CATALYTIC MECHANISM OF CARBONIC ANHYDRASE

统一的目标是阐明碳的催化机理 本文对该酶的三种同工酶和某些同工酶进行了动力学研究。 精心挑选的位点特异性突变体 一个共同的目标是 了解锌结合水的性质和作用， 脱水酶和质子转移的动力学， 锌结合的氢氧化物。 这些机制研究将基于18-O CO2与水的交换和同位素交换 催化作用 将使用膜入口质量测量交换 光谱分析并分析以确定在该过程中的中间步骤的速率。 催化作用 有关中间体的其他信息， Co（II）取代碳酸酯的可见吸收光谱研究 在CO2水合的进展曲线期间的脱水酶。 所有这些数据 将结合起来，给出一个定量的催化模型。 将制备碳酸酐酶的位点特异性突变体，以测试 假设碳酸酐酶II中His 64通过以下方式提高催化速率： 将质子从锌结合水穿梭到溶液中的缓冲液， 64在碳酸酐酶III中通过静电相互作用降低 降低了锌结合水的pKa值 到低于5的值。 水作为金属配体的氢/氘分馏因子 在解释同位素效应和这方面的信息方面很重要 水的属性缺乏。 将测量该分馏因子 使用本实验室开发的方法，并基于NMR弛豫时间， 水的质子在顺磁性金属离子的存在下。 值 对于简单的无机络合物，将确定分馏因子 以及Co（II）取代的碳酸酐酶和其它蛋白质。 这些研究将有助于表征水作为金属的配体， 金属蛋白和解释H/D溶剂同位素效应在酶 在这些反应中，偏结合水起作用。

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.