Investigation of Enzyme Catalysis: Mechanism at the Levels of Subunit Function and the Chemical Transformation

这个项目将促进对基片沟道的理解， 代谢，蛋白质功能的变构，以及 酶催化 将研究三个系统：（1）细菌 色氨酸合成酶复合物，（2）人肝醇 酶，和（3）羊肝醛脱氢酶。 色氨酸合酶双酶复合物催化两个连续的 代谢途径中的步骤;该酶表现出配体介导的 之间的变构相互作用。和>网站，这是 由一条25米长的隧道连接。 据推测， 代谢物吲哚通过这些位点之间转移， 隧道 本建议将探讨：（a） 代谢物转运中的隧道，（B）机制和功能 的变构效应，和（c）的化学决定因素， 底物特异性 通过基因工程，人类 肝脏乙醇脱氢酶同工酶可大量获得， 金额。 这一建议将确定蛋白质所起的作用 氢化物的构象变化及静电场作用 转移 乙醛酸脱氢酶催化氧化 醛通过酰基酶中间体产生。 如何 结合的辅酶的氧化态会影响酶的性质。 将研究酰基酶。 快速动力学技术， 将使用发色底物或类似物和1-H NMR。 一些色氨酸合成酶和乙醇脱氢酶的工作将 涉及通过定点突变制备突变酶的研究 诱变

This project will advance understanding of substrate channeling in metabolism, allosterism in protein function, and the chemistry of enzyme catalysis. Three systems will be studied: (1) the bacterial bienzyme tryptophan synthase complexes, (2) the human liver alcohol dehydrogenases, and (3) the sheep liver aldehyde dehydrogenase. The tryptophan synthase bienzyme complex catalyzes two sequential steps in a metabolic pathway; the enzyme exhibits ligand-mediated allosteric interactions between the . and > sites, which are connected by a 25 A-long tunnel. It is postulated that the common metabolite indole is transferred between these sites via the tunnel. This proposal will investigate: (a) the significance of the tunnel in metabolite transfer, (b) the mechanism and function of the allosteric effects, and (c) the chemical determinants of substrate specificity. Through genetic engineering, the human liver alcohol dehydrogenase isozymes are available in large amounts. This proposal will determine the roles played by protein conformation change and by electrostatic fields in hydride transfer. The aldehyde dehydrogenase-catalyzed oxidation of aldehydes occurs via an acyl-enzyme intermediate. How the oxidation state of bound coenzyme influences the properties of the acyl-enzyme will be studied. Rapid kinetic techniques, chromophoric substrates or analogues, and 1-H NMR will be used. Some of the tryptophan synthase and alcohol dehydrogenase work will involved studies of mutant enzymes prepared by site-directed mutagenesis.