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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 项目一：人胰腺α-淀粉酶(HPA；496A.A.‘S)是一种关键的消化酶，催化淀粉和其他长链碳水化合物的水解，而淀粉和其他长链碳水化合物是饮食中葡萄糖的主要来源。从机制上理解这一催化过程在治疗糖尿病和肥胖症方面将具有相当重要的意义，这两种慢性病在医疗保健结果方面都会造成沉重的代价。针对HPA活性的未来治疗开发在很大程度上取决于对该酶的催化残基如何发挥作用的结构理解，以及延长的结合裂缝中底物结合的方式。尽管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异型蛋白质的晶体，但由于使用我们自己的实验室X射线源(Rigaku RU-300)缺乏分辨率(~1.9？)，解释由底物和抑制剂形成的复合体的结构结果的成功有限。我们申请进入SSRL，以提高我们的结构结果的分辨率，以便能够解释迄今获得的令人费解的结果，并促进基于该机制数据的新疗法的开发。项目II：令人惊讶的是，只有革兰氏阴性细菌含有六聚体II型柠檬酸合成酶，这种合成酶具有代谢调节的特殊性质。相比之下，其他生物体(包括人类)的二聚体柠檬酸合成酶是不受监管的。由于许多革兰氏阴性菌是危险的人类病原体，II型柠檬酸合成酶的特殊性质可能是开发新型抗微生物药物的基础。开发这种抗微生物药物的关键是II型柠檬酸合成酶(CS；6x424A.A.‘S)在其各种变构和代谢控制状态下的结构特征。经过相当大的努力，我们的实验室已经将大肠杆菌酶结晶，这仍然是唯一一种获得可用晶体的这种酶。牛膝

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