Mechanistic Probes for Enzyme-Catalyzed Oxidations

描述（由申请人提供）：该计划旨在了解 酶催化各种氧化反应的机理细节 包括烃和胺的底物。烃氧化 酶包括含血红素的细胞色素P450酶、甲烷二铁 单加氧酶（MMO）和各种单核的非血红素铁酶。的 胺氧化酶包括含黄素辅因子的酶， 细胞色素P450酶该计划的一个前提是， 的机制将有医学上的好处，试图控制酶 行动上 涉及到几种研究方法。使用机械探针基底， 因为这种方法是唯一一种能产生定量的 P450和MMO羟基化中间寿命动力学信息 反应.探针是以一种特有的方式重新排列的化合物 当它们被转化为特定的中间体时， 关于中间体性质的信息。在激进的情况下， 中间体，确定重排的速率常数，和 自由基中间体的寿命可以由产物比率计算。的 探针设计和自由基反应速率常数的测定 重排，包括超快速重排，是生物学的组成部分。 程序. 其他研究方法包括低温下的光谱技术。 使用ESR和ENDOR光谱学对P450酶进行冷冻还原研究， 与另一组合作进行。冷冻氧化研究 铁卟啉和P450酶涉及瞬态的光学检测 通过激光闪光光解方法产生。MMO的停流动力学研究 还提出了酶。P450和MMO研究的范围 酶。 研究黄素辅因子酶氧化胺的探针在 先进的发展，研究将开始与黄连素桥 酶（BBE），一种催化氧化环化的FAD-辅因子酶 生物碱生物合成反应。单核非血红素铁探针 酶处于早期发展阶段，目前研究的目标是 期间将是探针的设计，这些探针是酶的底物， 例如克拉维马酸合酶。

DESCRIPTION (provided by applicant): This program is aimed at understanding the mechanistic details of enzymes that catalyze oxidation reactions of a variety of substrates including hydrocarbons and amines. The hydrocarbon-oxidizing enzymes include the heme-containing cytochrome P450 enzymes, the diiron methane monooxygenase (MMO) enzymes and various mononculear, non-heme iron enzymes. The amine-oxidizing enzymes include flavin cofactor-containing enzymes and cytochrome P450 enzymes. A premise of the program is that a good understanding of the mechanisms will have medicinal benefits in attempts to control enzyme action. Several methods of study are involved. Mechanistic probe substrates are used extensively because this method is the only one to have produced quantitative kinetic information about intermediate lifetimes in P450 and MMO hydroxylation reactions. The probes are compounds that rearrange in a characteristic manner when they are converted into a specific intermediate, and, in doing so, provide information about the nature of the intermediates. In the case of radical intermediates, the rate constants for rearrangement are determined, and lifetimes of radical intermediates can be computed from product ratios. The design of the probes and measurements of the rate constants for radical rearrangements, including ultra fast rearrangements, are integral parts of the program. Other methods of study include spectroscopic techniques at low temperature. Cryoreduction studies of P450 enzymes using ESR and ENDOR spectrsocopies will be conducted in collaboration with another group. Cryooxidation studies of iron-porphyrins and P450 enzymes involve optical detection of transients produced by laser flash photolysis methods. Stopped-flow kinetic studies of MMO enzymes are also proposed. The P450 and MMO studies are performed with a range of enzymes. Probes for studies of amine oxidations by flavin cofactor enzymes are in advanced development, and studies will be initiated with the berberine bridge enzyme (BBE), an FAD-cofactor enzyme that catalyzes an oxidative cyclization reaction in alkaloid biosynthesis. Probes for mononuclear, non-heme iron enzymes are in early development, and the objective in the current research period will be the design of probes that are substrates for the enzymes of interest such as clavimanate synthase.