Mechanistic Probes for Enzyme-Catalyzed Oxidations

描述（由申请人提供）：研究的主要重点是通过使用激光闪光光解（LFP）方法在模型和酶中产生反应性氧化剂，以及使用提供反应中形成的瞬态细节的机械探针底物的相关研究，在基础水平上研究氧化酶的反应机制。研究的主要目标是P450酶，它与许多疾病有关，是制药工业中最重要的酶之一。两个新的LFP方法，光诱导的配体裂解的铁（IV）络合物和光氧化的铁（IV）-氧物种，产生各种高价的铁-氧瞬变，是推定的反应物种的酶。这种方法允许在微秒时间尺度上进行动力学研究，这比快速混合实验快4-5个数量级。具体目的包括通过化合物II物质的光氧化在P450酶中产生难以捉摸的铁-氧代物质，在模型中以及如果可能的话在含血红素的酶中形成和表征高价铁-氧代物质，在模型和酶中动力学表征各种铁-氧代物质以评估P450催化的氧化反应中的动力学能力，将该方法扩展到含二铁的酶作为该计划的长期目标，以及设计用于测试酶催化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酶是参与癌症和肝病的更重要的酶之一，并且与许多其他疾病相关，并且P450酶的控制是重要的治疗目标和制药工业的关注点。更好地了解酶中形成的反应性物质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相关的进展。

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.