STRUCTURE-ACTIVITY RELATIONSHIPS OF ENDOPEPTIDASE 2415

哺乳动物锌金属内肽酶EC 3.4.24.15 [EP 24.15]活性为 对许多生物活性肽的形成和降解至关重要。 该酶已在体内定位，其cDNA克隆和 测序 EP 24.15的重要性通过以下方式得到证明： 十肽促性腺激素释放激素[GnRH]（也称为 促黄体激素释放激素），关键的神经肽 调节哺乳动物的繁殖。 EP 24.15裂解，使GnRH 无活性，是其细胞外加工的限速步骤 和退化。 EP 24.15还代谢小肽底物，如 如缓激肽、P物质和神经降压素， 前体蛋白 最近，这种酶也被牵连到 伤害感受生理学、血压调节和肺 响应能力。 因此，阐明 EP 24.15可能提供某些疾病的病理生理学的线索， 作为理解神经肽调节的范例， 其他肽类激素。 具体目标 这些项目是： 哪些关键残基参与了酶的催化作用 机制，活性位点和底物特异性？什么是负责任 对于一个小的基板偏好，这可以改变吗？定点 将进行诱变和酶测定。 圆二色谱 野生型/突变体以及内源性荧光的分析 变性剂尿素的存在和不存在将证实， 减弱的活性不是由于整体蛋白质构象的变化 （非天然折叠）。 临床相关的类似物的生理作用是什么？ GnRH对EP 24.15活性的影响，并且可以是良好表征的EP 24.15抑制剂 这些EP 24.15抑制剂对GnRH的影响是什么 退化？EP 24.15中的结构信息是否有助于设计新的 活性剂？可使用规定的药典 开发非甾体管理的男性和女性避孕药， 用于治疗各种疾病如不育症， 子宫内膜异位症、性类固醇依赖性乳腺癌和前列腺癌，以及 性早熟 EP 24.15的原子结构是什么？这些信息 指导未来合理的药物设计？结构测定将使用 同源金属蛋白酶建模，X射线衍射实验， EP 24的模拟退火分析和大分子模拟。15, 抑制剂和GnRH类似物。 该研究计划的实现目标将大大 有助于生物化学的综合方法的战略， 研究酶-配体相互作用的理论和结构方法 EP24.15在神经内分泌学中的应用。 在帮助未来合理用药的同时 设计，这些结果也应该适用于其他大分子。

Mammalian zinc metalloendopeptidase EC 3.4.24.15 [EP 24.15] activity is crucial to the formation and degradation of many bioactive peptides. This enzyme has been localized in vivo, and its cDNA cloned and sequenced. The importance of EP 24.15 is demonstrated with the decapeptide gonadotropin releasing hormone [GnRH] (also known as luteinizing hormone releasing hormone), the pivotal neuropeptide regulating mammalian reproduction. Cleavage by EP 24.15, renders GnRH inactive, and is the rate limiting step in its extracellular processing and degradation. EP 24.15 also metabolizes small peptide substrates such as bradykinin, substance P and neurotensin and generates enkephalins from precursor proteins. Recently, this enzyme has also been implicated in the physiology of nociception, blood pressure regulation and pulmonary responsiveness. Therefore, elucidating the function and structure of EP24.15 may yield clues to the pathophysiology of certain diseases, and act as a paradigm for understanding the regulation of neuropeptides and other peptide hormones by a peptidase. Specific objective addressed in this project are: Which critical residues are involved in the enzyme's catalytic mechanisms, active site and substrate specificity? What is responsible for a small substrate preference, and can this be altered? Site-directed mutagenesis and enzyme assays will be performed. Circular dichroism analysis of wild type/mutants as well as intrinsic fluorescence in the presence and absence of the denaturing agent urea will confirm that attenuated activity is not due to changes in global protein conformation (non-native folding). What are the physiological effects of clinically relevant analogues of GnRH on EP 24.15 activity and can a well characterized EP24.15 inhibitor be found, and what is the effect of these EP 24.15 inhibitors upon GnRH degradation? Can structural information in EP 24.15 aid in designing new pharmacologically active agents? The pharmacopoeia defined could be used to develop nonsteroidal managed male and female contraceptives and be used in the treatment of such diverse disorders as sterility, endometriosis, sex-steroid dependent mammary and prostate cancers, and precocious puberty. What is the atomic structure of EP 24.15? How will this information direct future rational drug design? Structural determination will use homologous metalloprotease modelling, X-ray diffraction experiments, simulated annealing analysis and macromolecular simulations of EP 24. 15, inhibitors and GnRH analogues. The realized goals of this research proposal will substantially contribute to the strategy of an integrated approach of biochemical, theoretical, and structural methods to study enzyme-ligand interactions of EP24.15 in neuroendocrinology. While aiding future rational drug design, these results should also be applicable to other macromolecules.