STRUCTURE-FUNCTION OF CYTOCHROME P450

哺乳动物细胞色素P450的结构-功能关系如下： 考察具体地说，我们正在阐明P450之间的相互作用 以及微粒体电子载体蛋白、底物和配体。 我们 评估了P450与NADPH细胞色素P450还原酶的相互作用， 确定这种蛋白质中涉及的特定氨基酸残基- 蛋白质相互作用结合P450序列的分子建模 比对用于预测还原酶的P450结合位点。 与P450 2B 1上的这些区域相对应的合成肽是： 制备并评估其抑制还原酶介导的 P450活动对应于C螺旋的肽（残基116- 118）可以是一种肽， 134)Arg-125对甲基苯丙胺脱甲基酶有抑制作用， 被Glu取代。 此外，表面模拟肽是 所制备的由在三级结构中接近的序列组成 结构，但在原生序列中距离较远。组成的肽 螺旋C和L的元件在抑制 重组和微粒体中的几种P450介导的活性 系统.因此，这些结果鉴定了特定区域和残基 参与P450与还原酶的结合。 的构象和动力学 用CO闪光光解技术检测P450。我们评估 一系列多环芳烃的影响， 大小和形状对大鼠肝微粒体CO结合动力学 P450 1A 1和杆状病毒表达的人P450 1A 1。结果表明 底物通过双重机制调节CO结合， 构象和空间效应。表达杆状病毒的实验 人P450 3A 4，一种代谢许多重要药物的形式， 不同的结构，表明这种P450由一群 它们的底物识别性质不同的构象异构体。这 这一发现有助于解释这种P450广泛的底物特异性。这些 结果表明CO结合动力学作为P450探针的实用性 在天然膜环境中的结构/动力学。

Structure-function relationships for the mammalian cytochromes P450 are examined. Specifically, we are elucidating the interactions between P450 and microsomal electron carrier proteins, substrates and ligands. We assessed the interaction of P450 with NADPH cytochrome P450 reductase to identify the specific amino acid residues involved in this protein- protein interaction. Molecular modeling in conjunction with P450 sequence alignments were used to predict the P450 binding site for reductase. Synthetic peptides corresponding to these regions on P450 2B1 were prepared and assessed for their ability to inhibit reductase-mediated P450 activities. A peptide corresponding to the C helix (residues 116- 134) inhibited benzphetamine demethylase, but had no effect when Arg-125 was replaced by Glu. In addition surface simulatory peptides were prepared which consist of sequences which are proximate in the tertiary structure but distant in primary sequence. A peptide consisting of elements of helices C and L was particularly effective in inhibiting several P450-mediated activities in both reconstituted and microsomal systems. These results thus identify both specific regions and a residue involved in P450 binding to reductase. The conformation and dynamics of P450 were examined with the CO flash photolysis technique. We evaluated the effect of a series of polycyclic aromatic hydrocarbons of varying sizes and shapes on the CO binding kinetics of both rat liver microsomal P450 1A1 and baculovirus expressed human P450 1A1. The results showed that substrates modulate CO binding by a dual mechanism involving both conformational and steric effects. Experiments with baculovirus expressed human P450 3A4, a form which metabolizes many important drugs with diverse structures, showed that this P450 consists of a population of conformers that differ in their substrate recognition properties. This finding helps explain the broad substrate specificity of this P450. These results demonstrate the utility of CO binding kinetics as a probe of P450 structure/dynamics in a native membrane environment.