Homotropic versus heterotopic cooperativity of cytochrome P450eryF: a substrate oxidation and spectral titration study.
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 alpha-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.
Khan
Kishore K
KK
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031, USA. kkkhan@utmb.edu
Liu
Hong
H
Halpert
James R
JR
D000409:Alanine / Q000235:genetics; D000735:Androstenedione / Q000378:metabolism; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001419:Bacteria; D001426:Bacterial Proteins; D001665:Binding Sites; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D047309:Flavones; D005419:Flavonoids / Q000378:metabolism; D066298:In Vitro Techniques; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011721:Pyrenes / Q000378:metabolism; D011804:Quinolines / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics; D017186:Titrimetry
C463480:1-pyrenebutanol; D001426:Bacterial Proteins; D047309:Flavones; D005419:Flavonoids; D011721:Pyrenes; D011804:Quinolines; C065956:7-benzyloxyquinoline; D013912:Threonine; D000735:Androstenedione; D009320:Atrial Natriuretic Factor; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D000409:Alanine; C043562:flavone