Tryptophan‐75 is a Potential Gating Residue of Cytochrome P450 2D6

Cytochrome P450s (CYPs) are responsible for the metabolism of an estimated 70 – 80% of pharmaceutical drugs in humans. Structurally, they have deeply buried active sites. Evidence suggests that access to the active site, and the resulting metabolism of substrates, is influenced by amino acid side chains ‐ a phenomenon referred to as tunnel‐gating. Our investigation hypothesized that tunnel‐gating in CYP2D6 exists and is mediated by a tryptophan‐75 (Trp‐75) residue. Analysis in silico and in vitro was conducted to understand gating in CYP2D6 and the role of plasticity in enzyme mechanisms. Molecular Dynamics simulations measured energy landscapes of Trp‐75 and simulated substrate passage within channel 2b. It was determined that Trp‐75 was able to obstruct both entry and exit of substrates from the enzyme within channel 2b, and that the residue alternates between three stable states. A Trp‐75 to alanine (*W75A) mutant of CYP2D6 was prepared along with a reference *1 enzyme and both were tested for their Michaelis‐Menten kinetics and ligand binding properties in vitro with the substrates bufuralol, dextromethorphan, and debrisoquine. Trp‐75 was not found to impact ligand binding by the CYP2D6 enzymes. The residue was observed to significantly impact K m and v max , but no predictable kinetic trends were found. Furthermore, Trp‐75 was observed to impact enzymatic efficiency, where the absence of the residue doubled overall efficiency. Thus, we propose that the range of conformations available to Trp‐75 function in mediating channel‐gating that is substrate specific and that can serve to trap substrates into channel 2b for shuttling into the active site. Support or Funding Information NIH 2R15GM086767‐03 and the Sherman Fairchild Foundation