CYTOCHROME P450 SUBSTRATE SITING AND MOTION

细胞色素P450羟化产物的合理化和预测对于了解该酶的作用机制和了解药物代谢是至关重要的，但总体上仍是一个谜。我们提出用新的磁共振方法研究底物在酶口袋中的取向，以及底物在酶口袋中的运动。一个长期存在的假说认为，与典型的酶学教条相反，许多结合到(哺乳动物或原核生物)细胞色素P450活性部位的底物在酶的周转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，甚至是周转所必需的，因为底物口袋似乎相当疏水，化学上“无特征”。如果这一运动真的发生了，它将对理解羟化偏好具有重要的意义。我们利用重离子幻角旋转(MAS)SS核磁共振谱初步研究了金刚烷与CP450cam休止态结合的底物位置和运动。这些初步数据支持大范围运动的假说。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。在这一初步研究中，利用各向同性位移的竞争位移和居里定律的温度依赖性来验证活性中心的位置。通过对重离子自旋边带强度的模拟，我们可以确定电子-核偶极超精细耦合的强度、有效的重离子四极分裂的强度(作为局域运动的量度)以及描述偶极张量和四极张量相互取向的欧拉角。最近的一份出版物报告了对模型化合物中类似数据的模拟；RMS协议可能达到约5%，并且模拟被用于确定距离，精度为0.5埃单位，最大“捕获半径”为7埃，角度被确定为在20度以内。对酶结合金刚烷数据的模拟表明，金属-氚的平均距离为6.0(+/-0.2)埃单位，并明确证明了快速高对称性运动的证据。对线形和旋转边带强度的分析提出了计算和实验上的改进。我们建议对额外的底物(樟脑、苯、甲苯、二甲苯、尼古丁和华法林)以及酶循环中额外的稳定中间体的底物运动进行表征。与血红素和底物运动有关的底物几何形状将使用膜模拟环境(DMPC/DHPC双列)研究哺乳动物P450酶；目前无法从结晶学研究中获得有关底物几何形状的信息。还计划进行具有类似化学机制的微生物解毒酶的研究。

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.