CYP2E1 substrate inhibition. Mechanistic interpretation through an effector site for monocyclic compounds

Abstract

In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min(-1)) with relatively low K(m) (24 microM); nevertheless, at concentrations of >100 microM, the rate of pNP oxidation gradually decreased as a second molecule bound to CYP2E1 through an effector site (K(ss) = 260 microm), which inhibited activity at the catalytic site. 4-Methylpyrazole (4MP) was a potent inhibitor for both sites through a mixed inhibition mechanism. The K(i) for the catalytic site was 2.0 microM. Although we were unable to discriminate whether an EIS or ESI complex formed, the respective inhibition constants were far lower than K(ss). Bicyclic indazole (IND) inhibited catalysis through a single CYP2E1 site (K(i) = 0.12 microM). Similarly, 4MP and IND yielded type II binding spectra that reflected the association of either two 4MP or one IND molecule(s) to CYP2E1, respectively. Based on computational docking studies with a homology model for CYP2E1, the two sites for monocyclic molecules, pNP and 4MP, exist within a narrow channel connecting the active site to the surface of the enzyme. Because of the presence of the heme iron, one site supports catalysis, whereas the other more distal effector site binds molecules that can influence the binding orientation and egress of molecules for the catalytic site. Although IND did not bind these sites simultaneously, the presence of IND at the catalytic site blocked binding at the effector site.

FIGURE 1

Substrate and inhibitors used in this study to probe CYP2E1 binding and activity toward monocyclic and bicyclic compounds.

FIGURE 2

Steady-state oxidation of pNP by CYP2E1 in the presence of 4MP (A) or IND (B). For reactions, 25 nM CYP2E1, 100 nM CPR-K56Q, and 50 nM cytochrome b₅ were reconstituted in 50 mM potassium phosphate, pH 7.4, 20 μM DLPC, pNP (varied from 5 to 750 μM), 2 units μl⁻¹ catalase, 0.04 μg μl⁻¹ superoxide dismutase, and an NADPH-regenerating system (2 microunits μl⁻¹ glucose-6-phosphate dehydrogenase, 10 mM glucose 6-phosphate, 2 mM MgCl₂, 500 μM NADP⁺) at 37 °C. To determine initial velocities, product p-nitrocatechol was quantitated as a function of time by HPLC as described (11). The reported values reflect the average from 2 to 4 experiments, including the mean ± S.D. A, for 4MP studies, 0, 1, 5, 25, and 125 μM inhibitor was added to reactions. Data were fit to model 2a in Scheme 2. B, IND studies were carried out in the presence of 0.1, 0.3, and 1.0 μM inhibitor at a final concentration of 0.25% methanol. Final data were fit to the single-site competition mechanism (model 1) shown in Scheme 2.

FIGURE 3. Type II difference spectra for CYP2E1 binding 4MP and IND

Titrations were performed using tandem cuvettes to correct for any solvent effects on CYP2E1 absorbance and possible contributions from titrant to observed changes in absorbance. In the process, we generated difference spectra by substrating the reference CYP2E1 sample with solvent from the absorbance of the sample with titrant present. Specifically, 0.1 μM CYP2E1 in 50 mM potassium phosphate, pH 7.4, 20 μM DLPC was titrated with increasing amounts of ligand at 25 °C. The reported values reflect the average from 6 experiments, including the mean ± S.D. A, typical type II binding spectra for these heterocyclic molecules. B, 4MP titration data were fit to the two-site model in Scheme 3. C, IND titrations were performed in the presence of 0.25% methanol. Data were fit to the single-site model in Scheme 3.

FIGURE 4. Surface depiction of catalytic and effector binding sites for molecules

Overlaid ligands are pNP (yellow), 4MP (blue), and IND (green). Molecular graphics were generated by PYMOL.

SCHEME 1. Possible reaction mechanisms for CYP2E1 activity toward pNP

E = CYP2E1; S = pNP.

SCHEME 2. Possible inhibition mechanisms for CYP2E1 pNP activity by 4MP and IND

E = CYP2E1; S = pNP; I = 4MP or IND.

SCHEME 3. Possible CYP2E1 binding modes for 4MP and IND

E = CYP2E1; L = 4MP or IND.