Mechanisms of Flavoproteins

描述（由申请人提供）：许多黄素蛋白通过转移相当于黄素的氢化物来催化碳氮和碳氧键的氧化，在整个代谢过程中发挥关键作用。许多这些酶是治疗重大疾病的靶点，因此更好地了解它们的机制可以为治疗剂的开发提供见解。这些酶也属于多个结构家族；目前尚不清楚这是否反映了催化本质上相同反应的多种策略，还是趋同进化的一个例子。我们已经解释了先前对醇、酮酸和氨基酸氧化酶的研究，认为它们都具有共同的氢化物转移机制，而CN和CO键氧化酶在后者中对活性位点碱基的需求不同。我们现在建议进行实验，以确定氧化简单胺和多胺的酶是否利用相同的机制，尽管蛋白质和底物结构不同。我们将确定L-羟基-尼古丁氧化酶的机制，这是单胺氧化酶结构家族中的一种酶，被提议催化碳-碳键而不是碳-氮键的氧化。我们将继续我们的研究多胺氧化酶，酶氧化相同的底物与不同的底物特异性。15N和13C的动力学同位素效应将用于确定胺氧化的机制，并结合晶体学分析位点定向突变的影响，以确定不同特异性的结构基础。我们将测量13C同位素对黄素胺氧化酶两种结构类型的影响，以更好地定义它们的过渡态结构，并为其他结构家族提供基线值。我们将确定三甲胺脱氢酶结构家族的成员是否使用与其他胺氧化酶结构家族相同的胺氧化机制。这些实验的结果将验证我们的假设，即不同的结构家族

DESCRIPTION (provided by applicant): The many flavoproteins that catalyze oxidation of carbon-nitrogen and carbon-oxygen bonds by transferring a hydride equivalent to the flavin play critical roles throughout metabolism. A number of these enzymes are targets for treatment of major diseases, so that better understanding of their mechanisms could provide insight for development of therapeutic agents. These enzymes also belong to multiple structural families; it is unclear if this reflects multiple strategies to catalyze essentially identical reactions or is a example of convergent evolution. We have interpreted previous studies of alcohol, keto acid, and amino acid oxidizing enzymes as consistent with a common hydride transfer mechanism for all, with CN and CO bond oxidizing enzymes differing in the need for an active site base in the latter. We now propose to carry out experiments to determine if enzymes that oxidize simple amines and polyamines utilize this same mechanism despite differences in protein and substrate structure. We will determine the mechanism of L- hydroxy-nicotine oxidase, an enzyme in the monoamine oxidase structural family that is proposed to catalyze the oxidation of a carbon-carbon bond rather than a carbon-nitrogen bond. We will continue our studies of polyamine oxidases, enzymes that oxidize the same substrates with different substrate specificities. 15N and 13C kinetic isotope effects will be used to determine the mechanism of amine oxidation, and analysis of the effects of site-directed mutations combined with crystallography will be used to determine the structural basis for the different specificities. We will measure 13C isotope effects for two structural classes of flavin amine oxidases to better define their transition state structures and to provide baseline values for other structural familis. We will determine if members of the trimethylamine dehydrogenase structural family use the same mechanism for amine oxidation as other structural families of amine-oxidizing enzymes. The results of these experiments will test our hypothesis that the different structural families of amine oxidizing flavoenzymes are examples of convergent evolution on a common catalytic mechanism, potentially providing a unifying mechanism for a structurally divergent group of enzymes. Finally, we will initiate studies of the oxidative reactions of flavin-dependent amine oxidases, to probe for intermediates in the reaction and for oxygen-binding sites.