Mechanisms of Flavoproteins

描述(申请人提供)：许多黄素蛋白通过转移等同于黄素的氢化物来催化碳-氮和碳氧键的氧化，在整个新陈代谢过程中发挥着关键作用。其中许多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洞察力。这些酶也属于多个结构家族；尚不清楚这是反映了催化基本上相同的反应的多种策略，还是收敛进化的例子。我们已经解释了以前对酒精、酮酸和氨基酸氧化酶的研究，认为它们都符合共同的氢化物转移机制，CN和CO键氧化酶的不同之处在于后者需要一个活性中心碱。我们现在建议进行实验，以确定氧化单胺和多胺的酶是否使用相同的机制，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，被认为催化碳-碳键而不是碳-氮键的氧化。我们将继续我们对多胺氧化酶的研究，这种酶能以不同的底物特性氧化相同的底物。将使用15N和13C动力学同位素效应来确定胺氧化的机制，并将使用定点突变和结晶学相结合的影响分析来确定不同特异性的结构基础。我们将测量两类黄素胺氧化酶的13C同位素效应，以便更好地确定它们的过渡态结构，并为其他结构家族提供基线数据。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结构家族的胺氧化酶相同的机制进行胺氧化。这些实验的结果将检验我们的假设，即不同的结构家族 胺氧化黄素酶是共同催化机制上收敛进化的例子，潜在地为结构上不同的一组酶提供了一个统一的机制。最后，我们将启动对黄素依赖的胺氧化酶的氧化反应的研究，以探索反应中的中间产物和氧结合部位。

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.