title: NMR studies of ligand binding to P450(eryF) provides insight into the mechanism of cooperativity.
abstract: Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450(eryF). UV/vis absorbance spectra of P450(eryF) demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of K(S) = 4 and 200 microM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450(eryF) with 9-AP and TST indicated binding on intermediate and fast chemical exchange time scales, respectively, which was consistent with the Hill-equation-derived K(S) values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands "contact" the same phenylalanines within the active site of P450(eryF). This finding is in agreement with X-ray crystal structures of bound P450(eryF) showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450(eryF) conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Box 357610, Seattle, Washington 98195, USA.
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Roberts
authors.forename: M Dolores
authors.initials: MD
authors.lastname: Díaz
authors.forename: Jed N
authors.initials: JN
authors.lastname: Lampe
authors.forename: Laura M
authors.initials: LM
authors.lastname: Shireman
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Grinstead
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Dabrowski
authors.forename: Josh T
authors.initials: JT
authors.lastname: Pearson
authors.forename: Michael K
authors.initials: MK
authors.lastname: Bowman
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
authors.forename: A Patricia
authors.initials: AP
authors.lastname: Campbell
mesh_terms: D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004917:Erythromycin / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010616:Phenanthrenes / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D008024:Ligands; D010616:Phenanthrenes; D013739:Testosterone; D010649:Phenylalanine; D004917:Erythromycin; C041014:9-aminophenanthrene; D003577:Cytochrome P-450 Enzyme System