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 对许多生物活性多肽的形成和降解至关重要。 该酶已在体内定位，并克隆了其基因。 已排序。EP 24.15的重要性通过以下几个方面得到了证明 十肽促性腺激素释放激素(又称 促黄体生成素释放激素)，关键神经肽 调节哺乳动物的繁殖。由EP 24.15切割，呈现GnRH 不活跃，并且是其细胞外处理的速率限制步骤 和退化。EP 24.15还代谢小肽底物，如 作为缓激肽、P物质和神经降压素，并从 前体蛋白。最近，这种酶也被认为与 伤害性、血压调节与肺的生理学 响应性。因此，阐明其功能和结构 EP24.15可能为某些疾病的病理生理学提供线索，以及 作为一个范例来理解神经肽和 其他由多肽酶产生的多肽荷尔蒙。中提出的具体目标 该项目包括： 酶的催化作用涉及哪些关键残基 作用机制、活性部位和底物专一性？谁要对此负责 对于一个小的底物偏好，这种情况可以改变吗？站点定向 将进行诱变和酶分析。圆二向色性 野生型/突变型的分析以及植物的本征荧光 变性剂尿素的存在和不存在将证实 活性减弱不是由于全球蛋白质构象的变化 (非本机折叠)。 临床上相关的类似物的生理作用是什么 促性腺激素释放激素对EP24.15活性的影响 以及这些EP 24.15抑制剂对GnRH有什么影响 退化？EP 24.15中的结构信息是否有助于设计新的 药理活性物质？所定义的药典可用于 开发非类固醇管理的男性和女性避孕药并 用于治疗各种疾病，如不孕症， 子宫内膜异位症、性激素依赖型乳腺癌和前列腺癌 性早熟。 EP 24.15的原子结构是什么？这些信息将如何 直接未来合理的药物设计？结构性决定将使用 同源金属蛋白酶模拟，X射线衍射实验， EP-24的模拟退火法分析和大分子模拟。15、 抑制剂和促性腺激素释放激素类似物。 这项研究提案实现的目标将实质上 促进生物化学综合方法战略， 研究酶-配体相互作用的理论方法和结构方法 EP24.15在神经内分泌学中的作用。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.