Secobarbital-mediated Inactivation of Cytochrome P450 2B1 and Its Active Site Mutants

Abstract

Secobarbital (SB) is a relatively selective mechanismbased inactivator of cytochrome P450 2B1, that partitions between epoxidation and heme and protein modification during its enzyme inactivation. The SB-2B1 heme adduct formed in situ in a functionally reconstituted system has been spectrally documented and structurally characterized as N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX. The SBprotein modification has been localized to 2B1 peptide 277-323 corresponding to the active site helix I of cytochrome P450 101. The targeting of heme and this active site peptide suggests that the 2B1 active site topology could influence the course of its inactivation. To explore this possibility, the individual SB epoxidation, heme and protein modification, and corresponding molar partition ratios of the wild type and seven structural 2B1 mutants, site-directed at specific substrate recognition sites, and known to influence 2B1 catalysis were examined after Escherichia coli expression. These studies reveal that Thr-302 is critical for SB-mediated heme Nalkylation, whereas Val-367 is a critical determinant of 2B1 protein modification, and Val-363 is important for SB epoxidation. SB docking into a refined 2B1 homology model coupled with molecular dynamics analyses provide a logical rationale for these findings.

Inhibition of Cytochrome P450 Enzymes

“…A variety of compounds containing an olefinic bond, such as ethylene, allylisopropylacetamide (AIA), and secobarbital, can form covalent adducts on the nitrogen of the porphyrin group of the prosthetic heme leading to inactivation [382][383][384][385] Secobarbital has been shown to completely inactivate CYP2B1 with only partial loss of the heme chromophore [384,386,387] Isolation of the modified CYP2B1 protein and the N-alkylated porphyrins indicates that the reactive compound partitions between protein modification, N-alkylation of the heme, and formation of an epoxide metabolite in the ratio of 02:08:59, respectively [387] (Fig 517) The formation of a heme adduct in the active site of CYP2B1 was confirmed spectrally based on its typical absorption maximum at ~ 445 nm, a characteristic fea- ture of iron complexed N-modified porphyrins [388] The modified CYP2B1 peptide has been isolated and shown to span residues 277-323 By sequence analogy, these residues correspond to the distal I helix in P450 cam [386,[389][390][391][392] Further digestion of the modified peptide has resulted in identification of the site for modification by secobarbital to a residue in the peptide G299-S304 [387] Although the identity of the adducted residue has not yet been determined, these results are consistent with modification of the CYP2B1 in the active site Specific mutations of CYP2B1 in the putative substrate-recognition site (SRS) 2,4,5, and 6, but not in SRS-1, cause a decrease in the inactivation by secobarbital Mutation of residue 367 from V to A in SRS-5 had a marked inhibitory effect on protein modification [387] Isolation of the N-modified porphyrins as the parent adducts as well as the corresponding dimethylesters and analysis by LC-MS demonstrated the formation of adducts of hydroxysecobarbital with protoporphyrin IX (Fig 517) [386] Like terminal olefins, terminal acetylenes can alkylate the P450 prosthetic heme However, compounds such as 10-undecynoic acid, 1-ethynylpyrene, 2-ethynylnaphthalene, 9-ethynylnaphthalene, 17β-ethynylprogesterone, and 17α-ethynylestradiol (EE) inactivate P450s primarily by covalently binding to the apoprotein with little or no effect on the heme group (Fig 518) [305, 310, 317, 318] Almost stoichiometric binding of 10-undecynoic acid to rat liver CYP4A1 (the ω-hydroxylase) as well as of 2-ethynylnaphthalene and 1-ethynylpyrene to CYP1A1 and -1A2, and of EE to CYP3A4 has been observed [305]…”

Covalent Modification of Thr302 in Cytochrome P450 2B1 by the Mechanism-Based Inactivator 4-tert-Butylphenylacetylene

“…Some examples include: 1) studies have demonstrated that Thr302 in CYP2B1 and Thr302 in CYP2B4 are critical determinants for secobarbital and 2EN inactivation, respectively (He et al, 1996;Roberts et al, 1996) and 2) a decrease in the inactivation of 2E1 by 5-phenyl-1-pentyne and several naturally occurring isothiocynates and the reversible formation of a novel heme adduct of tert-butylacetylene were observed in the Thr303A mutant of 2E1 (Roberts et al, 1998;Moreno et al, 2001;Blobaum et al, 2004).…”

Cation Modulation of Hemoglobin Interaction with Sodium n-Dodecyl Sulfate (SDS). II: Calcium Modulation at pH 5.0

“…Conversely, the 0.20 lM Ca 2? , causing less ionization of the indole moiety, resulted in heme adducts formation as suggested by the 448 nm peak [64,65].…”