STRUCTURE-FUNCTION OF CYTOCHROME P-450

我们正在研究细胞色素P450的结构-功能关系， 特别强调P450和其他混合物之间的相互作用， 功能加氧酶蛋白和底物和配体与 P450。 细胞色素P450与NADPH相互作用的研究进展 还原酶和细胞色素b5的延伸，以检查特定的氨基 酸残基参与这些蛋白质-蛋白质相互作用。分子 建模和多重序列比对被用来预测 分子间界面区，以及与这些相对应的肽 区域通过固相技术合成。结合 细胞色素b5对大鼠P450 2B 1的抑制率为75%， 对应于P450残基116-134。Glu取代Lys-122 几乎消除了这种抑制。这些结果表明， P450区域以及P450-细胞色素中的特定碱性残基 b5相互作用。 用一氧化碳（CO）检测P450的结构和动力学。 闪光光解技术调节CO结合到 微粒体P450通过P450效应子用于动力学定义 单个P450在内质网内。这种方法表明， 底物苄非他明使P450的CO进入通道扩大 2B 1以加速CO结合。多环芳烃 大小和形状同样增强CO与P450 1A 1的结合。最大的 速率增加，观察到与较小的三环，而 较大的四环素和五环素产生的增加幅度较小。Flash 光解研究也进行了杆状病毒表达的人 P450 3A4.至少有两个P450构象不同， 它们对常见底物的反应。因此，CO结合动力学是 有价值的探针的结构/动力学的个别P450在一个本地 膜环境

We are examining structure-function relationships for cytochrome P450, with particular emphasis on the interactions between P450 and other mixed function oxygenase proteins and binding of substrates and ligands to P450. Previous studies on the interactions of P450 with NADPH cytochrome P450 reductase and cytochrome b5 were extended to examine the specific amino acid residues involved in these protein-protein interactions. Molecular modeling and multiple sequence alignments were used to predict intermolecular interface regions, and peptides corresponding to these regions were synthesized by the solid phase technique. Binding of cytochrome b5 to rat P450 2B1 was inhibited (by 75%) by a peptide corresponding to P450 residues 116-134. Substitution of Glu for Lys-122 nearly abolished this inhibition. These results indicate a role for this P450 region as well as a specific basic residue in the P450-cytochrome b5 interaction. P450 structure and dynamics were examined with the carbon monoxide (CO) flash photolysis technique. Modulation of the kinetics of CO binding to microsomal P450 by P450 effectors was used to kinetically define individual P450s within the endoplasmic reticulum. This approach showed that the substrate benzphetamine enlarges the CO access channel of P450 2B1 to accelerate CO binding. Polycyclic aromatic hydrocarbons of varying sizes and shapes likewise enhanced CO binding to P450 1A1. The largest rate increases were observed with the smaller tricyclics, while the larger tetracyclics and pentacyclics yielded more modest increases. Flash photolysis studies were also carried out on baculovirus expressed human P450 3A4. At least two P450 conformers were identified which differed in their response to common substrates. The CO binding kinetics is thus a valuable probe of the structure/dynamics of individual P450s in a native membrane environment.