Capillary Electrophoretic Studies of Enzyme Inhibition

描述(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法的方法来研究酶的抑制作用，从而促进酶功能的研究。酶抑制剂是治疗多种人类疾病的主要靶点。本研究利用毛细管电泳法控制不同迁移率分子区带的混合和分离的独特能力来研究酶催化反应的动力学。在碱性磷酸酶方面的工作将集中于继续开发毛细管电泳酶抑制分析的定量模型，并了解酶和抑制剂之间的亲和力相互作用在这些实验中所起的作用。将探索EDTA在微摩尔浓度下意外地明显激活碱性磷酸酶，并将开发直接从非变性平板凝胶中提取全摩尔量的酶的分析方法。目前，高灵敏度的毛细管电泳酶1抑制分析仅限于具有荧光底物的酶。这一局限性将通过开发毛细管1和微制造设备中的光学门控空位毛细管电泳来克服。这一进展将使在极低浓度下研究任何可以合成荧光(而不是荧光)底物的酶成为可能，极大地扩大了这种毛细管电泳法用于酶抑制分析的潜在适用性。以NAD、tNADH和NADP/NADPH为辅因子的酶毛细管电泳酶抑制分析方法将进一步扩大可用该方法研究的酶的范围。最后，将对具有生物医学意义的特定酶和相关的抑制剂进行研究。将使用这些检测方法来研究腺苷脱氨酶的有效合成抑制剂。Kynurenase和相关的抑制剂也将被研究。

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.