Search

Treatment of transformed human skin fibroblasts stably expressing alcohol-inducible cytochrome P450 2E1 (CYP2E1) with the hsp90 inhibitor radicicol results in CYP2EI degradation that is inhibited by the proteasome inhibitor lactacystin. The way substrates regulate the interaction of CYP2E1 with the chaperones hsp90 and hsp70 profoundly alter enzyme turnover is shown.

The tert-Butyl acetylene (tBA) is a mechanism-based inactivator of cytochromes P450 2El and 2El T303A but the inactivation of the T303A mutant could be reversed by overnight dialysis. Insights into the reversible inactivation of the T303A mutant of P450 2El are presented.

Results show that tert-butyl isothiocyanate inactivates cytochrome P450 2E1 by binding to a critical amino acid residue(s) at the active site. Data point out that the modified residue binds heme, which interferes with the binding of oxygen, carbon monoxide and substrate.

The mechanism by which cytochrome P450 catalyzes hydroxylation reactions was studied through the oxidation of trans-2-phenyl-1-alkylcyclopropanes (alkyl = methyl, ethyl, 1-propyl, 1-methylethyl) and trans-2-(4-(trifluoromethyl)phenyl)-1-alkylcyclopropanes (alkyl = methyl, ethyl). Results were not in agreement with oxidation of a radical component in the transition state of an insertion reaction to form a cation. Findings also did not agree with the formation of cationic rearrangement products through solvolysis of first-formed protonated alcohol products formed by the insertion of the OH+ moiety from iron-complexed hydrogen peroxide.

Research was conducted to examine the interactions of lidocaine with related P450, 2B1 and 2B2 cytochromes using chimeric enzymes and single-point mutants from the two proteins. The mutated derivatives were expressed in Escherichia coli and purified using media supplements and purification procedures. Results showed two possible binding orientations for lidocaine within the active sites of 2B1 and 2B2. They also indicated that the inclusion of b5 in the initial reconstitution mixture differentially affected lidocaine oxidation.

A study was conducted to characterize the syntheses of racemic and enantiomerically enriched trans-1-methyl-2-(4-(trifluoromethyl)phenyl)cyclopropane and the oxidation products resulting from enzyme-catalyzed hydroxylation. Mechanisms associated with cytochrome P450 enzymes were also analyzed in the study. Results indicated that the formation of a radical during the oxygen rebound mechanism for cytochrome P450 catalyzed hydroxylation.

The impact of replacing Gly 478 with Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was studied. Results showed that this change eliminated the formation of the inactivating species of N-benzyl-1-aminobenzotriazole (BBT). An unusual observation was that the oxidation of BBT caused the formation of a dimer by both the wild-type and the mutant enzyme. These findings indicated that two molecules of BBT may be accommodated by P450 2B1.

The reactions of methylcubane with the tert-butoxyl radical, with cytochrome P-450 enzymes, and with a methane monooxygenase system are investigated. The major synthetic reactions are metalation and subsequent iodination of the 2-position of (diisopropylcarbamoyl)cubane to effect the initial functionalization, lithium-for-iodine exchange and methylation followed by reduction to produce 2-methyl-1-((diisopropylamino)methyl)-cubane, and dimethyldioxirane oxidation of this amine to produce 2-methylcubanecarboxylic acid.

The cytochrome P-450 catalyzed hydroxylation of a hypersensitive radical probe, (trans, trans-2-tert-butoxy-3-phenylcyclopropyl)methane, leads to unrearranged and rearranged products. The probe differentiates between the radical and cationic intermediates. The short-lived apparent radical intermediate is actually a reacting ensemble. Rearrangement of a cationic intermediate produces the major rearranged products. The lack of synchrony between the insertion reaction and the C-H bond scission is responsible for the small concentration of radically rearranged products.

Three different products are obtained by the oxidization of the ultrafast radical clock probe trans-1-methyl-2-phenylcyclopropane (1CH3) and to mono-di- and trideuteriomethyl analogues by phenobarbital-stimulated rat liver microsomal enzymes. The phenol trans-2-(p-hydroxyphenyl)-1-methylcyclopropane, the alcohol trans-(2-phenylcyclopropyl)methanol and the rearranged alcohol 1-phenylbut-3-en-1-ol are the three products. The analogous deuterated products are obtained in different amounts by further hydroxylation of the three deuteriomethy forms of the 1CH3.

3H-labeled, 2-ethynylnaphthalene-inactivated P450 2B1 and 2B4 each have a radiolabeled peptide detected by mass spectrometry and amino acid sequence analysis. Amino acid sequence studies revealed the N-terminal region in the peptide. Mass spectrometry indicate two mass peaks corresponding to the peptide. The mass difference of the peaks corresponds to the combination of peptide to the 2-naphthylacetyl group.

The first application of resonance Raman spectroscopy to study the inactivation of a truncated form of mammalian CYP2B4 by the acetylenic inhibitor 4-(tert-butyl)phenylacetylene is reported. The studies provide better insight into the structural basis for the altered functional properties of the mammalian cytochrome P450 2B4 enzyme and better understand MB inhibition.

Iron(IV)-oxo porphyrin radical cations (Compound I) of the mammalian hepatic P450 enzyme CYP2B4 and three mutants are prepared by laser flash photolysis of the Compound II species that, in turn, are prepared by reaction of the resting enzymes with peroxynitrite. The studies have shown that substrate binding constants for P450 Compound I are important for controlling overall rates of oxidation reactions and the intrinsic reactivities of Compounds I from various P450 enzymes are comparable.

The oxidation reactions of (S,S)-2-(p-trifluoromethylphenyl) cyclopropylmethane by the cytochrome P450 compounds I are analyzed to determine the various kinetic isotope effects. The analysis also demonstrates the necessity of a second electrophilic oxidant to obtain an effective oxidation reaction under the turnover conditions.

Oxidation reactions of norcarane were studied by seven iron-containing enzymes with careful attention to the identities of minor products. From the authentic samples of various oxidation products from 2-norcarene and 3-norcarene, it was found that the radical- and cation-derived rearrangement products from norcarane coelute with the norcarane oxidation products on low-polarity GC columns.

The product profiles from the oxidations of 2-norcarene and 3-norcarene were determined by using several enzymes. The findings from the matched oxidations suggest that 3-norcarene is a better substrate than 2-norcarene in terms of turnover.

The inactivations of P450 2B4 and the T302A mutant of 2B4 by tert-butyl acetylene (tBA) and the inactivation of 2B4 T302A by tert-butyl 1-methyl-2-propynyl ether (tBMP) are investigated. The results indicate that while T302 may play a role in proton delivery in the formation of the oxenoid-iron complex and in the stabilization of acetylene heme adducts in 2B4, it is not essential for proton delivery given the presence of E301 in the binding site.

The first experimental evidence is presented to demonstrate that the NADPH/O(sub 2-) and PhIO-supported P450 N-dealkylations are mechanistically distinct and thus, the P450/PhIO system may not be a good mechanistic model for P450/NADPH/O(sub 2-)-catalyzed n-dealkylations. The substrates provide a unique opportunity to further exploit the relative chemistries of these systems, which would be valuable in understanding the nature of their oxygen species.

Intramolecular and intermolecular kinetic isotopes effects (KIEs) were determined for hydroxylation of the enantiomers of trans-2(p-trifluoromethylphenyl)cyclopropylmethane by hepatic cytochrome P450 enzymes. The intra and intermolecular KIE results were combined to determine the relative rate constants for the unmasking and hydroxylation reactions, and a qualitative correlation was found for the unmasking reaction and release of hydrogen peroxide from four of the P450 enzymes in the absence of substrate.

Norcarane was employed as a mechanistic probe in oxidations catalyzed by hepatic cytochome P450 enzymes and by the soluble methane monooxygenase enzyme from methylococcus capsulatus. Generalizations regarding the intermediacy of radicals in P450 and soluble methane monooxygenase (sMMO) enzyme-catalyzed hydroxylations based on the norcarane results should be considered hypothetical until the origin of the unanticipated results can be determined.