Elucidation of Distinct Ligand Binding Sites for Cytochrome P450 3A4

Abstract

Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has broad selectivity for substrates. The enzyme can show marked catalytic regioselectivity and unusual patterns of homotropic and heterotropic cooperativity, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M(r) 614), a known substrate and inhibitor. Several C-terminal aminated peptides, including the model morphiceptin (YPFP-NH(2)), bind with spectral changes indicative of Fe-NH(2) bonding. The binding of the YPFP-NH(2) 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-NH(2) was not oxidized by P450 3A4 but blocked binding of the substrates testosterone and midazolam, with K(i) values similar to the spectral binding constant (K(s)) for YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of several typical P450 substrates with K(i) values 10-fold greater than the K(s) for binding YPFP-NH(2) and its K(i) for inhibiting substrate binding. The n values for cooperativity of these oxidations were not altered by YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of midazolam at two different positions (1'- and 4-) with 20-fold different K(i) values. The differences 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-NH(2) to ferrous P450 3A4 than to the ferric form. The ferrous protein can be considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH(2)) facilitate reduction of the ferric to ferrous enzyme. Our results with these peptides are considered in the context of several proposed models. A P450 3A4 model based on these peptide studies contains at least two and probably three distinct ligand sites, with testosterone and alpha-naphthoflavone occupying distinct sites. Midazolam appears to be able to bind to P450 3A4 in two modes, one corresponding to the testosterone binding mode and one postulated to reflect binding in a third site, distinct from both testosterone and alpha-naphthoflavone. The work with indinavir and YPFP-NH(2) also argues that room should be present in P450 3A4 to bind more than one smaller ligand in the "testosterone" site, although no direct evidence for such binding exists. Although this work with peptides provides evidence for the existence of multiple ligand binding sites, the results cannot be used to indicate their juxtaposition, which may vary through the catalytic cycle.

Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

“…Fitting the curve shown in Figure 2B with the Hill equation results in a K S and n-value of 6.0 ± 0.3 μM and 1.23 ± 0.07, respectively. These values are consistent with K S = 5.3-5.7 μM and n = 1.7 that were obtained previously [28,49]. However, in these titrations there appeared to be significant interference from the ANF absorbance shown as a rise at 350 nm [cf.…”

“…CYP3A4 has been modeled with two [11,12,14,15,[24][25][26], three [16,[27][28][29], and more ligand [13] binding sites. Since the x-ray crystal structures of CYP3A4 and P450 eryF have at most 2 ligands bound simultaneously [20,23], the data in this study were modeled with two ligand binding sites.…”

“…These compounds exhibit both homotropic and heterotropic cooperativity in steady state kinetic assays and optical equilibrium binding assays [14,15,25,28,[30][31][32][33][34][35][36]. Therefore, these compounds are ideally suited for studying the mechanism of heterotropic and homotropic cooperativity.…”

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

“…Initial "space-filling" models proposed that the large substrate-binding pocket of microsomal drug metabolizing cytochromes P450 sometimes requires more than one substrate molecule to assure a productive binding orientation of at least one of them [1][2][3][4][5][6][7][8][9]. However, this hypothesis fails to explain the entire body of observations obtained with cytochrome P450 3A4 (CYP3A4), the principal human drug-metabolizing enzyme [10].…”

“…However, this hypothesis fails to explain the entire body of observations obtained with cytochrome P450 3A4 (CYP3A4), the principal human drug-metabolizing enzyme [10]. Although the presence of at least two substrates in the same binding pocket is well established [7,[11][12][13][14][15][16]; the additive effects of different effectors and lack of competition between certain substrates possessing cooperativity [4,[7][8][9][17][18][19][20][21][22] have given rise to a set of complex and mutually incompatible models suggesting the presence of three or even more substrateselective binding sites per molecule of the enzyme [18,[23][24][25][26][27][28].…”