Capillary Electrophoretic Studies of Enzyme Inhibition

描述（由申请人提供）：本申请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的抑制作用，从而推进酶功能的研究。酶抑制剂是广泛的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能力来控制具有不同电泳迁移率的分子区域的混合和分离，以研究酶催化反应的动力学。与碱性磷酸酶的合作将集中于继续开发毛细管电泳酶抑制测定的定量模型，并了解酶和抑制剂之间的亲和相互作用在这些实验中所起的作用。我们将探索EDTA在微摩尔浓度下对碱性磷酸酶的意想不到的明显激活，并将开发从非变性板凝胶中直接取样的酶的原子量测定方法。目前，高灵敏度的毛细管电泳酶1抑制试验仅限于具有荧光底物的酶。这一限制将通过在毛细管和微制造装置中发展光学门控空位毛细管电泳来克服。这一进展将使任何酶的研究在非常低的浓度下，荧光（而不是荧光）底物可以合成，极大地扩展了这种毛细管电泳方法的潜在适用性酶抑制分析。毛细管电泳酶抑制试验将用于酶，使用NAD+tNADH和NADP+/NADPH作为辅助因子，进一步扩大酶的范围，可以使用这种方法进行研究。最后，研究具有生物医学意义的特定酶和相关抑制剂。有效的合成抑制剂腺苷脱氨酶将使用这些试验进行研究。犬尿氨酸酶和相关抑制剂也将被研究。

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.