CATALYTIC MECHANISM OF CARBONIC ANHYDRASE

统一的目标是阐明碳的催化机理。 脱水酶三种同工酶及其部分同工酶的动力学研究 精心挑选的特定位点突变体。同时的目标是 了解碳酸盐中锌结合水的性质和作用 无水合酶及其质子转移的动力学 结合锌的氢氧化物。这些机理研究将基于18-O 二氧化碳与水的交换和位置同位素交换 催化作用。交换将使用膜入口质量来测量。 光谱分析以确定中间步骤的速率 催化作用。有关中间体的其他信息将通过以下方式获得 Co(II)取代碳的可见吸收光谱研究 在CO2水合的进程曲线过程中的硬水酶。所有这些数据 将结合在一起，给出一个催化的定量模型。 碳酸酐酶的定点突变体将被准备用于测试 他的在碳酸酐酶II中的假说是通过 将质子从锌结合水中穿梭到溶液中的缓冲液中，赖斯 在碳酸酐酶III中通过静电相互作用还原 CO2水合的催化速率和降低锌结合水的pKa 设置为低于5的值。 水作为金属配位体的氢/氚分馏系数 在解释同位素效应和有关这方面的信息方面很重要 缺乏水的属性。这个分馏系数将被测量 使用本实验室开发的方法并基于核磁共振弛豫时间 在顺磁性金属离子存在的情况下水的质子。的价值 将测定简单无机络合物的分馏系数。 以及Co(II)取代的碳酸氢酶和其他蛋白质。 这些研究将有助于表征水作为金属的配体。 金属蛋白及其在酶中的H/D溶剂同位素效应 金属结合水在其中起作用的反应。

The unifying goal is to elucidate the catalytic mechanism of carbonic anhydrase by kinetic studies on three isozymes of this enzyme and some carefully selected site-specific mutants. A concurrent goal is to understand the properties and role of zinc-bound water in carbonic anhydrase and the kinetics of the proton transfer which converts it to zinc-bound hydroxide. These mechanistic studies will be based on 18-O exchange between CO2 and water and positional isotope exchange during catalysis. The exchanges will be measured using membrane-inlet mass spectrometry and analyzed to determine rates of intermediate steps in the catalysis. Additional information on intermediates will be obtained by studies of the visible absorption spectrum of Co(II)-substituted carbonic anhydrase during the progress curve for CO2 hydration. All of these data will be combined to give a quantitative model of catalysis. Site-specific mutants of carbonic anhydrase will be prepared to test the hypothesis that His 64 in carbonic anhydrase II enhances catalytic rate by shuttling protons from zinc-bound water to buffer in solution, and that Lys 64 in carbonic anhydrase III through electrostatic interactions decreases the catalytic rate of CO2 hydration and lowers the pKa of zinc-bound water to a value below 5. The hydrogen/deuterium fractionation factor of water as a ligand of metals is important in interpreting isotope effects and information on this property of water is lacking. This fractionation factor will be measured using a method developed in this lab and based on NMR relaxation times of the protons of water in the presence of paramagnetic metal ions. Values of the fractionation factor will be determined for simple inorganic complexes as well as for Co(II)-substituted carbonic anhydrase and other proteins. These studies will help to characterize water as a ligand of metals in metalloproteins and to interpret H/D solvent isotope effects in enzymatic reactions in which meta-bound water plays a role.