Abstract
The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (+/-0.13) microM and Vmax of 6.34 nmol/nmol P450/min (36-fold variation) in microsomal preparations from a panel of 12 human livers. Substrate depletion was avoided during the kinetic determinations. 8-Methoxypsoralen (8-MOP) is a potent mechanism-based inactivator of human liver P450 2A6 and reconstituted purified recombinant P450 2A6 based on the following evidence: 1) 8-MOP causes time, concentration, and NADPH-dependent loss of P450 2A6 activity that is not reversed by potassium ferricyanide or extensive dialysis, 2) loss of P450 2A6 activity is associated with a loss of spectrally observable P450, 3) addition of nucleophiles or reactive oxygen scavengers to the incubations does not prevent inactivation of P450 2A6, and 4) 8-MOP-dependent P450 2A6 inactivation is inhibited (concentration dependent) by the addition of a competitive inhibitor (pilocarpine). Inactivation is selective for P450 2A6 at low concentrations of 8-MOP (2.5 microM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 microM in a reconstituted and microsomal system, respectively, and a kinact of 1 min-1 and 2 min-1 in a reconstituted and microsomal system, respectively. A substrate depletion partition ratio of 21 was calculated for the inactivation of recombinant P450 2A6. Potency and selectivity suggest that 8-MOP could be a useful tool in vitro for evaluating P450 2A6 activity in various enzyme preparations.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adolescent
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Adult
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Aryl Hydrocarbon Hydroxylases*
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Child
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Coumarins / metabolism
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Cytochrome P-450 CYP1A2
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Cytochrome P-450 CYP1A2 Inhibitors
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Cytochrome P-450 CYP2A6
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Cytochrome P-450 CYP2C19
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Cytochrome P-450 CYP2D6 Inhibitors
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Cytochrome P-450 CYP2E1 Inhibitors
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Cytochrome P-450 CYP3A
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Cytochrome P-450 Enzyme Inhibitors*
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Cytochrome P-450 Enzyme System / genetics
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Cytochrome P-450 Enzyme System / metabolism*
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Cytochromes b5 / metabolism
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Enzyme Activation / drug effects
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Female
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Humans
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Kinetics
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Male
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Methoxsalen / pharmacology*
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Microsomes, Liver / drug effects
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Microsomes, Liver / enzymology*
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Microsomes, Liver / metabolism
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Middle Aged
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Mixed Function Oxygenases / antagonists & inhibitors*
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Mixed Function Oxygenases / genetics
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Mixed Function Oxygenases / metabolism*
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NADP / metabolism
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NADPH-Ferrihemoprotein Reductase / metabolism
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Spectrophotometry
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Steroid 16-alpha-Hydroxylase*
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Steroid Hydroxylases / antagonists & inhibitors
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Time Factors
Substances
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Coumarins
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Cytochrome P-450 CYP1A2 Inhibitors
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Cytochrome P-450 CYP2D6 Inhibitors
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Cytochrome P-450 CYP2E1 Inhibitors
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Cytochrome P-450 Enzyme Inhibitors
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Recombinant Proteins
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NADP
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Cytochromes b5
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Cytochrome P-450 Enzyme System
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coumarin
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Mixed Function Oxygenases
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Steroid Hydroxylases
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Aryl Hydrocarbon Hydroxylases
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CYP1A2 protein, human
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CYP2C19 protein, human
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CYP3A protein, human
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Cytochrome P-450 CYP1A2
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Cytochrome P-450 CYP2A6
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Cytochrome P-450 CYP2C19
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Cytochrome P-450 CYP3A
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Steroid 16-alpha-Hydroxylase
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NADPH-Ferrihemoprotein Reductase
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Methoxsalen