CATALYTIC MECHANISM OF CARBONIC ANHYDRASE

描述（改编自申请人摘要）：生理反应 碳酸酐酶（CA）催化的一个重要作用是碳的水合 二氧化物：CO（2）+ H（2）O <->HCO（3-）+ H（+）。这种催化作用需要攻击 通过锌结合的氢氧化物，然后从 将活性位点转移到溶液中以再生锌结合的氢氧化物。有效 动物CA的同工酶利用His 64作为分子内质子穿梭; 该残余物通过网状结构从锌结合的水接受质子， 氢键沃茨的周转率为106 s（-1），并将它们转移到 溶液这一建议的统一目标是扩大对 碳酸酐酶来理解限速质子步骤， 扩展到其他蛋白质。一个共同的目标是应用马库斯率理论 既要了解碳酸酐酶中的质子转移， Marcus理论中质子转移参数的重要性 酶或蛋白质。西尔弗曼博士将使用位点特异性突变， 化学修饰，将质子转移基团置于 CA的三个广泛和遗传上不同的类别：阿尔法（动物），贝塔 （植物）和伽马（古细菌）CA。西尔弗曼博士还将利用外源性 质子供体从溶液扩展到分子间质子 转移停流分光光度法与CO2-水间18 O交换 通过质谱法测量的速率常数将用于获得 和分子内质子转移。重要突变体的晶体结构 将被确定。一个目标是确定距离， 位置和环境影响质子的速率 转移西尔弗曼博士将应用马库斯率理论，以确定和 解释的内在能量障碍和热力学成分的 质子转移，并将其与结构和化学特征的 CA活性位点。

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.