PREFERRED ORIENTATIONS IN THE BINDING OF 4'-HYDROXYACETANILIDE (ACETAMINOPHEN) TO CYTOCHROME-P450 1A1 AND 2B1 ISOFORMS AS DETERMINED BY C-13-NMR AND N-15-NMR RELAXATION STUDIES

The widely used analgesic/antipyretic agent 4'-hydroxyacetanilide (ace taminophen, APAP) is oxidized by cytochromes P450 to a potent cytotoxi n, N-acetyl-p-benzoquinone imine (NAPQI), and a nontoxic catechol, 3', 4'-dihydroxyacetanilide (3-hydroxyacetaminophen, 3-OH-APAP). There are marked differences in the ratios of these two products formed from di fferent isoforms of cytochrome P450. For example, the ratio of NAPQI t o 3-OH-APAP formed by rat liver CYP1A1 was found to be approximately 8 :1, whereas the ratio of the same two products formed by rat liver CYP 2B1 was approximately 1:5. Investigations of the binding of APAP to CY P1A1 and CYP2B1 were carried out to assess the possibility that differ ent preferred orientations of APAP in the active sites of these isofor ms may, in part, by responsible for their different product selectivit ies. Although the spectral dissociation constants (K(s) congruent-to 0 .85 mM) and UV-vis binding spectra (type I; absorption minimum congrue nt-to 420 nm, absorption maximum congruent-to 390 nm) were similar for interactions of APAP with the two P450 isoforms, NMR longitudinal rel axation times (T1) of APAP nuclei were significantly different. Two is otopically substituted analogs of APAP, [2,3',5'-C-13(3)]-4'-hydroxyac etanilide and 4'-hydroxyacet-[N-15]-anilide, were synthesized, and the ir binding to purified CYP1A1 and CYP2B1 was examined by NMR spectrosc opy. Paramagnetic relaxation times (T1p) for each of the labeled nucle i were calculated from the T1 values obtained before (ferric) and afte r (ferrous-CO) treatment with Na2S2O4 and CO. The Solomon-Bloembergen equation was then used to calculate distances of the isotopically labe led nuclei from the heme iron of each P450 isoform. The results were t hat the amide nitrogen approaches relatively close to the heme iron in CYP1A1 (3.64 +/- 0.51 angstrom) whereas it is significantly further a way (>4.5 angstrom) in CYP2B1. In contrast, the aryl carbon atoms orth o to the phenolic group of APAP approach closer to the heme iron of CY P2B1 (3.19 +/- 0.12 angstrom) than to the heme iron of CPY1A1 (3.66 +/ - 0.30 angstrom). The results are consistent with the hypothesis that CYP1A1 produces NAPQI preferentially because of closer proximity of th e heme iron to the amide nitrogen, whereas CYP2B1 produces 3-OH-APAP p referentially because of closer proximity of the heme iron to the phen olic oxygen in this isoform.