title: Flexibility of human cytochrome P450 enzymes: molecular dynamics and spectroscopy reveal important function-related variations.
abstract: To gain more complete insight into flexibility and malleability of five forms of human liver cytochrome P450 enzymes, which play major roles in drug metabolism (CYPs 1A2, 2A6, 2C9, 2D6 and 3A4), we employed UV/VIS and resonance Raman spectroscopy in combination with all-atomic molecular dynamics simulations under normal and high pressure conditions (300 MPa). In general, the high pressure reduces the flexibility of CYPs, which become more dense and compact as their radii of gyration and temperature B-factors diminish. The flexibility of CYPs spans the regions, which are localized in solvent exposed loops. A considerable degree of flexibility is also observed at amino-acids making the pw2 and solvent channels, which are suggested to serve for substrate access and/or product release. The number of water molecules as well as the number of protein backbone atoms of the active site in close proximity of heme cofactor generally increases under high pressure. This finding provides new insights regarding the interpretation of pressure-related Soret band red shifts. Presented results also point towards considerable differences between the CYP forms studied: CYP2A6 and CYP1A2 have the least malleable active sites while those of CYP2D6, CYP2C9 and CYP3A4 have considerably greater degrees of flexibility or malleability. In addition, the number of water molecules in the active site cavity of CYP3A4 anomalously decreases under high pressure due to opening of the active site. These results correlate with the known substrate promiscuity of the respective CYP forms, with CYP3A4 displaying the highest substrate promiscuity, corresponding to the most open and malleable active site, whereas CYP1A2 and CYP2A6 show a high substrate-specificity and have a small and rigid active sites.
authors.affiliation: Department of Physical Chemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic.
authors.forename: Tereza
authors.initials: T
authors.lastname: Hendrychová
authors.forename: Eva
authors.initials: E
authors.lastname: Anzenbacherová
authors.forename: Jiří
authors.initials: J
authors.lastname: Hudeček
authors.forename: Josef
authors.initials: J
authors.lastname: Skopalík
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lange
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.forename: Michal
authors.initials: M
authors.lastname: Otyepka
authors.forename: Pavel
authors.initials: P
authors.lastname: Anzenbacher
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006874:Hydrostatic Pressure; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry / Q000379:methods; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C585710:CYP2A6 protein, human; D019388:Cytochrome P-450 CYP1A2; D065637:Cytochrome P-450 CYP2A6; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human