Mechanism-Based Inactivation of Cytochrome P450 2B1 by 9-Ethynylphenanthrene

doi:10.1006/abbi.1995.9961

Archives of Biochemistry and Biophysics

Volume 323, Issue 2, 10 November 1995, Pages 295-302

https://doi.org/10.1006/abbi.1995.9961 Get rights and content

Abstract

The 7-ethoxycoumarinO-deethylase activity of rat cytochrome P450 (P450) 2B1 was inactivated by 9-ethynylphenanthrene (9EPh) in a time- and NADPH-dependent manner, and the loss of activity followed pseudo-first-order kinetics. At 20°C, the extrapolated maximal rate constant of inactivation (k_inactivation) was 0.45 min⁻¹and the inactivator concentration required for half-maximal inactivation (K_I) was 138 nM. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) and HPLC analysis demonstrated that [2′-³H]- 9EPh was irreversibly bound to the protein moiety of P450 2B1 and the stoichiometry of binding was determined to be 0.82 mol of inactivator bound per mole of P450 2B1. A radiolabeled peptide of approximately 3.0 kDa was identified by autoradiography after Tricine SDS–PAGE analysis of the peptides generated from a cyanogen bromide cleavage of [2′-³H]9EPh-inactivated P450 2B1. After HPLC separation of these peptides, the fraction containing the most radioactivity was analyzed by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS) and peaks at m/z 2720.9 and 2939.9 were detected. The lower mass peak represents the molecular ion (MH⁺) for the peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the MH⁺of the modified peptide (theoretical 2940.5). The difference in mass (ap proximately 219) would correspond to the addition of a phenanthrylacetyl group to the peptide. When the fraction containing the modified and unmodified peptides was further digested with pepsin and reanalyzed by MALDI–MS, the site of attachment could be assigned to one of the amino acids contained in the peptide Phe297 to Leu307.

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2011, Archives of Biochemistry and Biophysics
Citation Excerpt :
MALDI-MS analyses of the [3H]9EPh-labeled peptides following cleavage with CNBr indicated the addition of phenanthrylacetyl group to the peptide. Peptide mapping of the pepsin-digest demonstrated that the radiolabeled peptide 294–307 was the site of covalent modification [30,31]. The functional importance of Thr302 in the inactivation of CYP2B4 by 2EN was confirmed when the T302A variant was shown to exhibit a significantly slower rate of inactivation by 2EN compared to CYP2B4 WT [32].
Cytochromes P450 (CYPs or P450s) contain a highly conserved threonine residue in the active site, which is referred to as Thr302 in the amino acid sequence of CYP2B4. Extensive biochemical and crystallographic studies have established that this Thr302 plays a critical role in activating molecular oxygen to generate Compound I, a putative iron(IV)-oxo porphyrin cation radical, that carries out the preliminary oxygenation of CYP substrates. Because of its proximity to the center of the P450 active site, this Thr302 is susceptible to mechanism-based inactivation under certain conditions. In this article, we review recent studies on the mechanism-based inactivation of three mammalian P450s in the 2B family, CYP2B1 (rat), 2B4 (rabbit) and 2B6 (human) by tert-butylphenylacetylene (tBPA). These studies showed that tBPA is a potent mechanism-based inactivator of CYP2B1, 2B4 and 2B6 with high k_inact/K_I ratios (0.23–2.3 min⁻¹ μM⁻¹) and low partition ratios (0–5). Furthermore, mechanistic studies revealed that tBPA inactivates these three CYP2B enzymes through the formation of a single ester adduct with the Thr302 in the active site. These inhibitory properties of tBPA allowed the preparation of a modified CYP2B4 where the Thr302 was covalently and stoichiometrically labeled by a reactive intermediate of tBPA in quantities large enough to permit spectroscopic and crystallographic studies of the consequences of covalent modification of Thr302. Molecular modeling studies revealed a unique binding mode of tBPA in the active site that may shed light on the potency of this inhibition. The results from these studies may serve as a basis for designing more specific and potent inhibitors for P450s by targeting this highly conserved threonine residue which is present in the active sites of most mammalian P450s.
Inhibition of CYP2B4 by the mechanism-based inhibitor 2-ethynylnaphthalene: Inhibitory potential of 2EN is dependent on the size of the substrate
2007, Archives of Biochemistry and Biophysics
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As an initial experiment, the effect of 2EN on 7-pentoxyresorufin-O-dealkylation (PROD) was examined (Fig. 1a). Preincubation with 2EN for 10 min led to an 85% decrease in PROD activity, a response typically observed with mechanism-based inhibitors [2,7,8,25]. However, a significant amount of inhibition of PROD was also observed without 2EN preincubation, which is consistent with a simple reversible interaction of 2EN with the enzyme.
2-Ethynylnaphthalene (2EN) is a mechanism-based inhibitor of CYP2B4 with two components to the inhibition, (1) enzyme inactivation, which requires covalent binding of the 2EN metabolite, and (2) reversible inhibition by 2EN itself. Both inhibitory components were examined using several different CYP2B4 substrates. Preincubation of CYP2B4 with 2EN led to a time-dependent inactivation of each of the CYP2B4-dependent activities examined; however, the ability of 2EN to reversibly inhibit CYP2B4 depended on the substrate employed, which is inconsistent with classical inhibition patterns. The degree 2EN’s reversible inhibition was shown not to correlate with the substrate affinity for the active site, but with parameters related to the molecular size of the substrate. The results are consistent with 2EN and the smaller substrates simultaneously fitting in the CYP2B4 active site, leading to very little inhibition. Larger substrates exhibited greater degrees of inhibition because of their inability to co-bind with inhibitor in the active site.
The mechanism-based inactivation of P450 2B4 by tert-butyl 1-methyl-2-propynyl ether: Structural determination of the adducts to the P450 heme
2004, Archives of Biochemistry and Biophysics
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Adduct C gives rise to signals with a slight downfield shift compared to adduct B. For example, the tert-butyl group has a chemical shift of −1.17 ppm compared to −0.91 ppm for adduct B (Fig. 4F). A number of acetylenic compounds have been identified as mechanism-based inactivators of various P450 2B enzymes [7,13,15,16,31–33]. The mechanism, as well as the rate of inactivation, differs significantly among the P450s and between different inactivators.
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Mechanistic studies of cytochrome P450 2B1 inactivation by xanthates
2000, Archives of Biochemistry and Biophysics
Xanthates have previously been shown to inactivate the phenobarbital-inducible rat cytochrome P450 2B1 as well as its human homologue P450 2B6. The inactivation was mechanism-based and the loss in enzymatic activity was due to covalent binding of a reactive xanthate intermediate to the P450 2B1 apoprotein. In this report, we investigated various mechanistic events to elucidate the individual step(s) in the P450 catalytic cycle that are compromised due to the inactivation by xanthates. Different xanthates displayed typical type I binding spectra and the spectral binding constants were in the low-millimolar range. A dramatic loss in 7-ethoxy-4-(trifluoromethyl)coumarin activity was observed when P450 2B1 was incubated with five different xanthates in the presence of NADPH. With the exception of the C14 xanthate, virtually no loss of absorbance at 418 or 450 nm in the reduced-CO complex was observed. Long-chain xanthates were able to affect the rate of the first electron transfer in the P450 catalytic cycle by stabilizing the heme in its low-spin state. n-Octyl xanthate (C8) metabolism led to very little observable oxy–ferro intermediate complex formation. The alternate oxidant tert-butyl hydroperoxide was able to support the inactivation reaction of C8 in the absence of reductase or NADPH. The rates of reduction of native, C8-exposed, and C8-inactivated P450 2B1 were measured. The C8-inactivated P450 had a 62% lower rate of reduction in the absence or presence of benzphetamine compared to the native enzyme. Product formation of the three enzyme preparations was quantified with benzphetamine as the substrate. The C8-inactivated P450 2B1 exhibited a much lower rate of NADPH consumption and formation of formaldehyde. However, the ratio of H₂O₂ to formaldehyde production increased from 1:1 for the native enzyme to 2.8:1 for the inactivated P450. Together these observations indicate that the covalent modification of P450 2B1 by a reactive intermediate of xanthates reduces the rate of the first electron transfer by the reductase and also leads to uncoupling of electron transfer from product formation by diverting a greater proportion of the electrons to H₂O₂ formation.
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REGULAR ARTICLEMechanism-Based Inactivation of Cytochrome P450 2B1 by 9-Ethynylphenanthrene

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REGULAR ARTICLE
Mechanism-Based Inactivation of Cytochrome P450 2B1 by 9-Ethynylphenanthrene