Allosteric behavior in cytochrome P450-dependent in vitro drug-drug interactions: A prospective based on conformational dynamics
Allosteric behavior in cytochrome P450-dependent in vitro drug-drug interactions: A prospective based on conformational dynamics
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DOI：
10.1021/tx0002132
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发表时间：
2001-04-01
影响因子：
4.1
通讯作者：
Lu, AYH
中科院分区：
文献类型：
--
作者：
Atkins, WM;Wang, RW;Lu, AYH
细胞色素 P450 (P450) 共同参与我们接触到的几乎所有药物的代谢。因此，大量学术和工业实验室对这些酶进行了深入研究（参见参考文献 1-4 的评论）。然而，我们定量预测 P450 活性位点基于代谢的药物间相互作用的能力是有限的。最近受到越来越多关注的细胞色素 P450 依赖性代谢的一个相关特征是一些 P450 同工酶/底物/效应物组合的明显“变构”行为，其中术语变构用于指获得非双曲线底物浓度与速度曲线的情况，或者当通过添加第二配体改变给定底物的动力学参数时 (5, 6)。事实上，随着人们对这种行为可能性的认识不断增强，我们有理由期待在 P450 依赖性代谢中发现更多变构例子。因此，在这一潜在“趋势”的早期阶段建立有用的模型来概念化这些过程并确定有助于定量的实验参数至关重要。基于哺乳动物 P450 和一种或多种结晶细菌 P450 之间的结构同源性，已经提出了几种肝微粒体细胞色素 P450 的同源模型 (7, 8)。这些模型以及定点诱变研究已被证明是研究各种 P450 的结构功能关系以及底物与酶之间相互作用的有用工具。例如，Harlow 和 Halpert (6) 报道，P450 3A4 的 L211F/D214E 双突变体在低底物浓度（即缺乏同向协同性）下表现出睾酮和孕酮 6β-羟基化速率增加，以及 R-萘黄酮引起的异向刺激水平降低。这些结果表明，P450 3A4 中的 Leu-211 和 Asp-214 在引发该酶催化的类固醇羟基化中的同向和异向协同性方面发挥着重要作用。此外，最近报道的嵌合哺乳动物 P450 (9) 的 X 射线结构为最终确定其他结构提供了乐观的信息，从而可以直接了解 P450 变构现象。尽管如此，许多哺乳动物细胞色素 P450 同工型的基于实验的结构模型不太可能很快出现，并且有关这些酶特性的几个基本问题仍未得到解答。本概述的目的是在变构定量观点的背景下评估已经提出的几种 P450 依赖性变构模型，并提出一个适应最大范围实验观察的通用模型。为了实现这些目标，考虑更传统的变构系统的特征，并确定经过充分研究的变构酶与迄今为止记录的 P450 的有限变构行为之间的相似性或差异是有用的。绝大多数变构蛋白都是寡聚的，在不连续的肽亚基上具有不同的配体结合位点。相比之下，P450 是单体酶，仅这一特征就应该引起人们对 P450 假定的变构行为的怀疑和兴奋。
The cytochrome P450s (P450s), collectively, contribute to the metabolism of nearly every drug to which we are exposed. As a result, these enzymes are studied intensely by an enormous number of academic and industrial labs (see refs 1-4 for reviews). However, our ability to quantitatively predict metabolism-based drug-drug interactions in P450 active sites is limited. A relevant characteristic of cytochrome P450-dependent metabolism that recently has received increased attention is the apparent “allosteric” behavior of some P450 isozyme/substrate/effector combinations, where the term allosteric is used to refer to cases where nonhyperbolic substrate concentration versus velocity curves are obtained, or when kinetic parameters for a given substrate are altered by addition of a second ligand (5, 6). In fact, with increased awareness of the possibility of such behavior, it is reasonable to expect additional examples of allosterism in P450-dependent metabolism to be discovered. Therefore, it is critically important to establish at an early stage in this potential “trend” useful models for conceptualizing these processes, and to identify experimental parameters that will aid in quantitation. Homology models have been proposed for several liver microsomal cytochrome P450s based on the structural homology between the mammalian P450s and one or several crystallized bacterial P450s (7, 8). These models, along with site-directed mutagenesis studies, have proved to be useful tools for the investigation of structurefunction relationship of various P450s and the interactions between substrates and enzymes. For example, Harlow and Halpert (6) reported that the L211F/D214E double mutant of P450 3A4 displays an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations (ie, absence of homotropic cooperativity) and a decreased level of heterotropic stimulation elicited by R-naphthoflavone. These results indicate that Leu-211 and Asp-214 in P450 3A4 play an important role in eliciting both homotropic and heterotropic cooperativity in steroid hydroxylation catalyzed by this enzyme. Also, the recently reported X-ray structure of a chimeric mammalian P450 (9) provides optimism for the eventual determination of additional structures which yield directly insight into P450 allosterism. Still, experimentally based structural models for many mammalian cytochrome P450 isoforms are not likely to be available soon, and several fundamental questions concerning the properties of these enzymes remain unanswered.The goals of this overview are to evaluate several models already proposed for P450-dependent allosterism in the context of quantitative views of allosterism, and to suggest a general model that accommodates the greatest range of experimental observations. To achieve these goals, it is useful to consider the characteristics of more traditional allosteric systems, and to identify similarities or differences between the well-studied allosteric enzymes and the limited allosteric behavior documented so far for the P450s. The vast majority of allosteric proteins have been oligomeric, with distinct ligand binding sites on noncontiguous peptide subunits. In contrast, P450s are monomeric enzymes, and this feature alone should prompt both skepticism and excitement about the putative allosteric behavior of the P450s.