title: tert-Butylphenylacetylene is a potent mechanism-based inactivator of cytochrome P450 2B4: inhibition of cytochrome P450 catalysis by steric hindrance.
abstract: We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a K(I) of 0.44 microM and k(inact) of 0.12 min(-1). The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by approximately 20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.
authors.affiliation: Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109-5632, USA.
authors.forename: Haoming
authors.initials: H
authors.lastname: Zhang
authors.forename: Hsia-lien
authors.initials: HL
authors.lastname: Lin
authors.forename: Vyvyca J
authors.initials: VJ
authors.lastname: Walker
authors.forename: Djemel
authors.initials: D
authors.lastname: Hamdane
authors.forename: Paul F
authors.initials: PF
authors.lastname: Hollenberg
mesh_terms: D000114:Acetylene / Q000031:analogs & derivatives* / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D001589:Benzphetamine / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D020134:Catalytic Domain / Q000187:drug effects; D000072467:Cytochrome P450 Family 2; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D001589:Benzphetamine; C044736:phenylacetylene; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D000114:Acetylene