Mechanisms of Flavoproteins

描述（由申请人提供）：黄素蛋白催化广泛的生物学关键反应。其中许多涉及碳-氧或碳-氮键的两电子氧化。尽管这些反应的重要性，这些酶的机制仍然存在争议。在下一个资助期，我们建议把重点放在氧化碳和氮之间的键的黄素酶。我们建议研究两种酶参与代谢的多胺：多胺氧化酶，它优先氧化乙酰化的精胺和亚精胺，和精胺氧化酶，它喜欢非乙酰化的精胺。多胺对细胞生长至关重要，多胺氧化酶和精胺氧化酶的替代底物可防止癌细胞的生长。将研究这两种酶的催化机制和底物特异性的结构基础。我们还将继续研究硝基烷氧化酶。这种酶是一种不寻常的黄素蛋白，通过形成碳负离子中间体来氧化硝基烷烃的碳-氮键。该酶作为黄素蛋白催化的质子从碳中去除的模型。所有三种酶的方法将结合联合收割机稳态和快速反应动力学，包括动力学同位素效应，与结构的方法。在硝基烷烃氧化酶的情况下，非酶反应也将进行研究，以了解酶增加质子提取率的机制。公共卫生相关性：多胺氧化酶和精胺氧化酶都参与多胺的再循环。多胺是细胞生长所需要的，并且破坏多胺代谢的化合物已经显示出防止癌细胞的生长。 硝基烷烃氧化酶是研究蛋白质从碳中去除质子的机制的模型酶。

DESCRIPTION (provided by applicant): Flavoproteins catalyze a wide range of biologically critical reactions. Many of these involve the two electron oxidation of a carbon-oxygen or carbon-nitrogen bond. Despite the importance of such reactions, the mechanisms of these enzymes continue to be controversial. In the next grant period we propose to focus on flavoenzymes which oxidize bonds between carbon and nitrogen. We propose to study two enzymes involved in metabolism of polyamines: polyamine oxidase, which preferentially oxidizes acetylated spermine and spermidine, and spermine oxidase, which prefers unacetylated spermine. Polyamines are critical for the growth of cells, and alternate substrates for polyamine oxidase and spermine oxidase prevent growth of cancer cells. The catalytic mechanisms and structural bases for substrate specificity in these two enzymes will be studied. We will also continue study of nitroalkane oxidase. This enzyme is an unusual flavoprotein which oxidizes the carbon-nitrogen bond of nitroalkanes by forming a carbanion intermediate. The enzyme serves as a model for flavoprotein-catalyzed proton removal from carbon. The approaches to all three enzymes will combine steady-state and rapid-reaction kinetics, including kinetic isotope effects, with structural approaches. In the case of nitroalkane oxidase, the nonenzymatic reaction will also be studied to gain insight into the mechanism by which the enzyme increases the rate of proton abstraction. PUBLIC HEALTH RELEVANCE: Polyamine oxidase and spermine oxidase are both involved in recycling of polyamines. Polyamines are needed for the growth of cells, and compounds which disrupt}polyamine metabolism have been shown to prevent the growth of cancer cells. Nitroalkane oxidase is a model enzyme for studying the mechanisms of proteins which remove a proton from carbon.