CYTOCHROME P450 SUBSTRATE SITING AND MOTION

细胞色素P450羟基化产物的合理化和预测对于理解该酶的机制和理解药物代谢至关重要，但通常仍然是谜。我们提出用新的磁共振方法研究酶袋中底物的取向和底物在酶袋中的运动。一个长期存在的假设认为，与典型的酶学规律相反，许多与（哺乳动物或原核生物）细胞色素P450活性位点结合的底物在酶周转的时间尺度上快速旋转；这种假定的运动实际上可能是合理的，甚至是周转所需要的，因为基板口袋似乎相当疏水，化学上“无特征”。如果这种运动确实发生，它将对理解羟基化偏好具有重要意义。我们利用氘魔角旋转（MAS） SSNMR光谱技术，对金刚烷与CP450cam静息态结合的底物定位和运动进行了初步研究。这些初步数据支持广泛运动的假设。在此初步基础上，我们建议对医学相关系统进行更详细的研究。在本初步研究中，采用竞争位移和各向同性位移的居里定律温度依赖来验证活性位点的位置。氘自旋边带强度的模拟使我们能够确定电子-核偶极超精细耦合的强度，有效氘四极分裂的强度（作为局部运动的测量）以及描述偶极张量和四极张量相互取向的欧拉角。在最近的出版物中报告了模型化合物的类似数据的模拟；有效值约为5%是可能的，模拟用于确定距离的精度为0.5埃，最大“捕获半径”为7埃，角度确定在20度以内。对酶结合的adamantane数据的模拟表明，平均金属-氘距离为6.0（+/- 0.2）埃单位，并明确证明了快速的高对称性运动。提出了线形和旋转边带强度分析的计算和实验改进方法。我们建议表征其他底物（樟脑，苯，甲苯，二甲苯，尼古丁和华法林）的底物运动，以及酶循环的其他稳定中间体。利用膜模拟环境（DMPC/DHPC双胞体）研究哺乳动物P450酶与血红素相关的底物几何形状和底物运动；关于衬底几何形状的信息目前还无法从晶体学研究中获得。还计划对具有类似化学机制的微生物解毒酶进行研究。

Rationalization and prediction of hydroxylation products of cytochrome P450 is crucial for understanding the mechanism of this enzyme and for understanding drug metabolism, but remains generally enigmatic. We propose studies of orientation of substrates in the enzyme pocket, and motion of substrates in the enzyme pocket, by new magnetic resonance methods. A longstanding hypothesis states that, counter to typical enzymological dogma, many substrates bound to the active site of (mammalian or prokaryotic) cytochrome P450 rotate rapidly on the timescale of enzymatic turnover; this putative motion might actually be reasonable and even needed for turnover, since the substrate pocket appears to be rather hydrophobic and chemically "featureless". If this motion does indeed occur, it would have important implications for understanding hydroxylation preferences. We have performed preliminary studies of substrate siting and motion for adamantane bound to the resting state of CP450cam, using deuterium magic angle spinning (MAS) SSNMR spectroscopy. These preliminary data support the hypothesis of extensive motion. On this preliminary basis we propose to conduct more detailed studies with medicinally relevant systems. In this preliminary study, competitive displacement and Curie-law temperature dependence of isotropic shifts were used to verify location at the active site. Simulation of deuterium spinning sideband intensities allowed us to determine the strengths of the electron-nuclear dipolar hyperfine coupling, the strength of the effective deuterium quadrupolar splitting (which serves as measure of local motion) and the Euler angles describing the mutual orientation of the dipolar and quadrupolar tensors. Simulations of analogous data from model compounds are reported in a recent publication; RMS agreements of approximately 5 percent are possible, and the simulations are used to determine distances to a precision of 0.5 Angstrom units up to a maximum "capture radius" of 7 Angstrom units, and angles were determined to within 20 degrees. Simulations of the data for enzyme-bound adamantane indicated an average metal-deuterium distance of 6.0 (+/- 0.2) Angstrom units and clear-cut evidence of a rapid high- symmetry motion. Computational and experimental improvements for analysis of the line-shape and spinning side-band intensities are proposed. We propose to characterize substrate motion for additional substrates (camphor, benzene, toluene, xylene, nicotine and warfarin), and an additional stable intermediate of the enzymatic cycle. Substrate geometry relative to the heme and substrate motion will be studies for the mammalian P450 enzyme using a membrane-mimic environment (DMPC/DHPC bicelles); information about substrate geometries is unavailable from crystallographic studies at present. Studies involving microbial detoxification enzymes with similar chemical mechanisms are also planned.