STRUCTURAL STUDIES OF HUMAN PANCREATIC A-AMYLASE AND GRAM-NEGATIVE TYPE II CITRA

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 项目I：人胰腺α-淀粉酶（HPA; 496 a.a.是催化淀粉和其它长链碳水化合物水解的关键消化酶，淀粉和其它长链碳水化合物是膳食葡萄糖的主要来源。对这种催化过程的机械理解在糖尿病和肥胖症的治疗中具有相当重要的意义，这两种慢性疾病在医疗保健结果方面都造成了严重的损失。未来针对HPA活性的治疗开发在很大程度上取决于对这种酶的催化残基如何起作用以及存在于伸长的结合裂缝中的底物结合模式的结构理解。虽然我们已经成功地应用了定点突变技术并生长了野生型和变体蛋白的晶体，但由于使用我们的家庭实验室X射线源（Rigaku RU-300）缺乏分辨率（约1.9 <$），对底物和抑制剂形成的复合物的结构结果的解释取得了有限的成功。我们申请进入SSRL，以提高我们的结构结果的分辨率，以允许解释迄今为止获得的令人困惑的结果，并促进基于该机制数据的新型疗法的开发。 项目二：令人惊讶的是，只有革兰氏阴性细菌含有六聚体II型柠檬酸脱氢酶，其具有被代谢调节的特殊性质。相反，其他生物体（包括人类）的二聚柠檬酸脱氢酶是不受调控的。由于许多革兰氏阴性菌是危险的人类病原体，II型柠檬酸脱氢酶的特殊性质可能是开发新型抗微生物剂的基础。开发这种抗微生物剂的关键是II型柠檬酸合酶（CS; 6 X 424 a.a.的）处于其各种变构和代谢控制状态。经过相当大的努力，我们的实验室已经结晶了E。大肠杆菌酶，并且这仍然是唯一已经获得可用晶体的这种酶。Thes

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project I: Human pancreatic alpha-amylase (HPA; 496 a.a.'s) is a critical digestive enzyme catalyzing the hydrolysis of starch and other long chain carbohydrates, which represent a major source of dietary glucose. A mechanistic understanding of this catalytic process would be of considerable importance in the treatment of diabetes and obesity, both chronic diseases exacting a heavy toll in term of health care outcomes. Future therapeutic development directed at HPA activity is largely dependent on a structural understanding of how catalytic residues of this enzyme function and the mode of substrate binding in the elongated binding cleft present. Although we have successfully applied site directed mutagenesis techniques and grown crystals of both wild-type and variant proteins, interpretation of the structural results of complexes formed by substrates and inhibitors has had limited success due to a lack of resolution (~1.9 ¿) using our home laboratory X-ray source (Rigaku RU-300). We apply for access to the SSRL to enhance the resolutions of our structural results to allow for the interpretation of the puzzling results obtained thus far and to facilitate the development of novel therapeutics based on this mechanistic data. Project II: Surprisingly, only Gram-negative bacteria contain the hexameric Type II citrate synthases that have the special property of being metabolically regulated. In contrast, the dimeric citrate synthases of other organisms (including humans) is unregulated. Since many Gram-negative bacteria are dangerous human pathogens, the special properties of Type II citrate synthases could be a basis for the development of novel anti-microbials. Key to the development of such anti-microbials is the structural characterization of a Type II citrate synthase (CS; 6 x 424 a.a.'s) in its various allosteric and metabolically controlled states. After considerable effort our laboratory has crystallized the E. coli enzyme and this remains the only such enzyme for which usable crystals have been obtained. Thes