Probing the cytochrome P-450 2B1 active site with diamantoid compounds.
Hydrocarbone diamantane has been shown to be a specific substrate with a high affinity for the binding site of PB-inducible cytochrome P-450 2B1 (Hodek et al. 1988). Using a difference spectroscopy approach, a battery of diamantane analogues and diamantane oxygen containing derivatives were examined for their interaction with P-450 2B1 active site. Of the compounds (diamantane and its analogues, adamantane and triamantane) tested, diamantane had the lowest value of a spectral dissociation constant Ks = 0.5 mumol/l, indicating that diamantane was accommodated well to the cytochrome P-450 2B1, hence values of 0.46 nm and 0.66 nm for the width and length of the diamantane molecule, respectively, were used to describe of the dimensions the cytochrome P-450 binding site. Adamantane (Ks = 1.3 mumol/l) is relatively small and thus it binds loosely whereas triamantane (Ks = 4.3 mumol/l) is bulky enough to fit the binding site. This conclusion has been confirmed by spectral competition experiments as well as metabolic studies. Of all oxygen containing derivatives diamantane 1,6-dicarboxylic acid dimethylester only exhibited a pronounced ligand interaction with cytochrome P-450. Using molecular dimensions of this derivative the distance of 0.56 nm from the heme iron to the center of the substrate binding site was estimated.
Hodek
P
P
Charles University, Department of Biochemistry, Praha, Czech Republic.
Burkhard
J
J
Janků
J
J
D000218:Adamantane / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000632:Aminopyrine / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D001667:Binding, Competitive; D055592:Biophysical Phenomena; D001703:Biophysics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D015394:Molecular Structure; D051381:Rats; D017208:Rats, Wistar; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000378:metabolism
D000632:Aminopyrine; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase; D000218:Adamantane