Allosteric activation of cytochrome P450 3A4 by α-naphthoflavone: branch point regulation revealed by isotope dilution analysis

Abstract

Cytochrome P450 3A4 (CYP3A4) is the dominant xenobiotic metabolizing CYP. Despite great interest in CYP enzymology, two in vitro aspects of CYP3A4 catalysis are still not well understood, namely, sequential metabolism and allosteric activation. We have therefore investigated such a system in which both phenomena are present. Here we report that the sequential metabolism of Nile Red (NR) is accelerated by the heterotropic allosteric effector α-naphthoflavone (ANF). ANF increases the rates of formation for NR metabolites M1 and M2 and also perturbs the metabolite ratio in favor of M2. Thus, ANF has as an allosteric effect on a kinetic branch point. Co-incubating deuterium-labeled NR and unlabeled M1, we show that ANF increases k(cat)/k(off) ~1.8-fold in favor of the k(cat) of M2 production. Steady-state metabolic experiments are analyzed using a kinetic model in which the enzyme and substrates are not in rapid equilibrium, and this distinction allows for the estimation of rates of catalysis for the formation of both the primary (M1) and secondary (M2) products, as well as the partitioning of enzyme between these states. These results are compared with those of earlier spectroscopic investigations of NR and ANF cooperativity, and a mechanism of ANF heteroactivation is presented that involves effects on substrate off rate and coupling efficiency.

Figure 1

Incubations of NR (a) and M1 (b) were quenched at successive time points. M2 formation from NR (triangles) shows signs of a lag in formation rate, indicative of the sequential steps required for M2 production from NR. When incubating M1 with CYP3A4 no lag in M2 formation is evident (squares). When 12 μM ANF is included in the NR incubation (circles, dotted line) the lag is seen to decrease as judged by the R² value and the amount of M2 produced is increased more than two-fold at six minutes.

Figure 2

Plots of spin-state perturbation vs. ligand concentration of NR and metabolites all yield sigmoidal binding titrations. Recovered S₅₀’s from fits to the Hill equation are 7.3, 68, and 58 μM for NR, M1, and M2 respectively.

Figure 3

Steady-state metabolism of Nile Red. The first panel shows total metabolite production (M1 + M2) for 0 μM ANF (black) and 12 μM ANF (green). The fit is to the M-M equation and illustrates that the ANF effect is on V_Max while K_M is not significantly different. The K_M values were used to parameterize the global fits. The second and third panels show M1 (pink), M2 (orange), and total metabolite (black or green) formation rates are plotted vs. NR concentration. The three data sets in each panel are fitted globally to the Steady-State velocity equations as detailed in Materials and Methods. The ANF stimulatory effect on M2 production is larger than the effect on M1, indicating the possible role of branch point modulation. See Table 2 for extracted rate constants.

Figure 4

The branching ratio (left) and inverse branching ratio (right; for ease of fitting, the lowest ratio was by set at zero) of NR sequential metabolism as determined by Isotope Dilution are plotted. ANF has the effect of increasing the sequential vs. dissociative M2 production. The inverse branching ratio (right) is fit to a sequential-binding substrate inhibition equation to give two affinity constants.

Figure 5

Simulated NR metabolism. M1 formation is represented in pink, M2 formation in orange, and total metabolite in black. The kinetic constants (min⁻¹) for the simulation on the left are in reference to Scheme 2 and are k₁=60 k₋₁=60 k₂=25 k₃=60 k₋₃=60 k₄=25 k₅=60 and k₋₅=60. Bimolecular rate constants have units of μM⁻¹min⁻¹. Values for the graph on the right are k₁=60 k₋₁=60 k₂=25 k₃=20 k₋₃=20 k₄=25 k₅=60 and k₋₅=60. The values that change are in bold for emphasis. Importantly, this simulation shows that the M1/M2 metabolite ratio can change by modulating only the off-rate of M1, and even if the M1 K_D does not change. In this scenario, however, the total metabolite production does not increase, and so we conclude that ANF must be modulating catalysis rates in addition to M1 dissociation rates.

Scheme 1

NR and ANF metabolites. NR is sequentially N-deethylated to its mono-ethyl and di-desethyl metabolites. ANF is metabolized to the 5,6-oxide.

Scheme 2

(a) Below is an illustration of the general case of sequential metabolism of substrate A to two products, primary metabolite B and secondary metabolite C. For the specific case of NR metabolism, NR = A, M1 = B, and M2 = C. The scheme assumes irreversible chemical steps and reversible binding. Equations for global analysis of NR metabolism were derived using the steady-state approximation according to the King-Altman method (26). Because of the sequential nature of NR metabolism, the concentration of the [EB] species is not in rapid equilibrium; it thus depends on the catalysis rates k₂ and k₄, in addition to the dissociation rate constant for B, K_d,B = (k₋₃/k₃). (b) Estimated rate constants for NR sequential metabolism with and without ANF. Catalytic rate constants are not microscopic rate constants because they include each of the rates in the P450 cycle, and so represent a flux through a pathway with multiple branching points rather than a rate constant.