CATALYTIC MECHANISM OF CARBONIC ANHYDRASE

这个提议的统一目标是在分子细节上理解 碳酸酐酶(CA)同工酶的催化机理。 对人类中效率最高和效率最低的人的调查 碳酸氢酶，同工酶II和III，将决定如何 有源位腔的性质影响基本步长nf 锌结合氢氧化物对二氧化碳的攻击和随后的质子转移 解决方案。仔细挑选锌附近的残留物，并将其放置在 影响催化，以及CA III独有的残基，将是 被定点突变所取代。每种催化剂的催化性能 将用包括二氧化碳在内的一系列动力学方法来研究突变体 水合作用和酯酶活性在稳定状态下，交换180 二氧化碳和水的化学平衡，缓冲催化，溶剂 氚同位素效应，以及底物、阴离子和 磺胺类抑制剂。第二个目标是利用磁共振 以帮助定义活性中心中水的性质。核磁 将使用松弛来衡量平均汇率和平均 主体溶剂间交换的水质子与金属的距离 和Co(II)取代的CA II和III(野生型)的活性中心腔 类型和突变体)。碳酸氢酶IV，一种膜结合型和主要 碳酸酐酶在分泌组织中的功能形式，将是 克隆并表达了该同工酶的基因，就像我们在 同工酶II和III.活性中心的结构-功能关系 残基及其在CA IV催化机理中的作用 测定并与同工酶II和III进行比较。 对质子转移和活性中心的作用有一个基本的了解。 水，包括碳酸氢酶中的锌结合水和 在其他酶中也是类似的。更好地了解活动站点， 将增强合理设计抑制剂的能力 CA在控制青光眼和脑积水方面的作用。

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.