CATALYTIC MECHANISM OF CARBONIC ANHYDRASE

这个提议的统一目标是从分子的细节上理解 碳酸酐酶（CA）同工酶的催化机制。 调查最有效率和最没有效率的人类 碳酸酐酶，同工酶II和III，将决定如何 活性部位腔的性质影响基本步骤NF 锌结合的氢氧化物对CO2的攻击和随后的质子转移， 溶液 仔细选择锌附近的残留物， 影响催化作用，以及CA III特有的残留物， 被定点诱变取代。 每种催化剂的催化性能 将在一系列动力学方法中研究突变体，包括CO2 稳定状态下的水合和酯酶活性， 化学平衡下的CO2和水，缓冲催化，溶剂 氘同位素效应，以及底物、阴离子和 磺酰胺抑制剂。 第二个目标是利用磁共振 以帮助确定活性部位中的水的性质。 核磁 放松将被用来衡量平均汇率和平均 水与本体溶剂之间质子交换的金属距离 和Co（II）取代的CA II和III（野生型）的活性位点空腔 类型和突变体）。 碳酸酐酶IV，一种膜结合的主要 碳酸酐酶在分泌组织中的功能形式，将是 克隆并表达这种同工酶的基因，正如我们对 同工酶II和III。 活性中心的结构-功能关系 残基及其在CA IV催化机制中的作用将被 并与同工酶Ⅱ、Ⅲ进行比较。 这项工作将导致 对质子转移和活性中心的作用有基本的了解。 水，包括碳酸酐酶中的锌结合水， 在其他酶中的相似性。 更好地了解活性部位， 将导致抑制剂的合理设计能力的增强 CA在青光眼和脑积水的控制。

The unifying goal of this proposal is to understand in molecular detail the catalytic mechanism of the carbonic anhydrase (CA) isozymes. Investigations of the most efficient and least efficient of the human carbonic anhydrases, isozymes II and III, will determine how the properties of the active-site cavity affect the fundamental steps nf attack of zinc-bound hydroxide on CO2 and subsequent proton transfer to solution. Carefully selected residues near the zinc and in a position to influence catalysis, as well as residues unique to CA III, will be replaced by site-directed mutagenesis. The catalytic properties of each mutant will be studied in an array of kinetic methods including C02 hydration and esterase activity at steady state, exchange of 180 between C02 and water at chemical equilibrium, buffer catalysis, solvent deuterium isotope effects, and the binding of substrates, anions, and sulfonamide inhibitors. A secondary goal is to use magnetic resonance to help define properties of water in the active-site. Nuclear magnetic relaxation will be used to measure the average exchange rate and average distance from the metal of water protons exchanging between bulk solvent and the active-site cavity of Co(II)-substituted CA II and III (wild type and mutants). Carbonic anhydrase IV, a membrane-bound and major functional form of carbonic anhydrase in secretory tissues, will be cloned and the gene for this isozyme expressed, as we have done for isozymes II and III. Structure-function relationships of active site residues and their role in the catalytic mechanism of CA IV will be determined and compared with isozymes II and III. This work will lead to a basic understanding of proton transfer and the role of active-site. water including zinc-bound water in the carbonic anhydrases and by analogy in other enzymes. A better understanding of the active site, will lead to an enhanced capability in the rational design of inhibitors of CA in the control of glaucoma and hydrocephalus.