Kinetics and Thermodynamics of Ligand Binding by Cytochrome P450 3A4

Abstract

Cytochrome P450 (P450) 3A4, the major catalyst involved in human drug oxidation, displays substrate-and reaction-dependent homotropic and heterotropic cooperative behavior. Although several models have been proposed, these mainly rely on steadystate kinetics and do not provide information on the contribution of the individual steps of P450 catalytic cycle to the observed cooperativity. In this work, we focused on the kinetics of substrate binding, and the fluorescent properties of bromocriptine and ␣-naphthoflavone allowed analysis of an initial ligand-P450 3A4 interaction that does not cause a perturbation of the heme spectrum. The binding stoichiometry for bromocriptine was determined to be unity using isothermal titration calorimetry and equilibrium dialysis methods, suggesting that the ligand bound to the peripheral site during the initial encounter dissociates subsequently. A three-step substrate binding model is proposed, based on absorbance and fluorescence stopped-flow kinetic data and equilibrium binding data obtained with bromocriptine, and evaluated using kinetic modeling. The results are consistent with the substrate molecule binding at a site peripheral to the active site and subsequently moving toward the active site to bind to the heme and resulting in a low to high spin iron shift. The last step is attributed to a conformational change in the enzyme active site. The later steps of binding were shown to have rate constants comparable with the subsequent steps of the catalytic cycle. The P450 3A4 binding process is more complex than a two-state system, and the overlap of rates of some of the events with subsequent steps is proposed to underlie the observed cooperativity.Cytochrome P450 (P450) 2 enzymes are found throughout nature, from bacteria to humans. These enzymes generally catalyze mixed function oxidation reactions that have similar chemistry or else utilize parts of the general catalytic mechanism for reductions and rearrangements (2, 3). The wide diversity of substrates of these enzymes and the basis of catalytic selectivity is a topic of considerable interest in the context of both basic biochemistry and practical applications (4). P450 3A4 is one of the most widely studied of the 57 human P450s (5), mainly due to its role in the metabolism of more than one-half of the drugs on the market as well as various endogenous and exogenous molecules (6, 7). In addition, P450 3A4 is the major P450 expressed in liver (8) and in the intestine (9). Recently solved P450 3A4 crystal structures (10 -12) demonstrate the presence of a large active site, consistent with the broad range of substrates that P450 3A4 can accommodate (7, 13), including (in order of increasing size) acetaminophen (14) (M r 151), testosterone (15) (M r 288), bromocriptine (16) (M r 655), and cyclosporin (17) (M r 1201). Despite its seemingly flexible substrate selectivity, P450 3A4 displays a high degree of regio-and stereoselectivity in many substrate oxidations (18,19).One of the important features of P450 3A4 is its cooperativ...

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

“…Similar to the wild type enzyme [44,48,57], CYP3A4 FEW showed very high affinity, a substantial spin shift, and no cooperativity with this substrate (Table 1). …”

“…Work by Baas and co-authors deduced a similar conclusion from studies of the testosterone-induced spin shift in monomeric CYP3A4 incorporated into a nanoscale lipid bilayer (Nanodiscs) [42]. Recent work by Isin and Guengerich proposed a three-step binding model in which the first step does not perturb the heme spectrum and involves binding at a site peripheral to the active site [44]. This hypothesis is consistent with the observation of a peripheral progesterone binding site in a CYP3A4 X-ray crystal structure [54].…”

“…Although most analysis of P450 cooperativity has been based on steady-state kinetics of substrate oxidation, more recent attention has focused on investigating the substrate binding step in the CYP3A4 catalytic cycle [20,[40][41][42][43][44][45][46][47][48][49]. In general, cooperativity in substrate binding is revealed by a sigmoidal curve of the substrate-induced displacement of the spin equilibrium of the heme iron, which is known to be an important determinant of the catalytic efficiency and coupling of cytochromes P450 [50][51][52].…”

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.…”