Structure and Dynamics of Metal-Containing Proteins

描述（由申请人提供）：细胞色素P450单加氧酶在许多生理过程中是至关重要的，包括类固醇生物合成，药物代谢和激活，以及异种生物降解。有关这些酶的结构和动态信息对于了解它们的活性（通常是非活性碳的氢氧基化）至关重要，并且作为药物设计的一部分，在预测抑制剂和底物的行为方面将发挥至关重要的作用。尽管在生物体中发现了大量的P450酶，但相对较少的P450酶已经结晶，并且缺乏快速表征P450活性位点及其与底物和抑制剂相互作用的方法。本提案描述了高分辨率多维核磁共振（NMR）方法在P450酶上的首次应用。在细胞色素P450cam （CYP101）中进行了广泛的序列1H， 15N和13C共振分配。CYP101活性位点的残基可以通过比较酶的顺磁性和反磁性形式来快速区分。活性位点共振只在抗磁形式下观察到。分配已被用于监测CP101与其生理氧化还原伙伴和效应体——腐胺氧还蛋白（Pdx）的相互作用。在此基础上，提出了Pdx效应活性的新模型。

DESCRIPTION (provided by applicant): Cytochrome P450 monooxygenases are critical in many physiological processes, including steroid biosynthesis, drug metabolism and activation, and xenobiotic degradation. Structural and dynamic information concerning these enzymes is critical for understanding their activity (typically hdryoxylation of unactivated carbons), and will play a vital role in predicting the behavior of inhibitors and substrates as a part of drug design. Despite the large numbers of P450 enzymes found in living organisms, relatively few have been crystallized, and methodology for rapid characterization of active sites of P450s and their interactions with substrates and inhibitors is lacking. This proposal describes the first application of high-resolution multidimensional nuclear magnetic resonance (NMR) methods to P450 enzymes. Extensive sequential 1H, 15N and 13C resonance assignments have been made in cytochrome P450cam (CYP101). Residues in the active site of CYP101 can be rapidly distinguished by comparison of paramagnetic and diamagnetic forms of the enzyme. Active site resonances are observed only in the diamagnetic form. Assignments have been used to monitor the interactions of CP101 with its physiological redox partner and effector, putidaredoxin (Pdx). Based on these observations, a new model for effector activity of Pdx has been proposed. During the next period of this project, the sequential assignments of CYP101 will be completed in multiple forms of the enzyme, local dynamics will be characterized as a function of oxidation state, substrate binding and effector binding in order to shed further light on the mechanism of "uncoupling", in which reducing equivalents are lost as superoxide, peroxide and water without turnover of substrate. Methodology for applying NMR to the characterization of active sites of other P450 enzymes will be tested, with CYP102 (cytochrome P450 BM-3) and CYP3A4, an important human P450, as test cases. Methodology developed during the current period for NMR structural characterization of metalloproteins will be further refined. This includes two-dimensional NMR methods that can be used near paramagnetic centers in proteins, and extraction of structural information from magnetic anisotropy data such as residual dipolar couplings and dipolar shifts.