A REFILLED SUBSTRATE MODEL FOR HUMAN CYTOCHROME-P450 2D6

Cytochromes P450 (P450s) constitute a large superfamily of heme-contai ning enzymes, capable of oxidizing and reducing a variety of substrate s. Cytochrome P450 2D6 is a polymorphic member of the P450 superfamily and is absent in 5-9% of the Caucasian population as a result of a re cessive inheritance of gene mutations. Recently, the importance of asp artic acid 301 (Asp(301)) for the catalytic activity of P450 2D6, as i ndicated by a preliminary homology model, was confirmed by site-direct ed mutagenesis experiments. In this study, the heme moiety and the I-h elix containing Asp(301) were incorporated into the previously derived substrate model for P450 2D6, in the spatial orientations as derived from a recently improved protein model for P450 2D6, thereby incorpora ting steric restrictions and orientational preferences into the substr ate model. The direction of well-defined hydrogen bonds formed between Asp(301) and basic nitrogen atoms of P450 2D6 substrates was incorpor ated into the substrate model as well. Also, the position(s) of the ba sic nitrogen atom(s) of the substrates was/were allowed more flexibili ty. This was established through the attachment of an aspartic acid re sidue (representing Asp(301)) to the (protonated) basic nitrogen atom( s) of the substrates and superimposing the C(a)lpha- and C(b)eta-atoms of this aspartic acid residue in the fitting procedure instead of the basic nitrogen atoms. A variety of 8 substrates of P450 2D6 (comprisi ng 17 known P450 2D6 dependent metabolic pathways) has been incorporat ed successfully into this refined and more restrictive substrate model .