Koley A P, Robinson R C, Friedman F K
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochimie. 1996;78(8-9):706-13. doi: 10.1016/s0300-9084(97)82528-x.
The kinetics of CO binding to cytochrome P450, as measured by the flash photolysis technique, is a powerful probe of P450 structure-function relationships. The kinetics are sensitive to P450 conformation and dynamics and are modulated by P450 interactions with substrates and other components of the microsomal membrane. Application of a difference method to kinetic data analysis distinguishes the kinetic behavior of individual P450 forms in the microsomal membrane. This approach shows that substrates differentially modulate the kinetics via: 1) changes in P450 conformation/dynamics that either accelerate or reduce the binding rate; and/or 2) steric effects that reduce the rate. Both mechanisms are observed, the relative contributions of each varying in a substrate- and P450-dependent manner. In addition to microsomes, substrate interactions with individual P450s can be similarly probed using expressed P450s. Experiments with baculovirus-expressed human P450 3A4 show that this P450 consists of multiple conformers with distinct substrate specificities, an observation which provides a basis for its recognition of a wide array of structurally diverse substrates. These studies thus demonstrate the utility of CO binding kinetics in elucidating fundamental P450-substrate interactions in a biological membrane environment.
通过闪光光解技术测定的一氧化碳与细胞色素P450结合的动力学，是研究P450结构-功能关系的有力探针。该动力学对P450的构象和动力学敏感，并受到P450与底物及微粒体膜其他成分相互作用的调节。将差异法应用于动力学数据分析，可区分微粒体膜中单个P450形式的动力学行为。这种方法表明，底物通过以下方式差异调节动力学：1）P450构象/动力学的变化，加速或降低结合速率；和/或2）空间位阻效应降低速率。两种机制均被观察到，每种机制的相对贡献因底物和P450的不同而有所变化。除了微粒体，使用表达的P450同样可以探究底物与单个P450的相互作用。用杆状病毒表达的人P450 3A4进行的实验表明，这种P450由具有不同底物特异性的多个构象体组成，这一观察结果为其识别多种结构不同的底物提供了基础。因此，这些研究证明了CO结合动力学在阐明生物膜环境中基本的P450-底物相互作用方面的实用性。
