Homotropic Versus Heterotopic Cooperativity of Cytochrome P450eryF: A Substrate Oxidation and Spectral Titration Study

• Published in: Drug metabolism and disposition Vol. 31; no. 4; pp. 356 - 359
• Main Authors: KHAN, Kishore K, HONG LIU, HALPERT, James R
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Pharmacology and Experimental Therapeutics 01.04.2003
• Subjects: Alanine - genetics; Androstenedione - metabolism; Atrial Natriuretic Factor - metabolism; Bacteria; Bacterial Proteins; Binding Sites; Biological and medical sciences; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Dose-Response Relationship, Drug; Flavones; Flavonoids - metabolism; General pharmacology; In Vitro Techniques; Medical sciences; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Mutagenesis, Site-Directed; Oxidation-Reduction; Pharmacokinetics. Pharmacogenetics. Drug-receptor interactions; Pharmacology. Drug treatments; Pyrenes - metabolism; Quinolines - metabolism; Structure-Activity Relationship; Substrate Specificity; Threonine - genetics; Titrimetry; Drug; Steroid; Molecular cooperativity; Homotropic interaction; Flavones; Cytochrome P450; Metabolism; Effector; Spectral analysis; Substrate; Heterotropic interaction; Binding capacity; Titration curve; Affinity; Bacteria; Oxidation; Pharmacokinetics; Comparative study
• ISSN: 0090-9556; 1521-009X
• DOI: 10.1124/dmd.31.4.356

P450eryF is the only bacterial P450 to show cooperativity of substrate binding and oxidation. However, the studies reported so far have provided evidence only for homotropic cooperativity of P450eryF but not for heterotropic cooperativity. Therefore, oxidation of 7-benzyloxyquinoline (7-BQ) and 1-pyrenebutanol (1-PB) by P450eryF A245T and spectral binding of 9-aminophenanthrene (9-AP) to wild-type P450eryF were investigated in the presence of various effectors. The addition of steroids and flavones caused no stimulation but rather moderate inhibition of 7-BQ or 1-PB oxidation by P450eryF A245T. However, the binding affinity of 9-AP was significantly increased in the presence of androstenedione or α-naphthoflavone (ANF). A comparative study with CYP3A4 revealed a similar increase in the binding affinity of 9-AP for the enzyme at low ANF concentrations but some competition at higher ANF concentrations. These studies, to our knowledge, provide the first report of heterotropic cooperativity in P450eryF as well as spectroscopic evidence for simultaneous presence of two ligand molecules in the CYP3A4 active site.