RAPID KINETICS STUDY OF BIOLOGICAL OXYGENASES

本研究是从事瞬态的检测和表征 生物加氧酶中存在的含氧中间体，其目的是 学习氧分子在生物系统中是如何被激活的。 我们目前 研究将集中在两个基本的酶系统上。 第一种是在细菌中经常发现的非血红素铁双加氧酶。 这些酶裂解相邻酚基之间的芳环。 我们有 先前检测到一种这样的酶中涉及的几种中间体， 原儿茶酸双加氧酶 我们将a）仔细研究质子 通量，以确定在反应的哪个阶段释放质子 B）研究替代底物及其与所述底物的反应， 酶，和c）使用EPR和快速反应冷冻淬灭技术，以更好地 确定这些中间体的化学性质。 另一种类似的酶， 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化酶。 主要的努力将 与肝微粒体N，S-单加氧酶黄素蛋白有关。 我们有 已经表征了反应机理中涉及的中间体。 我们将 尝试使用不同的基质来扩展这些研究，其中许多基质是 毒理学和毒理学上重要。 此外，我们将努力， 用FAD类似物代替FAD辅酶，目的是系统地 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.