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

该项目将促进对代谢中的底物通道、蛋白质功能中的变构和酶催化化学的理解。将研究三种体系：(1)细菌双酶色氨酸合成酶复合物，(2)人肝脏乙醇脱氢酶，(3)羊肝脏醛脱氢酶。色氨酸合成酶双酶复合物在代谢途径中催化两个连续的步骤；酶表现出配体介导的变构相互作用。和b>站点，它们由一条25 a长的隧道连接。据推测，常见的代谢物吲哚通过隧道在这些位点之间转移。该提案将研究：(a)通道在代谢物转移中的重要性，(b)变构效应的机制和功能，以及(c)底物特异性的化学决定因素。通过基因工程，人类肝脏酒精脱氢酶同工酶可以大量获得。这一建议将确定蛋白质构象变化和静电场在氢化物转移中所起的作用。醛脱氢酶催化醛的氧化通过酰基酶中间体发生。结合辅酶的氧化状态如何影响酰基酶的性质将被研究。将使用快速动力学技术、显色底物或类似物以及1-H NMR。一些色氨酸合成酶和乙醇脱氢酶的工作将涉及到通过定点诱变制备的突变酶的研究。

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.