AbstractAs an important member of cytochrome P450 (CYP) enzymes, human CYP1A2 is associated with the metabolism of caffeine and melatonin and the activation of precarcinogens. Besides, this CYP protein also involves in metabolizing 5-10% of clinical medicines. Some peripheral mutations in CYP1A2 (P42R, I386F, R431W, and R456H) significantly decrease the enzyme activities, resulting in a vital reduction in substrate metabolisms. To explore the effects of these peripheral mutations, we constructed a membrane-binding model for the full-length human CYP1A2 and studied their dynamic behaviors on lipid membranes. Free energy calculations indicate that the peripheral mutations donot influence substrate binding. P42R is located in the N-terminal anchor, and its positive charged sidechain is adverse to membrane binding. I386F enhances the van der Waals contacts of the water channel bottleneck and R456H breaks the hydrogen bonding interactions that function to position the BC loop, both of which result in a significant inhibition on the water channel. R431W causes a sidechain conformational rearrangement for aromatic residues around the substrate channel, making it in a closed state in most cases. Our computational simulations demonstrate that pi-pi stacking interactions are essential for substrate binding and channel opening. We hope that these findings may be of general relevance to the mutation-induced activity changes for CYP proteins, providing useful information for understanding the CYP-mediated drug metabolism.
A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts
