Abstract
Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of ∼50% clinically used drugs. Midazolam (MDZ) is a commonly used sedative drug and serves as a marker substrate for the CYP3A4 activity assessment. MDZ is metabolized by CYP3A4 to two hydroxylation products, 1′-OH-MDZ and 4-OH-MDZ. It has been reported that the ratio of 1′-OH-MDZ and 4-OH-MDZ is dependent on the MDZ concentration, which reflects the homotropic cooperative behavior in MDZ metabolism by CYP3A4. Here, we used quantum chemistry (QC), molecular docking, conventional molecular dynamics (cMD), and Gaussian accelerated molecular dynamics (GaMD) approaches to investigate the mechanism of the interactions between CYP3A4 and MDZ. QC calculations suggest that C1′ is less reactive for hydroxylation than C4, which is a pro-chirality carbon. However, the 4-OH-MDZ product is likely to be racemic due to the chirality inversion in the rebound step. The MD simulation results indicate that MDZ at the peripheral allosteric site is not stable and the binding modes of the MDZ molecules at the productive site are in line with the experimental observations.
Highlights
Ligand cooperativity is frequently found in the reactions catalyzed by cytochrome P450 enzymes (P450s).[1−5] The cooperative binding of ligands can change the kinetics profile of the catalytic reactions, resulting in a hyperbolic or sigmoidal curve on the graph of reaction rate versus substrate concentration.[3]
In the case that the bound ligands are the same, it is referred to as homotropic cooperativity, and otherwise, it is referred to as heterotropic cooperativity.[3]
Studies have been carried out for unraveling the binding modes of these substrates to cytochrome P450 3A4 (CYP3A4).11,15,16 The cooperative ligand binding at the productive site of CYP3A4 has been observed in the available crystal structures.[17−19] Some other experimental and computational studies indicate that the cooperative binding may take place at the peripheral allosteric site, which is in the middle of the productive site and membrane interaction area.[20−22] there are three crystal structures of CYP3A4 with a steroid molecule bound at this site.[23,24]
Summary
Ligand cooperativity is frequently found in the reactions catalyzed by cytochrome P450 enzymes (P450s).[1−5] The cooperative binding of ligands can change the kinetics profile of the catalytic reactions, resulting in a hyperbolic or sigmoidal curve on the graph of reaction rate versus substrate concentration.[3]. Studies have been carried out for unraveling the binding modes of these substrates to CYP3A4.11,15,16 The cooperative ligand binding at the productive site of CYP3A4 has been observed in the available crystal structures (see PDB: 2V0M, 4K9U, and 4D6Z).[17−19] Some other experimental and computational studies indicate that the cooperative binding may take place at the peripheral allosteric site, which is in the middle of the productive site and membrane interaction area.[20−22] there are three crystal structures of CYP3A4 with a steroid molecule bound at this site (see PDB: 1W0F, 5A1P, and 5A1R).[23,24] These efforts provide a vast amount of information for understanding the aberrant catalytic kinetics caused by the cooperative ligand binding
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