Elucidation of distinct ligand binding sites for cytochrome P450 3A4

Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has b road selectivity for substrates. The enzyme can show marked catalytic regio selectivity and unusual patterns of homotropic and heterotropic cooperativi ty, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M-r 614), a known subst rate and inhibitor. Several C-terminal aminated peptides, including the mod el morphiceptin (YPFP-NH2), bind with spectral changes indicative of Fe-NH2 bonding. The binding of the YPFP-NH2 N-terminal amine and the influence of C-terminal modification on binding argue that the entire molecule (M-r 521 ) fits within P450 3A4. YPFP-NH2 was not oxidized by P450 3A4 but blocked b inding of the substrates testosterone and midazolam, with K-i values simila r to the spectral binding constant (K-s) for YPFP-NH2. YPFP-NH2 inhibited t he oxidations of several typical P450 substrates with K-i values 10-fold gr eater than the K-s for binding YPFP-NH2 and its K-i for inhibiting substrat e binding. The n values for cooperativity of these oxidations were not alte red by YPFP-NH2, YPFP-NH2 inhibited the oxidations of midazolam at two diff erent positions (1'- and 4-) with 20-fold different K-i values. The differe nces in the K-i values for blocking the binding to ferric P450 3A4 and the oxidation of several substrates may be attributed to weaker binding of YPFP -NH2 to ferrous P450 3A4 than to the ferric form. The ferrous protein can b e considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH2) facilitate reduction of the ferric to f errous enzyme. Our results with these peptides are considered in the contex t of several proposed models. A P450 3A4 model based on these peptide studi es contains at least two and probably three distinct ligand sites, with tes tosterone and alpha-naphthoflavone occupying distinct sites. Midazolam appe ars to be able to bind to P450 3A4 in two modes, one corresponding to the t estosterone binding mode and one postulated to reflect binding in a third s ite, distinct from both testosterone and alpha-naphthoflavone, The work wit h indinavir and YPFP-NH2 also argues that room should be present in P450 3A 4 to bind more than one smaller ligand in the "testosterone" site, although no direct evidence for such binding exists. Although this work with peptid es provides evidence for the existence of multiple ligand binding sites, th e results cannot be used to indicate their juxtaposition, which may vary th rough the catalytic cycle.