Activation of CYP3A4: Evidence for the Simultaneous Binding of Two Substrates in a Cytochrome P450 Active Site

Abstract

A unique characteristic of the CYP3A subfamily of cytochrome P450 enzymes is their ability to be activated by certain compounds. It is reported that CYP3A4-catalyzed phenanthrene metabolism is activated by 7,8-benzoflavone and that 7,8-benzoflavone serves as a substrate for CYP3A4. Kinetic analyses of these two substrates show that 7,8-benzoflavone increases the Vmax of phenanthrene metabolism without changing the Km and that phenanthrene decreases the Vmax of 7,8-benzoflavone metabolism without increasing the Km. These results suggest that both substrates (or substrate and activator) are simultaneously present in the active site. Both compounds must have access to the active oxygen, since neither phenanthrene nor 7,8-benzoflavone can competitively inhibit the other substrate. These data provide the first evidence that two different molecules can be simultaneously bound to the same P450 active site. Additionally, structure-activity relationship studies were performed with derivatives of 7,8-benzoflavone structure. The effects of 13 different compounds on the regioselectivity of phenanthrene, chrysene, and benzo[a]pyrene metabolism were determined. Of the 13 compounds studied, 6 were activators, 2 were partial activators, and 5 were inhibitors. Analyses of the data suggest that (1) naphthalene substituted with a ketone in the 2-position can activate 3A4 and (2) the presence of an activator results in a narrower effective substrate binding site. Since the CYP3A enzymes are very important in drug metabolism, the possibility of activation, and autoactivation, must be considered when in vitro-in vivo correlations are made and when possible drug interactions are considered.

Surface Plasmon Resonance Analysis of Antifungal Azoles Binding to CYP3A4 with Kinetic Resolution of Multiple Binding Orientations

“…It is now widely accepted that 'allosteric' steady state CYP kinetics may result from multiple drugs binding to a single CYP molecule [4][5][6][12][13][14][15] Figure 9.…”

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].…”

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

“…There are three prevailing models of cooperativity for cytochrome P450s (CYPs): the multisubstrate binding site model [11][12][13], the peripheral effector binding site model [14][15][16], and the conformational heterogeneity model [17][18][19]. With the multi-substrate binding model, multiple substrates are bound at unproductive binding sites within the active site that can modulate the apparent K m , k cat and V max of substrate that transiently occupies a metabolically productive position.…”

“…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.…”