Raag R, Li H, Jones B C, Poulos T L
Center for Advanced Research in Biotechnology, Maryland Biotechnology Institute, University of Maryland, Rockville 20850.
Biochemistry. 1993 May 4;32(17):4571-8. doi: 10.1021/bi00068a013.
The X-ray crystal structures of cytochrome P-450CAM complexed with both enantiomers of a chiral, multifunctional inhibitor have been refined to R-factors of 21.0% [(+)-enantiomer] and 19.6% [(-)-enantiomer] at approximately 2.1-A resolution. Binding of either enantiomer, both considerably larger than the natural substrate camphor, results in similar, dramatic structural changes in the enzyme. In contrast to all previous P-450CAM crystallographic structures, the Tyr96 side chain is not pointing "down" toward the heme but is rather directed "up" into the proposed substrate access channel. This conformational change is accompanied by the displacement of the Phe193 side chain out into the solvent at the enzyme surface. These changes are consistent with the assignment of this region of the enzyme as the access channel [Poulos et al. (1986) Biochemistry 25, 5314-5322] and suggest that several aromatic residues lining the channel may be involved in substrate recognition and channeling to the active site. The cation usually observed coordinated to the Tyr96 carbonyl oxygen is missing in the presence of the (+)-enantiomer but is present with the (-)-enantiomer. The Phe87 side chain, located near the inhibitor binding site, adopts different orientations depending upon which enantiomer is bound. Finally, electron density reveals that although the inhibitor enantiomers were dichlorinated as provided, when bound to P-450CAM the chlorine atoms are present at only 0-20% occupancy, probably reflecting selective binding of impurities in the samples. Coordinates of these inhibited P-450CAM complexes have been deposited in the Brookhaven Protein Data Bank [Bernstein et al. (1977) J. Mol. Biol. 112, 535-542].
细胞色素P-450CAM与一种手性多功能抑制剂的两种对映体复合的X射线晶体结构，在约2.1埃分辨率下已精修至R因子分别为21.0%[（+）-对映体]和19.6%[（-）-对映体]。两种对映体的结合，二者都比天然底物樟脑大得多，导致酶中出现相似的显著结构变化。与之前所有的P-450CAM晶体结构不同，Tyr96侧链不是指向血红素“下方”，而是指向“上方”进入推测的底物通道。这种构象变化伴随着Phe193侧链向酶表面的溶剂中位移。这些变化与将酶的该区域指定为通道[普洛斯等人（1986年）《生物化学》25卷，5314 - 5322页]一致，并表明通道内衬的几个芳香族残基可能参与底物识别以及向活性位点的转运。通常观察到与Tyr96羰基氧配位的阳离子在（+）-对映体存在时缺失，但在（-）-对映体存在时存在。位于抑制剂结合位点附近的Phe87侧链，根据结合的对映体不同而采取不同取向。最后，电子密度显示，尽管抑制剂对映体按提供的是二氯化的，但与P-450CAM结合时氯原子的占有率仅为0 - 20%，这可能反映了样品中杂质的选择性结合。这些受抑制的P-450CAM复合物的坐标已存入布鲁克海文蛋白质数据库[伯恩斯坦等人（1977年）《分子生物学杂志》112卷，535 - 542页]。
