Oxidations of <i>p</i>-Alkoxyacylanilides Catalyzed by Human Cytochrome P450 1A2: Structure−Activity Relationships and Simulation of Rate Constants of Individual Steps in Catalysis

Yun, Chul‐Ho; Gp, Miller; Fp, Guengerich

doi:10.1021/bi002906n

Cited by 39 publications

(62 citation statements)

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“…Moreover, the observed kinetic isotope effects can very considerably with different P450s and different reactions even though all are of low efficiency (31,34). The variability of observed kinetic isotope effects in various P450 reactions (28,29,31,34,94) is further extended in further work with human P450s 2E1 and 3A4 (95,96). Thus, the branched nature of the P450 reactions can contribute to the higher expression of kinetic isotope effects (92, 93) but is not a sufficient explanation in the absence of further kinetic details about particular reactions.…”

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confidence: 87%

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Yun

Kim

Calcutt

et al. 2005

Journal of Biological Chemistry

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115

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Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b 5 (b 5 ). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k on ϳ 10 6 M ؊1 s ؊1 ), and the k off rates range from 5.7 to 36 s ؊1 (at 23°C). Reduction of ferric P450 2A6 is rapid (7.5 s ؊1 ) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O 2 is rapid (k > 10 6 M ؊1 s ؊1 ), and the putative Fe 2؉ ⅐O 2 complex decayed at a rate of ϳ0.3 s ؊1 at 23°C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b 5 . Kinetic analyses showed that ϳ 1 ⁄3 of the reduced b 5 was rapidly oxidized in the presence of the Fe 2؉ ⅐O 2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6 -10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s. P4501 enzymes are involved in the oxygenation of a variety of natural products and xenobiotic chemicals in microbial systems (3, 4). Much is known about the structure, function, and catalytic features of some of the P450s, particularly the more extensively studied of the bacterial P450s (4, 5). In mammalian systems P450s oxidize many drugs, steroids, carcinogens, fatty acids and eicosanoids, fat-soluble vitamins, and other endobiotic and xenobiotic chemicals (6). Less information is available about the biochemical details of most of the 57 human P450s (7). In particular, the basis of the inherently lower catalytic activities of these and other mammalian P450s relative to some of the microbial forms is not clear.P450 2A6 is a low-to-medium abundance P450 in human liver (7-9) and is also expressed in some extrahepatic tissues (10). The history of this gene/protein goes back to Phillips et al. (11), who identified a human P450 cDNA as a relative of rat P450 2B1. The 7-hydroxylation of coumarin has long been used as an assay of P450 activity in animal and human liver microsomes (12, 13), and Yamano et al. (14) isolated a P450 2A6 cDNA (then termed 2A3) and first showed that the protein derived from heterologous expression had coumarin 7-hydroxylation activity. Miles et al. (15) also provided similar evidence for this particular sequence being associated with coumarin 7-hydroxylation. Our group purified a pr...

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confidence: 87%

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Yun

Kim

Calcutt

et al. 2005

Journal of Biological Chemistry

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115

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“…12 and 15), but the evidence indicates that this is not the common case, at least with purified P450 systems (16). Some of the steps in the generally accepted catalytic cycle (Scheme 1) shown to be ratelimiting in certain instances include (2) introduction of the first electron into ferric P450 (16), (4) introduction of the "second" electron, as judged by the stimulation of reactions by the action of cytochrome b 5 in some cases (18,19), (5) protonation of the fully reduced (Fe 2ϩ O 2 Ϫ ) complex (20,21), (6) C-H bond breaking (11,22) or 1-electron oxidation (23,24), (7) a step following product formation (25,26), and (10, 11) competition with rates of abortive O 2 reduction (27).…”

Section: P450mentioning

confidence: 99%

“…The step in which C-H bond cleavage occurs can be studied in detail in O-demethylation reactions due to (i) general considerations about using non-competitive intermolecular experiments to assess the rate-limiting contributions of C-H bond cleavage (3,38); (ii) the ease of synthesis of the labeled substrates; (iii) the lack of an issue of prochirality of hydrogens; and (iv) the lack of complications of base-catalyzed proton abstraction encountered in N-dealkylation reactions (39). We analyzed several of the individual reaction steps in the P450 2D6-catalyzed O-demethylations of 3-and 4-methoxyphenethylamine and, as in earlier work with P450 1A2-catalyzed O-dealkylation of 4-alkoxyacylanilides (27), applied kinetic simulation analysis to the results. The work collectively implicates both the accumulation of an oxygenated iron complex and C-H bond breaking in contribution to the rate-limiting step(s) of the reactions.…”

Section: P450mentioning

confidence: 99%

“…HCHO was recovered from an incubation (5 min, 37°C) as the 2,4-dinitrophenylhydrazone and analyzed by combined gas chromatography/MS (positive ion mode) as described in detail previously (27). The [ 2 H 1 ]formaldehyde hydrazone was monitored at m/z 211, and the [ 2 H 2 ]formaldehyde hydrazone was monitored at m/z 212.…”

Section: Estimation Of Kinetic Isotope Effectsmentioning

confidence: 99%

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Oxidation of Methoxyphenethylamines by Cytochrome P450 2D6

Guengerich

Miller²,

Hanna³

et al. 2002

Journal of Biological Chemistry

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Cytochrome P450 (P450) 2D6 is involved in the oxidation of a large fraction (ϳ30%) of drugs used by humans and also catalyzes the O-demethylation of the model substrates 3-and 4-methoxyphenethylamine followed by subsequent ring hydroxylation to dopamine. Burst kinetics were not observed; rate-limiting step(s) must occur prior to product formation. Rates of reduction of ferric P450 2D6 were stimulated by 3-or 4-methoxyphenethylamine or the inhibitor quinidine; reduction is not the most rate-limiting step. The non-competitive intramolecular deuterium isotope effect, an estimate of the intrinsic isotope effect, for 4-methoxyphenethylamine O-demethylation was 9.6. Intermolecular noncompetitive deuterium isotope effects of 3.1-3.8 were measured for k cat and k cat /K m for both O-demethylation reactions, implicating at least partially rate-limiting C-H bond breaking. Simulation of steady-state kinetic data yielded a catalytic mechanism dominated by the rates of (i) Fe 2؉ O 2 ؊ protonation (plus O-O bond scission) and (ii) C-H bond breaking, consistent with the appearance of the spectral intermediates in the steady state, attributed to iron-oxygen complexes. However, all the rates of individual steps (or rates of combined steps) are considerably higher than k cat , and the contributions of several steps must be considered in understanding rates of the P450 2D6 reactions.

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“…No new metabolites were detected. Yun et al (2001) have shown significant water formation by human CYP1A2; thus, this particular attribute does not appear to be caffeine-specific.…”

Section: Discussionmentioning

confidence: 93%

Oxidation of Caffeine by CYP1A2: Isotope Effects and Metabolic Switching

Regal

Kunze

Peter

et al. 2005

Drug Metabolism and Disposition

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ABSTRACT:Caffeine (1,3,7-trimethylxanthine) has previously been shown to undergo metabolic switching in vivo when the N-1 or the N-7 methyl groups were trideuteromethylated [Horning et al. (1976) Proceedings of the Second International Conference on Stable Isotopes, pp 41-54]. We have examined the effect of replacing the N-3 methyl group with a trideuteromethyl group. The corresponding isotope effects can then be used to distinguish the kinetic mechanism by which four primary metabolites can be formed from one substrate by one cytochrome P450 (P450). We have synthesized 3-CD 3 -caffeine and 3-CD 3 -7-CD 3 -caffeine as well as trideuteromethylated analogs of each of the in vitro metabolites formed by cytochrome P4501A2. The observed competitive isotope effects for the metabolites, which do not result from deuterium abstraction (theobromine, theophylline), demonstrate that the nondissociative mechanism applies to caffeine metabolism by cytochrome P4501A2. Thus, there must be equilibration of the kinetically distinguishable activated P450-substrate complexes at rates competitive with hydrogen abstraction. The true isotope effects for the N-3 demethylation of caffeine were derived from the ratios of the amount of paraxanthine relative to the amount of theobromine or theophylline. The resultant ratios indicate that these isotope effects are essentially intrinsic. Observation of the isotope effects on N-3 demethylation was facilitated by branching to the minor in vitro metabolites as well as water formation. Product release is not rate-limiting for this system. Cytochrome P4501A2 (CYP1A2) is estimated to be responsible for 90% of the primary metabolism of caffeine in humans (Tassaneeyakul et al., 1994) and is believed to be largely responsible for formation of the major metabolite, paraxanthine (PX), in vivo. The four in vitro metabolites formed by human CYP1A2 are PX (80%), theobromine (TB; 11%), theophylline (TP; 4%) and 1,3,7-trimethyluric acid (TMU; 1%) ( Fig. 1; Gu et al., 1992). Presumably, multiple binding orientations exist within the active site of CYP1A2 since the entire periphery of caffeine is available for metabolism.Experimental evidence suggests that when metabolically susceptible hydrogen(s) are replaced with deuterium atoms, there can be a decrease in the oxidation rate at the labeled site with no concomitant change in the overall extent of metabolism (Harada et al., 1984;Atkins and Sligar, 1986;Jones et al., 1986). Thus, the net effect of deuteration is an increased rate of formation of one or all of the alternate metabolites, a phenomenon known as "isotopically sensitive branching" or "metabolic switching." On the basis of studies that exhibited classical metabolic switching (Harada et al., 1984), it was hypothesized that switching occurs at the level of the activated P450 or "EOS complex," and not earlier in the catalytic cycle. Theoretical studies have supported this concept (Korzekwa et al., 1989;Nelson and Trager, 2003). Hence, deuterium-induced metabolic switching indicates that isomeric EOS co...

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Oxidations of p-Alkoxyacylanilides Catalyzed by Human Cytochrome P450 1A2: Structure−Activity Relationships and Simulation of Rate Constants of Individual Steps in Catalysis

Cited by 39 publications

References 58 publications

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Oxidation of Methoxyphenethylamines by Cytochrome P450 2D6

Oxidation of Caffeine by CYP1A2: Isotope Effects and Metabolic Switching

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