RAPID KINETICS STUDY OF BIOLOGICAL OXYGENASES

本研究致力于瞬变信号的检测与表征 存在于生物加氧酶中的含氧中间体，其目的是 了解分子氧如何在生物系统中被激活。我们目前的情况 研究将集中在两个基本的酶系统。 第一种是细菌中常见的非血红素铁双加氧酶。 这些酶在相邻的酚类基团之间裂解芳环。我们有 之前检测到几种中间体参与了一种这样的酶， 原儿茶酸双加氧酶。我们将是)仔细研究质子 通量，以便确定在反应的哪些阶段释放质子 或采用；b)研究替代底物及其与 酶，以及c)使用EPR和快速反应冷冻-淬火技术，以更好地 确定这些中间体的化学性质。第二种类似的酶， 儿茶酚双加氧酶也将通过类似的方法进行研究。 第二类系统是黄素蛋白羟基酶。主要的努力将是 关注肝微粒体N、S-单加氧酶黄素蛋白。我们有 已经表征了参与反应机理的中间体。我们会 尝试使用各种底物来扩展这些研究，其中许多是 具有重要的毒理和药理作用。此外，我们还将尝试和 用FAD类似物取代FAD辅酶，目的是系统性地 改变酶的反应性。

This study is engaged in the detection and characterization of transient oxygenated intermediates which occur in biological oxygenases, with the aim of learning how molecular oxygen is activated in biological systems. Our current studies will focus on two basic enzymatic systems. The first is that of nonheme iron dioxygenases frequently found in bacteria. These enzymes cleave aromatic rings between adjacent phenolic groups. We have previously detected several intermediates involved in one such enzyme, protocatechuate dioxygenase. We shall be a) carefully studying the proton flux, in order to establish at which stages of the reaction protons are release or taken up; b) investigating alternative substrates and their reaction with the enzyme, and c) using EPR and rapid reaction freeze-quench techniques to better establish the chemical nature of these intermediates. A second similar enzyme, catechol dioxygenase, will also be studied by similar approaches. The second type of system is flavoprotein hydroxylases. The major effort will be concerned with the liver microsomal N,S-monooxygenase flavoprotein. We have already characterized intermediates involved in the reaction mechanism. We will try and extend these studies using various substrates, many of which are toxicologically and pharmacologically important. In addition we shall try and replace the FAD coenzyme with FAD analogues, with the intent of systematically varying the reactivity of the enzyme.