CATALYTIC MECHANISM OF CARBONIC ANHYDRASE

描述(改编自申请者摘要)：生理反应 碳酸酐酶(CA)催化的意义是碳的水合作用 二氧化碳：CO(2)+H(2)O&lt；-&gt；HCO(3-)+H(+)。这种催化作用需要对 由锌结合的氢氧化物产生的二氧化碳，随后从 活性部位到溶液，以再生锌结合的氢氧化物。高效的 动物CA的同工酶利用His64作为分子内的质子穿梭； 这种残留物通过一个网络接受来自锌结合水的质子 氢键水，周转率为106%S(-1)，并将它们转移到 解决方案。这项建议的统一目标是扩大对 碳酸酐酶以一种可以 可以扩展到其他蛋白质。同时的目标是应用马库斯利率理论。 了解碳酸酐酶中的质子转移并阐明 马库斯理论参数对质子转移的意义 一种酶或蛋白质。西尔弗曼博士将使用定点突变技术 化学修饰将质子转移基团放置在 三种广泛且在基因上截然不同的CA：阿尔法(动物)、贝塔 (植物)和伽马(古生菌)CA的。Silverman博士还将利用外源 从溶液到质子供体将工作扩展到分子间质子 调职。停流分光光度法与二氧化碳与水的~(18)O交换 通过质谱学测量，将被用来获得相互作用的速率常数 和分子内质子转移。重要突变体的晶体结构 将会被确定。目标是具体确定距离有多远， 活化位的位置和环境影响质子的速率 调职。西尔弗曼博士将应用马库斯比率理论来确定和 解释本征能垒和热力学成分 质子转移并将它们与分子的结构和化学特征联系起来 CA活动站点。

DESCRIPTION (adapted from applicant's abstract): The reaction of physiological significance catalyzed by carbonic anhydrase (CA) is the hydration of carbon dioxide: CO(2) + H(2)O <-> HCO(3-) + H(+). This catalysis requires attack on CO2 by zinc-bound hydroxide followed by rate-limiting proton transfers from the active site to solution to regenerate the zinc-bound hydroxide. The efficient isozymes of the animal CA's utilize His64 as an intramolecular proton shuttle; this residue accepts protons from the zinc-bound water through a network of hydrogen-bonded waters at a turnover rate of 106 s(-1) and transfers them to solution. The unifying goal of this proposal is to expand the study of the carbonic anhydrases to understand rate-limiting proton steps in a way that can be extended to other proteins. A concurrent goal is to apply Marcus rate theory both to understand the proton transfers in carbonic anhydrase and to elucidate the significance of the parameters of the Marcus theory for proton transfer in an enzyme or protein. Dr. Silverman will use site-specific mutagenesis and chemical modification to place proton transfer groups at strategic locations in three broad and genetically distinct classes of CA's: the alpha (animal), beta (plant), and gamma (archaeal) CA's. Dr. Silverman will also utilize exogenous proton donors from solution to expand the work to intermolecular proton transfer. Stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry will be used to obtain rate constants for inter- and intramolecular proton transfer. Crystal structures of important mutants will be determined. A goal is to determine specifically how distances, location, and environment in the active site influence the rate of proton transfer. Dr. Silverman will apply Marcus rate theory to determine and interpret the intrinsic energy barriers and thermodynamic components for the proton transfers and relate them to the structural and chemical features of the CA active site.