CARBONIC ANHYDRASE--CATALYSIS, INHIBITION AND REDESIGN

该提案概述了描绘结构的研究工作 催化效率的决定因素；抑制剂结合；和金属 锌金属酶、人碳酸的结合和反应性 脱水酶 II (CA II)。 然后将测试和扩展这些原则 通过重新设计两个 CA 活性位点的功能库 II和锌金属蛋白酶、羧肽酶。 催化效率 抑制剂的效力将通过以下方式与蛋白质结构相关： 诱变、光谱学、动力学分析、结构的结合 测定和理论计算。 实验旨在： 1) 检查CA II的底物特异性； 2）界定角色 金属结合、特异性和反应性中的蛋白质配体； 3）定义 金属蛋白质背景的结构和功能作用 配位多面体，包括第二壳氢键和 疏水壳； 4）研究氢键对 锌溶剂配体确定催化机制； 5）转换 CA II 中的锌结合位点转化为蓝色铜位点和水解位点 与锌蛋白酶相似的位点； 6）重新设计活性位点 羧肽酶 I 催化 CO2 水合； 7) 表征 CA II 生理血浆抑制剂的生化特性， 包括与 CA II 的结合相互作用、与转铁蛋白的相似性以及 初级序列； 8) 鉴定其他特异性抑制剂 同工酶。 获得的信息将影响我们对法规的理解和 碳酸酐酶同工酶的生理重要性及其设计 同工酶特异性抑制剂。 人类纯合 CA II 缺陷 对骨骼、肾脏和大脑有临床影响 骨石症、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。结构剖析 CA II 和 CPA 中对于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 了解金属蛋白质工程的指导原则 结合位点并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和催化中不可或缺的作用 重要的生理反应，例如 DNA 和 RNA 聚合、CO2 水合作用、结缔组织和蛋白质降解以及中介 新陈代谢。 这些酶的故障和抑制具有影响 用于癌症、衰老、代谢疾病、关节炎、免疫缺陷和 青光眼。

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.