title: Structural analysis of mammalian cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene: insight into partial enzymatic activity.
abstract: A combined structural and computational analysis of rabbit cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene (tBPA) has yielded insight into how the enzyme retains partial activity. Since conjugation to tBPA modifies a highly conserved active site residue, the residual activity of tBPA-labeled 2B4 observed in previous studies was puzzling. Here we describe the first crystal structures of a modified mammalian P450, which show an oxygenated metabolite of tBPA conjugated to Thr 302 of helix I. These results are consistent with previous studies that identified Thr 302 as the site of conjugation. In each structure, the core of 2B4 remains unchanged, but the arrangement of plastic regions differs. This results in one structure that is compact and closed. In this conformation, tBPA points toward helix B', making a 31° angle with the heme plane. This conformation is in agreement with previously performed in silico experiments. However, dimerization of 2B4 in the other structure, which is caused by movement of the B/C loop and helices F through G, alters the position of tBPA. In this case, tBPA lies almost parallel to the heme plane due to the presence of helix F' of the opposite monomer entering the active site to stabilize the dimer. However, docking experiments using this open form show that tBPA is able to rotate upward to give testosterone and 7-ethoxy-4-trifluoromethylcoumarin access to the heme, which could explain the previously observed partial activity.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, USA. scgay@ucsd.edu
authors.forename: Sean C
authors.initials: SC
authors.lastname: Gay
authors.forename: Haoming
authors.initials: H
authors.lastname: Zhang
authors.forename: P Ross
authors.initials: PR
authors.lastname: Wilderman
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Roberts
authors.forename: Tong
authors.initials: T
authors.lastname: Liu
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Hsia-Lien
authors.initials: HL
authors.lastname: Lin
authors.forename: Qinghai
authors.initials: Q
authors.lastname: Zhang
authors.forename: Virgil L
authors.initials: VL
authors.lastname: Woods
authors.forename: C David
authors.initials: CD
authors.lastname: Stout
authors.forename: Paul F
authors.initials: PF
authors.lastname: Hollenberg
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D000114:Acetylene / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D019295:Computational Biology; D018360:Crystallography, X-Ray; D000072467:Cytochrome P450 Family 2; D041961:Deuterium Exchange Measurement; D019281:Dimerization; D008958:Models, Molecular; D011487:Protein Conformation; D011817:Rabbits
chemical_list: C545118:4-tert-butylphenylacetylene; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D000114:Acetylene