Mechanism-based inactivation of CYP2B1 and its F-helix mutant by two tert-butyl acetylenic compounds: covalent modification of prosthetic heme versus apoprotein

Abstract

The mechanism-based inactivation of cytochrome CYP2B1 [wild type (WT)] and its Thr205 to Ala mutant (T205A) by tert-butylphenylacetylene (BPA) and tert-butyl 1-methyl-2-propynyl ether (BMP) in the reconstituted system was investigated. The inactivation of WT by BPA exhibited a k(inact)/K(I) value of 1343 min(-1)mM(-1) and a partition ratio of 1. The inactivation of WT by BMP exhibited a k(inact)/K(I) value of 33 min(-1)mM(-1) and a partition ratio of 10. Liquid chromatography/tandem mass spectrometry analysis (LC/MS/MS) of the WT revealed 1) inactivation by BPA resulted in the formation of a protein adduct with a mass increase equivalent to the mass of BPA plus one oxygen atom, and 2) inactivation by BMP resulted in the formation of multiple heme adducts that all exhibited a mass increase equivalent to BMP plus one oxygen atom. LC/MS/MS analysis indicated the formation of glutathione (GSH) conjugates by the reaction of GSH with the ethynyl moiety of BMP or BPA with the oxygen being added to the internal or terminal carbon. For the inactivation of T205A by BPA and BMP, the k(inact)/K(I) values were suppressed by 100- and 4-fold, respectively, and the partition ratios were increased 9- and 3.5-fold, respectively. Only one major heme adduct was detected following the inactivation of the T205A by BMP. These results show that the Thr205 in the F-helix plays an important role in the efficiency of the mechanism-based inactivation of CYP2B1 by BPA and BMP. Homology modeling and substrate docking studies were presented to facilitate the interpretation of the experimental results.

Fig. 1.

Structures of the two tert-butyl acetylenic compounds: BMP and BPA.

Fig. 2.

Time- and concentration-dependent loss of EFC de-ethylation activity for the WT and T205A mutant of CYP2B1 during inactivation by BPA. The reconstituted system containing the WT was incubated with 0.31 μM (●), 0.62 μM (○), 1.25 μM (▾), and 2.5 μM (▿) BPA, and the T205A mutant was incubated with 3.1 μM (■), 6.2 μM (□), 12.5 μM (▴), and 25 μM (▵) BPA. Aliquots were removed at the times indicated and assayed for residual EFC de-ethylation activity as described under Materials and Methods. The insets show the double reciprocal plots of the kinetic constants obtained from the initial plots. The K_I and k_inact values were determined from the double reciprocal plots. The data represent the average of three separate experiments done in duplicate that did not differ by >10%.

Fig. 3.

Determination of partition ratios for the inactivation of the WT and T205A mutant P450s by BPA (A) and BMP (B). The percentage of catalytic activity remaining was determined as a function of the molar ratio of BPA or BMP to the P450s as described under Materials and Methods. The partition ratios were estimated from the intercept of the linear regression line from the lower ratios of each inactivator to the P450s and the straight line obtained from higher ratios of each inactivator to the P450s.

Fig. 4.

HPLC elution profiles for the reconstituted system after incubating the WT (A) or T205A mutant (B) 2B1 with BPA or BMP as described under Materials and Methods. WT/control and T205A/control are the reaction mixtures incubated without NADPH. WT/BMP, WT/BPA, T205A/BMP, and T205A/BPA are the reaction mixtures incubated with inactivator and NADPH. Native heme, peak 1, and peak 2 eluted at 20, 24, and 26 min, respectively.

Fig. 5.

Analysis of CYP2B1 heme adducts after incubating the reaction mixture with BMP and NADPH. After inactivation of WT by BMP, as described under Materials and Methods, the reaction mixture was injected directly onto a reverse-phase HPLC column, and the effluent was analyzed using a photodiode-array detector and a Thermo Fisher Scientific LTQ linear ion trap mass spectrometer as described under Materials and Methods. A, HPLC elution profile for peaks a, b, c, d, e, and f monitored at 405 nm. B, the extracted ion chromatograms for each of the peaks observed in the HPLC elution profile. C, the full mass spectra for peak a having m/z 616.2, peaks b, c, and f having m/z values 705.5, and peaks d and e having m/z 758.5.

Fig. 6.

ESI/LC/MS analysis of CYP2B1 apoprotein inactivated by BPA or BMP. The incubation conditions, HPLC, and MS analysis conditions were as described under Materials and Methods. A, representative deconvoluted mass spectrum from a control incubation of CYP2B1 with either BPA or BMP in the absence of NADPH. B, representative deconvoluted mass spectrum of P450 2B1 incubated with BPA in the presence of NADPH. C, representative deconvoluted mass spectrum of CYP2B1 incubated with BMP in the presence of NADPH.

Fig. 7.

LC/MS/MS analysis of a GSH conjugate of BPA formed by WT CYP2B1. A reaction mixture containing CYP2B1 was incubated with BPA in the presence of NADPH; the reactive intermediate was trapped with GSH; and the GSH conjugate was analyzed as described under Materials and Methods. A, extracted ion chromatogram of a GSH conjugate eluting at 25.8 min with the MH⁺ ion at m/z 482. B, MS/MS spectrum of the GSH conjugate eluting at 25.8 min. C, proposed structure of the GSH conjugate. The dashed lines indicate the sites of fragmentation and are explained in the text. The MS/MS spectra were obtained in the positive mode and analyzed using the Xcalibur software package (Thermo Fisher Scientific).

Fig. 8.

LC/MS/MS analysis of GSH conjugates of BMP formed by WT CYP2B1. A reaction mixture containing CYP2B1 was incubated with BMP in the presence of NADPH, and the reactive intermediates were trapped with GSH. The GSH conjugates were analyzed as described under Materials and Methods. A, extracted ion chromatogram of the GSH conjugates with the MH⁺ ion at m/z 450 eluting at 11.9 and 13.5 min. B, MS/MS spectrum of the GSH conjugate eluting at 11.9 min. C, MS/MS spectrum of the GSH conjugate eluting at 13.5 min. D, proposed structure of the GSH conjugates. The dashed lines indicate the sites of fragmentation. The MS/MS spectra were obtained in the positive mode and analyzed using the Xcalibur software package (Thermo Fisher Scientific).

Fig. 9.

Homology modeling and docking studies of BPA (A) and BMP (B) into the CYP2B1 active site. Leu201, Thr205, and Phe206 are F-helix residues. Phe297, Glu301, and Thr302 are I-helix residues. Two hydrogen bonds are indicated by blue dashed lines: 1) between Thr205 and Leu201; 2) between Thr205 and Asn237. Residues within 3.5 Å of BPA are Ile101, Ile104, Ile114, Phe115, Phe297, Thr302, Val367, and Ile477. Residues within 3.5 Å of BMP are Arg98, Ile114, Glu301, Val363, and Val367. Shown in color are BPA and BMP (cyano), F-helix (orange), I-helix (green), heme (red), and the axial Cys436 (yellow).