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`0090-9556/06/3408-1288–1290$20.00
`DRUG METABOLISM AND DISPOSITION
`Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 34:1288–1290, 2006
`
`Vol. 34, No. 8
`10280/3128274
`Printed in U.S.A.
`
`Short Communication
`KINETIC ISOTOPE EFFECTS IMPLICATE A SINGLE OXIDANT FOR CYTOCHROME P450-
`MEDIATED O-DEALKYLATION, N-OXYGENATION, AND AROMATIC HYDROXYLATION OF
`6-METHOXYQUINOLINE
`
`Received March 23, 2006; accepted May 12, 2006
`
`ABSTRACT:
`
`One major point of controversy in the area of cytochrome P450
`(P450)-mediated oxidation reactions is the nature of the active-
`oxygen species. A number of hypotheses have been advanced
`which implicate a second oxidant besides the iron-oxo species
`designated as compound I (Cpd 1). This oxygen is thought to be
`either an iron-hydroperoxy species (Cpd 0) or a second spin-state
`of Cpd 1. Very little information is available on what fraction of
`P450 oxidations is mediated by the two different oxidants. Herein,
`we report results on three cytochrome P450-mediated reactions:
`O-dealkylation, N-oxygenation, and aromatic hydroxylation, which
`occur by three distinct chemical mechanisms. We have used ki-
`
`netic isotope effects to test for branching from O-demethylation to
`N-oxygenation
`and
`aromatic
`hydroxylation,
`using
`6-me-
`thoxyquinoline and 2H3-6-methoxyquinoline as substrates for
`P4501A2. Identical large inverse isotope effects on Vmax/Km are
`obtained for the formation of both the N-oxide and the phenol. This
`indicates that all three reactions occur through the same enzyme-
`substrate complex and, thus, through a single iron-oxygen spe-
`cies. The nature of the iron-oxygen species is less certain but is
`more likely to be iron-oxo Cpd 1, given the energetics of these
`reactions.
`
`The ubiquitous cytochrome P450 enzymes (P450s) are a superfam-
`ily of monooxygenases that function to metabolize a variety of en-
`dogenous and xenobiotic compounds (Ortiz de Montellano, 1995;
`Meunier et al., 2004). A great deal of effort has been expended on
`probing the oxygenation mechanisms and identifying the intermediate
`species formed in the catalytic cycle of the enzyme. Until recently, a
`consensus seemed to have been reached that the high-valent iron-oxo
`species (Cpd 1) is the sole active oxygenating species in P450 (Groves
`et al., 1978; Guengerich and MacDonald, 1990; Ortiz de Montellano,
`1995). This mechanism was first elaborated by Groves et al. (1978)
`for aliphatic substrates as being an initial hydrogen atom abstraction
`followed by recombination to produce an alcohol. However, many
`recent publications implicate an iron-hydroperoxo species (Cpd 0) as
`a second electrophilic oxidant (Vaz et al., 1998; Hutzler et al., 2003;
`Chandrasena et al., 2004). The hypothesis that Cpd 0 is a second
`oxidant in the P450 catalytic cycle began with a series of site-directed
`mutagenesis experiments performed by Coon and coworkers (Vaz et
`al., 1998) in which an active-site threonine is replaced by an alanine.
`The mutation is thought to affect proton delivery that converts Cpd 0
`to Cpd 1. The results indicate that some of the mutant enzymes will
`perform olefin epoxidation at enhanced rates while decreasing ali-
`phatic C-H hydroxylation (Vaz et al., 1998). Radical probe and kinetic
`isotope effect experiments were used by Newcomb and coworkers
`(Chandrasena et al., 2004) to conclude that Cpd 0 is the predominant
`
`This research was supported by National Institute of Environmental and Health
`Sciences Grant 09122.
`1 Current affiliation (T.S.D.): Department of Medicinal Chemistry and Pharma-
`cognosy, University of Illinois at Chicago.
`Article, publication date, and citation information can be found at
`http://dmd.aspetjournals.org.
`doi:10.1124/dmd.106.010280.
`
`oxidant affecting aliphatic hydroxylation of substituted cyclopropanes
`but that Cpd 1 oxygenates higher barrier reactions such as hydroxy-
`lation of the methyl group of straight-chain aliphatic compounds. In
`separate studies, Hutzler et al. (2003) have suggested that two differ-
`ent oxidants may be involved in O-dealkylation and N-dealkylation.
`Jin et al. (2003) came to the conclusion that Cpd 0 could epoxidize
`alkenes but not hydroxylate the high-energy aliphatic C-H bond on
`camphor, and Volz et al. (2002) suggested that Cpds 0 and 1 may be
`the sole oxidants involved in N-dealkylation and sulfoxidation, re-
`spectively.
`An alternative explanation for these results, as proposed by Shaik
`and coworkers (Ogliaro et al., 2002), is a two-state reactivity model in
`which Cpd 1 has two accessible spin states that can behave like two
`different oxidants. Shaik used density functional theory to conclude
`“. . . that sulfoxidation and N-dealkylation proceed largely via differ-
`ent spin states of Cpd 1,” consistent with the two oxidations occurring
`by different pathways (Sharma et al., 2003). Furthermore, Shaik and
`coworkers (Ogliaro et al., 2002) used density functional theory to
`conclude that Cpd 0 is a weak oxidant in comparison to Cpd 1. This
`has been confirmed with model systems (Park et al., 2006). In con-
`trast, Bach (Bach and Dmitrenko, 2006) used theoretical calculations
`to conclude that the peroxy species was the active oxidant.
`Given the controversy, and the conflicting data, a simple experi-
`ment is required to determine whether, and when, multiple oxidants
`are catalyzing a reaction. One such experiment involves the use of
`isotopically sensitive branching, which can be used to determine
`whether products come from rapidly interchanging enzyme-substrate
`complexes, or whether they arise from kinetically independent en-
`zyme-substrate complexes (Jones et al., 1986; Atkins and Sligar,
`1988; Wagschal et al., 1991). Three things led us to consider the
`possibility that N-oxygenation may occur through Cpd 0. 1) Sulfoxi-
`
`ABBREVIATIONS: P450, cytochrome P450; Cpd, compound; LC/MS, liquid chromatography/mass spectroscopy.
`
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`SINGLE OXIDANT FOR P450
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`SCHEME 1. Metabolism of 6-methoxyquinoline (1) by P4501A2 to produce 6-hy-
`droxyquinoline (2), 6-methoxyquinoline N-oxide (3), and 6-methoxy-3-quinolinol
`(4).
`
`three products can be formed, as shown in Scheme 1. Substitution of
`deuterium on the methoxy group should lead to a significant normal
`isotope effect on the O-dealkylation pathway, leading to lower prod-
`uct formation. If aromatic oxidation and N-oxide formation occur
`from enzyme-substrate complexes that are rapidly interchangeable
`with the O-dealkylation enzyme-substrate complex, an inverse isotope
`effect will be observed on the aromatic oxidation and N-oxide prod-
`ucts. The overall magnitude of the inverse isotope effect depends on
`the amount of each product formed and the magnitude of the isotope
`effect for O-dealkylation. If aromatic oxidation or N-oxide formation
`occur from enzyme-substrate complexes that cannot interchange with
`the O-dealkylation enzyme-substrate complex, no isotope effect will
`be observed on the aromatic oxidation or N-oxide products. The latter
`result was observed for N-dealkylation and sulfoxidation (Volz et al.,
`2002) of N,N-dimethyl-4-(methylthio)aniline, indicating that these
`two products do not come from interchangeable enzyme-substrate
`complexes and most likely come from two different oxidants. Given
`previous experiments that indicated that aromatic oxidation occurred
`from rapidly interchanging enzyme-substrate complexes, we antici-
`pated two possible outcomes upon deuteration of the methoxy group:
`1) a decrease in 2 as a result of a normal isotope effect, an increase in
`aromatic oxidation to form 4, and the same amount of N-oxide
`formation, or 2) a decrease in product 2 and an increase in products 3
`and 4 as a result of branching. Situation 1 would implicate a second
`oxidant for N-oxide formation, and situation 2 would indicate a
`common oxidant for all three products.
`Large inverse isotope effects were observed on both N-oxygenation
`and aromatic hydroxylation of 6-methoxyquinoline (Table 1). The
`results are only consistent with all three products arising from a
`kinetically indistinguishable (rapidly interchanging) enzyme-substrate
`complex, i.e., the same oxidant. However, some fraction of the prod-
`ucts may still come from a second noninterchangeable enzyme-sub-
`strate complex. If this is the case, only a fraction of the intrinsic
`isotope effect would be observed.
`Because N-oxide formation and aromatic oxidation are minor path-
`ways, the majority of the isotope effect is observed on these pathways,
`with only a small isotope effect on O-dealkylation. The lower limit of
`the intrinsic isotope effect can be estimated to be 6.5 by dividing the
`isotope effect on O-dealkylation (1.3) by the inverse isotope effect for
`either N-oxide formation (0.20) or the aromatic oxidation product
`(0.17). Some of the observed intramolecular and branched isotope
`effects for P450 O-dealkylation reactions that have been reported are
`
`dation, a reaction closely related to N-oxygenation, has been shown by
`isotopically sensitive branching experiments to occur from a second
`oxidant (Volz et al., 2002). 2) N-Oxygenation possesses a relatively
`low energy barrier in comparison to aromatic hydroxylation (Dowers
`et al., 2004), and low barrier reactions have a higher probability of
`occurring through a reaction with Cpd 0, given its low reactivity.
`(Ogliaro et al., 2002). 3) The possibility that N-oxygenation occurs
`through Cpd 0 has been suggested in the past (Coon et al., 1998).
`Therefore, we aimed to test whether N-oxygenation occurs through
`the same active iron-oxygen species as O-dealkylation, a P450-medi-
`ated reaction that has a relatively high energy barrier in comparison to
`N-dealkylation (Jones et al., 2002) and is generally accepted to occur
`through Cpd 1 (Lindsay-Smith and Sleath, 1983; Harada et al., 1984;
`Vaz and Coon, 1994; Higgins et al., 2001; Meunier et al., 2004).
`
`Materials and Methods
`All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless
`otherwise noted. The aromatic hydroxylation product, 6-methoxy-3-quinoli-
`nol, was purchased from ChemBridge Corporation (San Diego, CA). 2H3-6-
`Methoxyquinoline was synthesized from 6-hydroxyquinoline and 2H3-methyl
`iodide, which was purchased from Cambridge Isotope Laboratories (Andover,
`MA). The product was purified on a gravity silica column and verified by
`liquid chromatography/mass spectroscopy (LC/MS). The deuterium content
`from the LC/MS was consistent with that in the 2H3-methyl iodide (99.5%),
`and no attempt was made to correct for incomplete incorporation. Incubations
`were performed using RECO system CYP1A2, purchased from Invitrogen
`(Carlsbad, CA). Invitrogen’s CYP1A2 enzyme mix consists of 0.5 ␮M
`CYP1A2, 0.2 ␮M NADPH P450 reductase, 0.5 ␮g/␮l 3-[(3-cholamidopro-
`pyl)dimethylammonio]propanesulfonate, 0.1 ␮g/␮l liposomes [dilauroyl phos-
`phatidylcholine, dioleoyl phosphatidylcholine, dilauroyl phosphatidylserine
`(1:1:1)], 3 mM reduced glutathione, and 50 mM HEPES/KOH, pH 7.4.
`Invitrogen’s buffer mix is in 1 M potassium/sodium phosphate (pH 7.4). The
`incubations consisted of 200 ␮l enzyme mix, 270 ␮l buffer mix, 1 mM
`NADPH, and 0.04 mg/ml catalase. Varying concentrations of substrate in
`methanol were added to the incubations in 5-␮l quantities. The samples were
`incubated in a shaker bath for 30 min at 37°C. The reactions were quenched
`with 3 ml of ethyl acetate, and 9.2 nmol of quinoline N-oxide was added as an
`internal standard. The samples were extracted with ethyl acetate, which was
`evaporated under nitrogen. The resulting residue was dissolved in approxi-
`mately 200 ␮l of methanol for LC/MS analysis. LC/MS analysis was per-
`formed as described previously (Dowers et al., 2004).
`
`Results and Discussion
`The use of 6-methoxyquinoline as a substrate for P4501A2 allowed
`us to determine whether aromatic hydroxylation, N-oxidation, and
`O-dealkylation come from a common active-oxygen species (Scheme
`1). Because isotopically sensitive branching from O-demethylation to
`aromatic hydroxylation has been shown previously (Lindsay-Smith
`and Sleath, 1983; Harada et al., 1984), we expected branching to
`occur to the aromatic hydroxylation metabolite of 6-methoxyquino-
`line (4), but not necessarily to the N-oxide (3). Obtaining an inverse
`isotope effect on the formation of the phenol 4 would ensure that the
`substrate’s motion in the active site is fast relative to the oxidation
`reaction. In an elegant study by Regal et al. (2005) on the kinetics of
`caffeine metabolism by CYP1A2, fast interchange was observed for
`caffeine N-dealkylation; thus, fast interchange is likely for 6-me-
`thoxyquinoline as well.
`Isotopically sensitive branching experiments test whether enzyme-
`substrate complexes for different products for a single substrate can
`rapidly interchange (Jones et al., 1986). If interchange is rapid, iso-
`topic substitution will result in branching from the product with an
`isotope effect to a product that has no isotope effect associated with its
`formation. If the enzyme-substrate complexes are slow to interchange,
`no branching will be observed. In the case of 6-methoxyquinoline,
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`DOWERS AND JONES
`
`TABLE 1
`Isotope effects for metabolism of protio and deuterio 6-methoxyquinoline (1)
`
`6-OH-Quinoline 2
`
`6-Methoxyquinoline N-Oxide 3
`
`6-Methoxy-3-Quinolinol 4
`
`a
`
`1994 ⫾ 138
`1552 ⫾ 151
`1.3 ⫾ 0.22
`
`(V/K)H
`(V/K)D
`(V/K)H/(V/K)D
`a The Vmax and Km values were obtained by plotting the rate of product formation versus substrate concentration. The data were fit to Michaelis-Menton kinetics using the program EnzFitter.
`At higher substrate concentrations, a change in the ratio of product 3 to product 4 was observed, indicating two substrates binding in the active site. Therefore, the Vmax and Km values were obtained
`at low substrate concentrations (3 ␮M to 50 ␮M). Possible errors associated with two substrates binding should affect the Vmax and Km values of the protio and deuterio substrates equally and,
`therefore, would have no effect on (V/K)H/(V/K)D.
`
`9.1 ⫾ 1.1
`45 ⫾ 15
`0.20 ⫾ 0.09
`
`24 ⫾ 1.1
`140 ⫾ 26
`0.17 ⫾ 0.04
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`7.3 for anisole (Lindsay-Smith and Sleath, 1983), 5.1 (Watanabe et
`al., 1982) and 8.4 (Guengerich et al., 2004) for 4-methoxyanisole, 7.0
`for methacetin (Guengerich et al., 2004), 8.4 for 4-cyanoanisole
`(Guengerich et al., 2004), 14.7 for 4-nitroanisole, and 5.5 for
`7-ethoxycoumarin (Miwa et al., 1985). Thus, the magnitude of this
`isotope effect is consistent with other isotope effects that have been
`measured for P450-mediated O-dealkylation reactions indicating that
`the majority, or all, of the product comes from rapidly interchangeable
`enzyme-substrate complexes and, therefore, from the same oxidant.
`Again, however, this does not prove that 100% of the N-oxide arises
`from a single oxidant.
`The strongest evidence that all the products come from the same
`oxidant is that the amount of branching to the N-oxide (3) and
`6-methoxy-3-quinolinol (4) is identical. Isotopically sensitive branch-
`ing requires that the relative amount of branching from a major
`pathway to alternate products will be the same for any two products;
`i.e., that the inverse isotope effects be the same for both products if the
`enzyme-substrate complexes are in rapid equilibrium (Korzekwa et
`al., 1995). However, if two oxidants catalyze the reactions that occur
`to form 3 and 4, the fraction of 3 and 4 that comes from a given
`oxidant will most likely be different, reflecting the different activation
`energies for the two reactions. If this is the case,
`the observed
`branching isotope effects would be different, with a smaller inverse
`isotope effect for the pathway that favors the second oxidant. Clearly,
`this not the result obtained (see Table 1). Thus, for both the reactions
`to occur from both oxidants, it is required that the relative energy of
`formation of 3 and 4 be identical for both oxidizing species. In fact,
`mutants that purportedly enhance Cpd 0 have been shown to alter
`product ratios and rates of reaction in every case, indicating a differ-
`ence in the energetics of the two oxidants (Vaz et al., 1998; Vatsis and
`Coon, 2002; Volz et al., 2002; Jin et al., 2003; Chandrasena et al.,
`2004) and consistent with computational results (Ogliaro et al., 2002).
`From these results it is apparent that for the three reactions in
`Scheme 1 a single oxidant is responsible for most, if not all, of the
`products formed. The nature of the oxidant remains a matter of debate.
`However, the consensus oxidant for O-dealkylation reactions is Cpd
`1. Since the results herein require a single oxidant for three energet-
`ically different reactions, it is most likely the iron-oxo species Cpd 1.
`
`Department of Chemistry
`Washington State University
`Pullman, Washington
`
`TAMARA S. DOWERS1
`JEFFREY P. JONES
`
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`Address correspondence to: Jeffrey P. Jones, Department of Chemistry, 477
`Fulmer Synthesis, Washington State University, Pullman, WA 99164. E-mail
`jpj@wsu.edu
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