Mechanism-based inactivation of cytochromes P450 2E1 and 2B1 by 5-phenyl-1-pentyne.
A series of acetylenic compounds whose structures were based on "P450 2E1-like" substrates was investigated for their ability to cause inactivation of P450 2E1-dependent p-nitrophenol hydroxylation. The most effective compound with liver microsomes from pyridine-treated rats or with rabbit P450 2E1 in a reconstituted system was 5-phenyl-1-pentyne. The inactivation of purified 2B1, 2E1, a truncated 2E1 lacking amino acids 3-29, 2E1(Delta3-29), or a truncated 2E1 in which threonine 303 was replaced with alanine, 2E1(Delta3-29, T303A), in a reconstituted system by 5-phenyl-1-pentyne was NADPH- and time-dependent and followed pseudo-first-order kinetics. The maximal rate constants for inactivation, the concentrations that gave half-maximal inactivation (KI), and the partition ratios (the number of 5-phenylvaleric acid molecules formed/inactivation event) were determined with each P450. The KI values for 2B1 and 2E1(Delta3-29, T303A) were twice those for 2E1 and 2E1(Delta3-29), and the partition ratios for 2B1 and 2E1(Delta3-29, T303A) were 5-10 times greater than those of 2E1 or 2E1(Delta3-29). During the incubation of P450 2E1 with 5-phenyl-1-pentyne, the loss of P450 as determined by the reduced-CO difference spectra was equal to the loss of catalytic activity. The formation of a heme adduct was demonstrated by HPLC analysis of reaction mixtures containing 5-[3H]phenyl-1-pentyne. HPLC analysis with diode-array detection showed that the Soret region of the proposed heme adduct was different from that of the unmodified heme. The HPLC peak containing the proposed heme adduct was further analyzed by matrix-assisted laser desorption ionization mass spectrometry, and the resulting peaks could result from the addition of a 2-oxo-5-phenylpentyl group to the heme.
Roberts
E S
ES
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, 48109-0632, USA.
Alworth
W L
WL
Hollenberg
P F
PF
D000480:Alkynes / Q000494:pharmacology; D000818:Animals; D001555:Benzene Derivatives / Q000494:pharmacology; D019362:Cytochrome P-450 CYP2B1 / Q000037:antagonists & inhibitors*; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000201:enzymology; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley
D000480:Alkynes; D001555:Benzene Derivatives; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004791:Enzyme Inhibitors; C112922:5-phenyl-1-pentyne; D019362:Cytochrome P-450 CYP2B1