Capillary Electrophoretic Studies of Enzyme Inhibition

描述（由申请人提供）：本申请中提出的研究目标是通过开发和应用基于毛细管电泳的方法来研究酶1抑制，从而推进酶功能的研究。酶抑制剂是多种人类疾病的主要治疗靶点。本研究利用毛细管电泳方法的独特功能，即可以将具有不同电泳迁移率的分子区域混合和分离，来研究酶催化反应的动力学。碱性磷酸酶的工作将集中在继续发展毛细管电泳酶抑制试验的定量模型，并了解酶和抑制剂之间的亲和力相互作用在这些实验中的作用。将探索EDTA在微摩尔浓度下对碱性磷酸酶的意外表观活化，并将开发直接从非变性平板凝胶中采集的阿托摩尔量酶样品1的测定方法。目前，高灵敏度的毛细管电泳酶抑制试验仅限于荧光底物的酶。这一限制将通过毛细管1和微加工设备中的光门控空位毛细管电泳的发展来克服。这一进展将使任何酶的研究在非常低的浓度，荧光（相对于荧光）底物可以合成，大大扩展了这种毛细管电泳方法的酶抑制分析的潜在适用性。将开发酶的毛细管电泳酶抑制试验，其使用NAD+tNADH和NADP+/NADPH作为辅因子，以进一步扩大使用该方法可研究的酶的范围。最后，将研究具有生物医学意义的特定酶和相关抑制剂。将使用这些试验研究腺苷脱氨酶的有效合成抑制剂。还将研究犬尿氨酸酶和相关抑制剂。

DESCRIPTION (provided by applicant): The goal of the research proposed in this application is to advance the study of enzyme function through1 the development and application of capillary electrophoresis-based approaches for studying enzyme1 inhibition. Enzyme inhibitors are primary therapeutic targets for a wide range of human diseases. This research takes advantage of the unique capabilities of capillary electrophoretic methods for controlling1 the mixing and separation of zones of molecules with different electrophoretic mobilities to study the kinetics of enzyme catalyzed reactions. Work with alkaline phosphatase will focus on continuing the development of quantitative models of the capillary electrophoretic enzyme inhibition assays and understanding the role that affinity interactions between enzymes and inhibitors play in these1 experiments. The unexpected apparent activation of alkaline phosphatase by EDTA at micromolar concentrations will be explored, and assays will be developed for attomole quantities of enzyme sampled1 directly from non-denaturing slab gels. Currently, high sensitivity capillary electrophoretic enzyme1 inhibition assays are limited to enzymes for which fluorogenic substrates are available. This limitation will be overcome through the development of optically gated vacancy capillary electrophoresis in capillaries1 and microfabricated devices. This advance will enable the study of any enzyme at very low concentrations for which a fluorescent (as opposed to fluorogenic) substrate can be synthesized, greatly expanding the potential applicability of this capillary electrophoretic approach for enzyme inhibition analysis. Capillary electrophoretic enzyme inhibition assays will be developed for enzymes, which use NAD+tNADH and NADP+/NADPH as cofactors to further expand the range of enzymes that can be1 investigated using this methodology. Finally, specific enzymes of biomedical significance and related inhibitors will be investigated. Potent synthetic inhibitors of adenosine deaminase will be studied using these assays. Kynureninase and related inhibitors will also be investigated.