Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics

Abstract

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure-activity study from another laboratory using various effector-substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/ D214E double mutant displayed an increased rate of testosterone and progesterone 6␤-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by ␣-naphthof lavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6␤-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/ D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.Human cytochrome P450 3A4 (P450 3A4) is one of the most abundant xenobiotic-metabolizing enzymes in the human liver (1) and intestine (2, 3) and is capable of oxidizing a wide range of structurally diverse drugs, including antineoplastic, antihistaminic, cardiac, psychotropic, analgesic, hormonal, and immunosuppresant agents (4). In addition to its broad substrate specificity and abundance, another important aspect of the metabolic activity of P450 3A4 is its apparent allosteric nature. P450 3A4 demonstrates homotropic cooperativity toward a number of substrates, including progesterone (5-7), testosterone (8), 17␤-estradiol (8), aflatoxin B 1 (8), and amitriptyline (8). In addition, the activity of the enzyme can be influenced heterotropically. For example, ␣-naphthoflavone (ANF) stimulates the oxidation of progesterone (5-7), testosterone (7), estradiol (9), aflatoxins (10, 11), polycyclic aromatic hydrocarbons (12, 13), carbamazepine (14), and acetaminophen (15). Similarly, progesterone can heterotropically stimulate carbamazepine metabolism by P450 3A4 (14), and both progesterone and testosterone stimulate estradiol metabolism (9).The most extensively studied allosteric protein is probably hemoglobin. The molecular mechanisms of both the homotropic action of oxygen binding and the heterotropic control exerted by binding of CO 2 were initially determined by a combination of x-ra...

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

“…We expected this to be a result of the blockage of the effector site, as proposed initially [12,55]. To probe the stoichiometry of the binding we performed a Job's titration of CYP3A4 FEW with 1-PB.…”

“…This construct was derived from our initial observation of abolished homotropic cooperativity of testosterone and progesterone 6β-hydroxylation and of testosterone binding in L211F/D214E [12]. Interestingly, however, whereas heterotropic activation of testosterone hydroxylation by ANF was lost, progesterone hydroxylation by L211F/D214E was still simulated by ANF.…”

“…The enzyme was expressed as the His-tagged protein in Escherichia coli TOPP3, and the first purification was carried out using Ni-NTA resin (QIAGEN) under conditions described previously [12,55,56]. The protein was stored at −80 °C in 100 mM N-2-hydroxyethylpiperazine-N′-[2-ethanesulfonic acid] (HEPES) buffer (pH 7.4), containing 10% glycerol (v/v), 1 mM, dithiothreitol (DTT), and 1 mM ethylenediaminetetraacetic acid (EDTA) (buffer A).…”

“…Direct physical evidence for the presence of two ketoconazole molecules in the CYP3A4 active site was provided very recently by x-ray crystallography [11]. Inherent in most of the models is the assumption that a loose fit of a single substrate molecule requires the binding of a second ligand for efficient binding and/or catalysis [9,10,12], although recent evidence for effector-induced conformational rearrangements has emerged from our laboratory and others [13][14][15]. Strong support for an important conformational transition in P450 function is provided by several recent x-ray crystal structures of mammalian P450 enzymes including CYP3A4, which have revealed considerable flexibility [11,[16][17][18].…”

“…One means of elucidating mechanisms of cooperativity is through the use of site-directed mutants [12,55,56]. In the present study, we applied our recent spectroscopic approaches to the CYP3A4 mutant L211F/D214E/F304W (FEW), which displays no homotropic cooperativity of progesterone hydroxylation and abolished heterotropic activation by ANF [55].…”