CYTOCHROME P450 SUBSTRATE SITING AND MOTION

细胞色素P450的羟化产物的表征和预测对于理解这种酶的机制和理解药物代谢是至关重要的，但通常仍然是谜。 我们提出的酶口袋中的基板的方向，并在酶口袋中的基板的运动，通过新的磁共振方法的研究。 一个长期存在的假说指出，与典型的酶学教条相反，许多底物结合到（哺乳动物或原核生物）细胞色素P450的活性位点上，在酶促周转的时间尺度上迅速旋转;这种假定的运动实际上可能是合理的，甚至需要周转，因为底物口袋似乎是相当疏水的，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。 我们已经进行了初步的研究，衬底的位置和运动的金刚烷绑定到静止状态的CP 450 cam，使用氘魔角旋转（MAS）SSNMR光谱。 这些初步数据支持广泛运动的假设。 在这个初步的基础上，我们建议对与医学有关的系统进行更详细的研究。在这项初步研究中，竞争位移和居里定律的温度依赖性的各向同性位移被用来验证在活性位点的位置。 氘自旋边带强度的模拟使我们能够确定的电子核偶极超精细耦合的强度，有效的氘四极分裂的强度（作为衡量本地运动）和欧拉角描述的偶极和四极张量的相互取向。在最近的出版物中报道了来自模型化合物的类似数据的模拟;大约5%的RMS一致性是可能的，并且模拟用于确定精确度为0.5埃单位的距离，直到最大“捕获半径”为7埃单位，并且角度被确定为在20度内。 对酶结合的金刚烷的数据的模拟表明6.0（+/-0.2）埃单位的平均金属-氘距离和快速高对称运动的明确证据。 对线型和自旋边带强度的分析提出了计算和实验上的改进。 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，和一个额外的稳定的中间体的酶循环。 底物几何形状相对于血红素和底物运动将研究哺乳动物P450酶使用膜模拟环境（DMPC/DHPC bicelles）;有关底物几何形状的信息是目前无法从晶体学研究。 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.