MUTAGENESIS TO PROBE CATALYSIS BY CARBONIC ANHYDRASE II

这项提案概述了一项研究工作，以描绘出 人碳酸氢酶II(CA II)活性部位氨基酸的研究 催化和药物(磺胺)结合。磺胺缓蚀剂 这种酶已被用于治疗青光眼、癫痫和 溃疡。单部位突变的影响(由 寡核苷酸定向突变)对CA II功能、稳定性的影响 和结构将被测量。拟议研究的目标是 是为了加深我们对……重要性的理解 蛋白质/底物和蛋白质/蛋白质相互作用 催化作用。设计实验的目的是：1)描绘路径 和CA II催化的分子内和分子内机制 质子转移反应；2)测量一般的重要性 CA II催化中的碱催化或质子穿梭机理； 3)测定活性部位氨基酸对大鼠肾小管上皮细胞PKA的影响 酶结合锌水配体；4)定量评价 疏水相互作用对结合和 催化作用；5)测定单一氨基酸变化的影响 关于过渡态结构；6)量化的重要性 同工酶活性位点变异；以及7)测量 利用1-和2-位点突变对CAⅡ结构的影响 三维核磁共振技术。 所获得的信息将影响我们对 碳酸酐酶和其他金属酶的作用机制，如 以及所有蛋白质中质子转移的机制。 定量剖析各种残留物对 碳酸酐酶的结合和催化有望 推广到其他酶，并将在 活性中心抑制剂和新型蛋白质催化剂的设计，AS 同时也加深了我们对酶催化剂的理解。

This proposal outlines a research effort to delineate the roles of active site amino acids in human carbonic anhydrase II (CA II) in catalysis and drug (sulfonamide) binding. Sulfonamide inhibition of this enzyme has been used to treat glaucoma, epilepsy and ulcers. The effect of single-site mutations (prepared by oligonucleotide-directed mutagenesis) on CA II function, stability and structure will be measured. The goal of the proposed studies is to further our understanding of the importance of protein/substrate and protein/protein interactions for binding and catalysis. Experiments are designed to: 1) delineate the pathway and mechanism of CA II-catalyzed inter- and intramolecular proton transfer reactions; 2) measure the importance of general base catalysis or a proton shuttle mechanism in catalysis by CA II; 3) determine the effect of active site amino acids on the pKa of the enzyme-bound zinc-water ligand; 4) quantitatively evaluate the importance of hydrophobic interactions for binding and catalysis; 5) determine the effects of single amino acid changes on transition state structure; 6) quantitate the importance of isozymic active site variations; and 7) measure the effect of single site mutations on the structure of CA II using one- and two- dimensional NMR techniques. Information gained will impact on our understanding of the mechanism of carbonic anhydrases and other metalloenzymes, as well as the mechanism of proton transfers in all proteins. Quantitative dissection of the contribution of various residues to binding and catalysis for carbonic anhydrase hopefully can be generalized to other enzymes and will play a significant role in the design of active site inhibitors and new protein catalysts, as well as enlarge our understanding of enzymic catalysts.