Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by 2-phenyl-2-(1-piperidinyl)propane.
2-Phenyl-2-(1-piperidinyl)propane (PPP), an analog of phencyclidine, was tested for its ability to inactivate cytochrome P450s (P450s) 2B1 and 2B6. PPP inactivated the 7-(benzyloxy)resorufin O-dealkylation activity of liver microsomes obtained from phenobarbital-induced rats with a K(I) of 11 microM. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified rat liver P450 2B1 and expressed human P450 2B6 was inactivated by PPP in a reconstituted system containing NADPH-cytochrome P450 reductase and lipid. In the presence of NADPH, the loss of activity was time- and concentration-dependent, and followed pseudo first order kinetics. The rate of inactivation for P450 2B1 was 0.3 min(-1), and the concentration of PPP required to achieve half-maximal inactivation was 12 microM. The time for 50% of the P450 2B1 to become inactivated at saturating concentrations of PPP was 2.5 min. P450 2B6 was inactivated with a k(inact) of 0.07 min(-1), a K(I) of 1.2 microM, and a t(1/2) of 9.5 min. The inactivated P450s 2B1 and 2B6 lost about 25 and 15%, respectively, of their ability to form a CO-reduced complex, suggesting that the loss of activity was caused by a PPP modification of the apoprotein rather than the heme. The estimated partition ratio for P450s 2B1 and 2B6 with PPP was 31 and 15, respectively. The inactivation was not reversible and reductase activity was not affected. Coincubation of P450 2B1 and 2B6 with PPP and NADPH in the presence of an alternate substrate protected both enzymes from inactivation. The exogenous nucleophile GSH did not affect the rate of inactivation. PPP-inactivated P450s 2B1 and 2B6 were recognized on Western blots by an antibody generated to phencyclidine that had been conjugated to BSA. Stoichiometries of 1.4:1 and 0.7:1 were determined for the binding of a [3H]PPP metabolite to P450 2B1 and 2B6, respectively.
Chun
J
J
Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan, USA.
Kent
U M
UM
Moss
R M
RM
Sayre
L M
LM
Hollenberg
P F
PF
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001667:Binding, Competitive; D015153:Blotting, Western; D003374:Coumarins / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000187:drug effects / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000187:drug effects / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D065702:Cytochrome P-450 CYP2B6; D019389:Cytochrome P-450 CYP2D6 / Q000187:drug effects / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000187:drug effects / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000378:metabolism; D003580:Cytochromes; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000187:drug effects* / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000494:pharmacology*; D051381:Rats; D011916:Rats, Inbred F344; D013237:Stereoisomerism; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity
C412835:2-phenyl-2-(1-piperidinyl)propane; D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D003580:Cytochromes; D004791:Enzyme Inhibitors; C056033:7-ethoxy-4-trifluoromethylcoumarin; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; C017170:Cyp1a2 protein, rat; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D019362:Cytochrome P-450 CYP2B1; D065702:Cytochrome P-450 CYP2B6; D019389:Cytochrome P-450 CYP2D6; D039181:Steroid 16-alpha-Hydroxylase; C022657:steroid hormone 6-beta-hydroxylase; D010089:Oxidoreductases, N-Demethylating; D010622:Phencyclidine