title: Allosteric mechanisms in P450eryF probed with 1-pyrenebutanol, a novel fluorescent substrate.
abstract: 1-Pyrenebutanol (1-PB) has been used as a new fluorescent substrate for P450eryF to explore the molecular mechanisms of cooperativity. Hydroxylation of 1-PB by P450eryF was detected by both fluorometric and chromatographic assays. Binding was monitored by a substrate-induced low-to-high spin shift, as well as by fluorescence resonance energy transfer (FRET) from 1-PB to the heme. Spectrophotometric titration showed that P450eryF has high affinity for 1-PB with distinct positive cooperativity (S(50) = 12.4 +/- 2.2 microM, n = 2.3 +/- 0.6), as also revealed in activity measurements. FRET analysis showed a different binding process obeying a simple bimolecular mechanism with a K(D) = 2.15 +/- 0.8 microM that suggests the presence of the higher affinity binding site. 1-PB binding at this site appears not to modulate the spin state directly but rather to facilitate the spin shift caused by the interactions of P450eryF with the other substrate molecule.
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston 77555-1031, USA. d.davydov@utmb.edu
authors.forename: Dmitri R
authors.initials: DR
authors.lastname: Davydov
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D000495:Allosteric Site; D001426:Bacterial Proteins; D001665:Binding Sites; D000440:Butanols / Q000494:pharmacology*; D002855:Chromatography, Thin Layer; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008956:Models, Chemical; D011721:Pyrenes / Q000737:chemistry* / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence
chemical_list: C463480:1-pyrenebutanol; D001426:Bacterial Proteins; D000440:Butanols; D005456:Fluorescent Dyes; D011721:Pyrenes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea