CARBONIC ANHYDRASE--CATALYSIS, INHIBITION AND REDESIGN

碳酸酐酶——催化、抑制和重新设计

• 批准号: 2180470
• 负责人: CAROL A FIERKE
• 金额: $ 17.1万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180470
• 关键词: active sites; carbonate dehydratase; carbonic anhydrase inhibitors; carboxypeptidase; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; hydrogen bond; isozymes; ligands; nuclear magnetic resonance spectroscopy; protein engineering; protein structure function; site directed mutagenesis; zinc

This proposal outlines a research effort to delineate the structural determinants of catalytic efficiency; inhibitor binding; and metal binding and reactivity in the zinc metalloenzyme, human carbonic anhydrase II (CA II). These principles will then be tested and expanded by redesigning the functional repertoire of the active sites of both CA II and the zinc metalloprotease, carboxypeptidase. Catalytic efficiency and inhibitor potency will be related to protein structure through a combination of mutagenesis, spectroscopy, kinetic analysis, structure determination and theoretical calculations. Experiments are designed to: 1) examine the substrate specificity of CA II; 2) delineate the role of protein ligands in metal binding, specificity and reactivity; 3) define the structural and functional role of the protein context of the metal coordination polyhedron, including second shell hydrogen bonds and a hydrophobic shell; 4) investigate the role of hydrogen bonds to the zinc-solvent ligand in determining the catalytic mechanism; 5) convert the zinc binding site in CA II into a blue copper site and a hydrolytic site similar to that of the zinc proteases; 6) redesign the active site of carboxypeptidase I to catalyze CO2 hydration; 7) characterize the biochemical properties of a physiological plasma inhibitor of CA II, including binding interactions with CA II, similarity to transferrin and primary sequence; and 8) identification of inhibitors specific for other isozymes. Information gained will impact on our understanding of the regulation and physiological importance of carbonic anhydrase isozymes and in the design of isozyme specific inhibitors. Homozygous CA II deficiency in humans has clinical consequences for bone, kidney and brain causing osteopetrosis, renal tubular acidosis and cerebral calcification, respectively. In addition, CA II is potently inhibited by sulfonamides which are used clinically to treat glaucoma. Dissection of structural motifs in CA II and CPA essential for stability and reactivity will increase our understanding of the catalytic mechanism of these and other metalloenzymes, as well as lead to substantial improvement in our understanding of the guiding principles for protein engineering of metal binding sites and designing active site inhibitors. Zinc proteins play indispensable roles in metabolism and gene expression and catalyze crucial physiological reactions such as DNA and RNA polymerization, CO2 hydration, connective tissue and protein degradation, and intermediary metabolism. Malfunction and inhibition of these enzymes has implications for cancer, aging, metabolic diseases, arthritis, immunodeficiency and glaucoma.

该提案概述了一项研究工作， 催化效率的决定因素;抑制剂结合;和金属 锌金属酶中的结合和反应性，人碳 脱水酶II（CA II）。 这些原则随后将得到检验和扩展 通过重新设计两个CA的活性位点的功能库， II和锌金属蛋白酶、羧肽酶。 催化效率 而抑制剂的效力将通过一种 诱变组合，光谱学，动力学分析，结构 测定和理论计算。 实验旨在： 1)检查CA II的底物特异性; 2）描述CA II的作用， 金属结合、特异性和反应性中的蛋白质配体; 3）定义 金属蛋白质的结构和功能作用 配位多面体，包括第二层氢键和 疏水壳; 4）研究氢键对疏水壳的作用。 锌-溶剂配体在确定催化机理中的作用; 5）转化 CA II中的锌结合位点转化为蓝铜位点， 与锌蛋白酶相似的位点; 6）重新设计活性位点 的羧肽酶I催化CO2水合作用; 7）表征 CA II的生理性血浆抑制剂的生物化学性质， 包括与CA II的结合相互作用，与转铁蛋白的相似性， 一级序列;和8）鉴定对其他 同工酶 获得的信息将影响我们对法规的理解， 碳酸酐酶同工酶的生理学重要性及其在设计中的应用 同工酶特异性抑制剂。 人类纯合子CA II缺乏症 对骨骼、肾脏和大脑有临床后果， 骨硬化、肾小管酸中毒和脑钙化， 分别 此外，磺胺类药物可有效抑制CA II 临床上用于治疗青光眼。结构解剖 CA II和CPA中对稳定性和反应性至关重要的基序将 增加我们对这些和其他催化机制的理解 金属酶，以及导致我们的实质性改善， 了解金属蛋白质工程的指导原则 结合位点和设计活性位点抑制剂。 锌蛋白发挥作用 在新陈代谢和基因表达中起着不可或缺的作用， 关键的生理反应，如DNA和RNA聚合，CO2 水化、结缔组织和蛋白质降解以及中间体 新陈代谢. 这些酶的功能障碍和抑制 癌症、衰老、代谢性疾病、关节炎、免疫缺陷和 青光眼