CARBONIC ANHYDRASE--CATALYSIS, INHIBITION AND REDESIGN

这项提案概述了一项研究工作，以描绘出结构 催化效率的决定因素；缓蚀剂结合；以及金属 人碳锌金属酶的结合和反应活性 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的活性位点的功能库 II和锌金属蛋白水解酶、羧基肽酶。催化效率 而抑制物的效力将通过一种 诱变、光谱、动力学分析、结构的组合 测定和理论计算。实验旨在： 1)检测CA II的底物专一性；2)描述 金属结合、特异性和反应性中的蛋白质配体；3)定义 金属的蛋白质背景的结构和功能作用 配位多面体，包括第二壳层氢键和一个 疏水壳层；4)研究氢键对壳层的作用 锌-溶剂配体在确定催化机理中的作用：5)转化 锌在CAⅡ中的结合部位转变为蓝铜部位和水解物 与锌蛋白水解酶相似的部位；6)重新设计活性部位 羧肽酶I催化二氧化碳水合作用；7)表征 CA II生理性血浆抑制物的生化性质 包括与CA II的结合作用、与转铁蛋白的相似性和 初级序列；以及8)鉴定其他特异性的抑制剂 同工酶。 所获得的信息将影响我们对该法规和 碳酸酐酶同工酶的生理意义及其在设计中的应用 同工酶特异性抑制物。人类CA II纯合子缺乏症 会对骨骼、肾脏和大脑造成临床后果 骨质疏松症，肾小管性酸中毒和脑钙化， 分别进行了分析。此外，磺胺类药物对CA II有很强的抑制作用 临床上用于治疗青光眼。对结构的剖析 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的催化机理的理解 金属酶，以及导致我们的 对金属蛋白质工程指导原则的理解 结合部位和设计活性部位抑制剂。锌蛋白发挥作用 代谢、基因表达和催化中不可或缺的作用 关键的生理反应，如DNA和RNA聚合，二氧化碳 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一定的意义 治疗癌症、衰老、代谢性疾病、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.