The Structural Basis for Homotropic and Heterotropic Cooperativity of Midazolam Metabolism by Human Cytochrome P450 3A4

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• 作者：Arthur G. Roberts ; Jing Yang ; James R. Halpert ; Sidney D. Nelson ; Kenneth T. Thummel ; William M. Atkins
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：December 20, 2011
• 年：2011
• 卷：50
• 期：50
• 页码：10804-10818
• 全文大小：712K
• 年卷期：v.50,no.50(December 20, 2011)
• ISSN：1520-4995

文摘

Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1鈥?OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1鈥?OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T₁ nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T₁ NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ molecules or an MDZ molecule and a CBZ molecule assume a stacked configuration within the CYP3A4 active site. In either case, the proton at position 4 of the MDZ molecule was closer to the heme than the protons of the 1鈥?CH₃ group. In contrast, molecular docking of a single molecule of MDZ revealed that the molecule was preferentially oriented with the 1鈥?CH₃ position closer to the heme than position 4. This study provides the first detailed molecular analysis of heterotropic and homotropic cooperativity of a human cytochrome P450 from an NMR-based model. Cooperativity of ligand binding through direct interaction between stacked molecules may represent a common motif for homotropic and heterotropic cooperativity.