Enzymic Detoxification of Organophosphate Nerve Agents

描述(由申请人提供)： 这项建议中描述的研究的长期目标是设计和建造一个强大的催化系统，该系统可用于去除、销毁和检测有机磷神经毒剂。神经毒剂沙林(GB)、梭曼(GD)和VX是迄今报道的毒性最大、最致命的神经毒剂。由于这些化合物易于合成和分配，它们对文明社会的健康和福祉构成了严重威胁。为了制备一种能够解毒有机磷神经毒剂的体系，我们将操纵和增强野生型蛋白的酶能力，以作为这些剧毒物质的识别和水解周转的催化剂。已经证明，嵌入细菌磷酸三酯酶和α-丙氨酸酯酶活性部位的催化机制能够在扩散控制的限度内降解某些有机磷神经毒剂，如对氧磷。这些酶的活性部位结构将通过合理的组合突变技术进行重新设计，以创建具有改变催化性质的突变酶文库。这些酶文库将通过高通量筛选方案，使用GB、GD和VX的手性类似物的荧光和可见光光谱进行有效的评估。通过与阿伯丁试验场的DeFrank小组直接合作，将优化受限神经毒剂的催化活性。野生型和突变型蛋白质的结构分析将由威斯康星州的Holden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.