Mechanistic Probes for Enzyme-Catalyzed Oxidations

描述(由申请者提供)：本课程旨在了解 催化各种氧化反应的酶的机理细节 包括碳氢化合物和胺在内的底物。碳氢化合物的氧化 酶包括含血红素的细胞色素P450酶，二铁甲烷 单加氧酶(MMO)和各种单核、非血红素铁酶。这个 胺氧化酶包括含有黄素辅因子的酶和 细胞色素P450酶。该计划的一个前提是，对 在试图控制酶的过程中，这些机制将具有医学上的好处 行动。 其中涉及到几种研究方法。使用机械探头基板 因为这种方法是唯一一种能够产生定量的 P450和MMO羟化中间寿命的动力学信息 反应。探针是以特有的方式重新排列的化合物 当它们被转换为特定的中间体时，并在这样做时提供 有关中间体性质的信息。在激进的情况下 中间体，确定重排的速率常数，以及 自由基中间体的寿命可以从产品比率计算出来。这个 探头的设计及自由基速率常数的测定 重新安排，包括超快的重新安排，是 程序。 其他研究方法包括低温下的光谱技术。 用ESR和Endor光谱分析研究P450酶的低温还原 将与另一个小组合作进行。超低温氧化技术的研究 铁-卟啉和P450酶涉及对瞬变的光学检测 采用激光闪光光解的方法制作。MMO停流动力学研究 还提出了酶制剂的概念。P450和MMO研究在一定范围内进行 酶的作用。 黄素辅酶催化胺氧化反应的研究进展 先进的开发，并将启动对黄连素桥的研究 酶(BBE)，一种催化氧化环化的FAD辅酶 生物碱合成中的反应。单核、非血红素铁的探针 酶还处于早期开发阶段，目前的研究目标是 Period将设计作为酶的底物的探针 兴趣，如克拉维那酸合成酶。

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.