered through rational and combinatorial mutagenesis techniques to create libraries of mutant enzymes with altered catalytic properties. These enzyme libraries will be efficiently evaluated with high throughput screening protocols using fluorescence and visible spectroscopy with chiral analogs of GB, GD, and VX. The catalytic activities with the restricted nerve agents will be optimized through a direct collaboration with the DeFrank group at the Aberdeen Proving Ground. The structural analyses of the wild type and mutant proteins will be conducted by the Holden group at Wisconsin.