Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe.
Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-(bromoacetyl)-2-(dimethylamino)naphthalene (BADAN), 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and alpha-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of the BADAN-modified enzyme is characterized by an S50 of 18.2 +/- 0.7, compared with the value of 2.2 +/- 0.3 for the ANF-induced spin transition, thus revealing an additional low-affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer.
Tsalkova
Tamara N
TN
Department of Pharmacology and Toxicology, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1031, USA.
Davydova
Nadezhda Y
NY
Halpert
James R
JR
Davydov
Dmitri R
DR
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D001571:Benzoflavones / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D013379:Substrate Specificity; D013997:Time Factors
C122155:6-bromoacetyl-2-dimethylaminonaphthalene; D001571:Benzoflavones; D003374:Coumarins; D005456:Fluorescent Dyes; C011512:alpha-naphthoflavone; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D003577:Cytochrome P-450 Enzyme System; D015081:2-Naphthylamine; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003545:Cysteine