Mechanistic Probes for Enzyme-Catalyzed Oxidations

描述(申请人提供)：该研究的主要重点是在基础水平上通过使用激光闪光光解(LFP)方法在模型和酶中产生活性氧化剂来研究氧化酶的反应机理，以及与提供反应中形成的瞬变细节的机械探针底物的相关研究。研究的主要目标是P450酶，它与许多疾病有关，是制药业较重要的酶之一。两种新的LFP方法，即铁(IV)络合物的光诱导配体裂解和铁(IV)-氧物种的光氧化，被用来产生各种高价铁-氧过渡产物，它们是酶中可能的反应物种。这种方法允许在微秒时间尺度上进行动力学研究，比快速混合实验快4-5个数量级。具体目标包括通过化合物II的光氧化产生P450酶中难以捉摸的铁氧物种，在模型中以及在可能的情况下在含血红素的酶中形成和表征高价铁氧物种，在模型中和在酶中对各种铁氧物种进行动力学表征以评估P450催化的氧化反应中的动力学能力，将方法扩展到含二铁的酶作为该计划的长期目标，以及旨在测试在酶催化的反应中形成的自由基和/或阳离子中间体的机械探针研究。P450酶是与癌症和肝脏疾病相关的较重要的酶之一，并与许多其他疾病有关，控制P450酶是一个重要的治疗目标，也是制药行业关注的问题。更好地了解酶中形成的活性物种的基本性质及其反应机制有望最终导致疾病治疗和药物设计方面与健康有关的进展。

DESCRIPTION (provided by applicant): The major focus of the research is the study of reaction mechanisms of the oxidizing enzymes at a fundamental level by using laser flash photolysis (LFP) methods to produce reactive oxidants in models and in the enzymes and related studies with mechanistic probe substrates that provide details about the transients formed in the reactions. The primary targets of study are the P450 enzymes, which have numerous disease relationships and are among the more important enzymes for the pharmaceutical industry. Two new LFP methods, photo-induced ligand cleavage of iron(IV) complexes and photo-oxidation of iron(IV)-oxo species, are employed to produce various high-valent iron-oxo transients that are the putative reactive species in the enzymes. This methodology permits kinetic studies on the microsecond time scale, which is 4-5 orders of magnitude faster than can be achieved in rapid mixing experiments. Specific aims include production of the elusive iron-oxo species in P450 enzymes via photo-oxidation of Compound II species, formation and characterization of high valent iron-oxo species in models and, if possible, in heme- containing enzymes, kinetic characterization of various iron-oxo species in models and in enzymes to evaluate the kinetic competency in P450-catalyzed oxidation reactions, extensions of the methods to di-iron containing enzymes as a long-range goal of the program, and mechanistic probe studies designed to test for radical and/or cationic intermediates formed in the enzyme-catalyzed reactions. The P450 enzymes are among the more important enzymes involved in cancer and liver disease and are associated with numerous other diseases, and control of P450 enzymes is an important therapeutic goal and a concern of the pharmaceutical industry. A better understanding of the fundamental nature of the reactive species formed in the enzymes and the mechanisms of their reactions is expected to lead to lead ultimately to health-related advances in disease treatment and drug design.