Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

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

2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês

10.1074/jbc.m411019200

ISSN

1083-351X

Autores

Chul‐Ho Yun, Keonhee Kim, M. Wade Calcutt, F. Peter Guengerich,

Tópico(s)

Computational Drug Discovery Methods

Resumo

Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b5 (b5). 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 (kon ∼ 106 m–1 s–1), and the koff 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 O2 is rapid (k ≥ 106 m–1 s–1), and the putative Fe2+ ·O2 complex decayed at a rate of ∼0.3 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 b5. Kinetic analyses showed that ∼1/3 of the reduced b5 was rapidly oxidized in the presence of the Fe2+·O2 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. Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b5 (b5). 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 (kon ∼ 106 m–1 s–1), and the koff 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 O2 is rapid (k ≥ 106 m–1 s–1), and the putative Fe2+ ·O2 complex decayed at a rate of ∼0.3 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 b5. Kinetic analyses showed that ∼1/3 of the reduced b5 was rapidly oxidized in the presence of the Fe2+·O2 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. P450 1The abbreviations used are: P450, cytochrome P450; OH, hydroxy; OMe, methoxy; OEt, ethoxy; OR, alkoxy; di-12:0 GPC, l-α-1,2-dilauroyl-sn-glycero-3-phosphocholine; MS, mass spectrometry; b5, cytochrome b5; HPLC, high performance liquid chromatography; Em,7, standard oxidation-reduction potential at pH 7.0. Conventions used for kinetic hydrogen isotope effects: Dk, intrinsic kinetic deuterium isotope effect, DV = Hkcat/Dkcat, and D(V/K) = (Hkcat/HKm)/(Dkcat/DKm) (1.Northrop D.B. Biochemistry. 1975; 14: 2644-2651Crossref PubMed Scopus (359) Google Scholar, 2.Northrop D.B. Methods Enzymol. 1982; 87: 607-625Crossref PubMed Scopus (108) Google Scholar). enzymes are involved in the oxygenation of a variety of natural products and xenobiotic chemicals in microbial systems (3.Munro A.W. Lindsay J.G. Mol. Microbiol. 1996; 20: 1115-1125Crossref PubMed Scopus (136) Google Scholar, 4.Cryle M.J. Stok J.E. De Voss J.J. Aust. J. Chem. 2003; 56: 749-762Crossref Scopus (100) Google Scholar). 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.Cryle M.J. Stok J.E. De Voss J.J. Aust. J. Chem. 2003; 56: 749-762Crossref Scopus (100) Google Scholar, 5.Mueller E.J. Loida P.J. Sligar S.G. Ortiz de Montellano P.R. Cytochrome P450 Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 83-124Crossref Google Scholar). In mammalian systems P450s oxidize many drugs, steroids, carcinogens, fatty acids and eicosanoids, fat-soluble vitamins, and other endobiotic and xenobiotic chemicals (6.Ortiz de Montellano P.R. Cytochrome P450 Structure, Mechanism, and Biochemistry. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005Crossref Scopus (2) Google Scholar). Less information is available about the biochemical details of most of the 57 human P450s (7.Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450 Structure, Mechanism, and Biochemistry. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2004: 377-531Google Scholar). 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.Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450 Structure, Mechanism, and Biochemistry. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2004: 377-531Google Scholar, 8.Yun C.-H. Shimada T. Guengerich F.P. Mol. Pharmacol. 1991; 40: 679-685PubMed Google Scholar, 9.Shimada T. Yamazaki H. Mimura M. Inui Y. Guengerich F.P. J. Pharmacol. Exp. Ther. 1994; 270: 414-423PubMed Google Scholar) and is also expressed in some extrahepatic tissues (10.Ding X. Kaminsky L.S. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 149-173Crossref PubMed Scopus (663) Google Scholar). The history of this gene/protein goes back to Phillips et al. (11.Phillips I.R. Shephard E.A. Ashworth A. Rabin B.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 983-987Crossref PubMed Scopus (56) Google Scholar), 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.Kerekjarto B.V. Kratz F. Staudinger H.J. Biochem. Z. 1964; 339: 460-468PubMed Google Scholar, 13.Creaven P.J. Parke D.V. Williams R.T. Biochem. J. 1965; 96: 390-398Crossref PubMed Scopus (86) Google Scholar), and Yamano et al. (14.Yamano S. Tatsuno J. Gonzalez F.J. Biochemistry. 1990; 29: 1322-1329Crossref PubMed Scopus (417) Google Scholar) 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.Miles J.S. McLaren A.W. Forrester L.M. Glancey M.J. Lang M.A. Wolf C.R. Biochem. J. 1990; 267: 365-371Crossref PubMed Scopus (204) Google Scholar) also provided similar evidence for this particular sequence being associated with coumarin 7-hydroxylation. Our group purified a protein from human liver microsomes, identified it as P450 2A6, and showed it to be the major coumarin 7-hydroxylase in human liver (8.Yun C.-H. Shimada T. Guengerich F.P. Mol. Pharmacol. 1991; 40: 679-685PubMed Google Scholar). Subsequently P450 2A6 has been studied extensively, in large part because of its role in the metabolism of nicotine and carcinogenic N-alkylnitrosamines (16.Yamazaki H. Inui Y. Yun C.-H. Mimura M. Guengerich F.P. Shimada T. Carcinogenesis. 1992; 13: 1789-1794Crossref PubMed Scopus (389) Google Scholar, 17.Patten C.J. Smith T.J. Friesen M.J. Tynes R.E. Yang C.S. Murphy S.E. Carcinogenesis. 1997; 18: 1623-1630Crossref PubMed Scopus (95) Google Scholar). Genetic polymorphisms have been identified (18.Daly A.K. Cholerton S. Gregory W. Idle J.R. Pharmacol. Ther. 1993; 57: 129-160Crossref PubMed Scopus (200) Google Scholar, 19.London S.J. Idle J.R. Daly A.K. Coetzee G.A. Lancet. 1999; 353: 898-899Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and may be of relevance to cancer risk; (i) impaired metabolism of nicotine has been proposed to reduce cigarette smoking in P450 2A6-deficient individuals (20.Pianezza M.L. Sellers E.M. Tyndale R.F. Nature. 1998; 393: 750Crossref PubMed Scopus (380) Google Scholar); (ii) impaired metabolism can yield reduced levels of activation of the N-nitrosamines found in tobacco (21.Ariyoshi N. Miyamoto M. Umetsu Y. Kunitoh H. Dosaka-Akita H. Sawamura Y. Yokota J. Nemoto N. Sato K. Kamataki T. Cancer Epidemiol. Biomark. Prev. 2002; 11: 890-894PubMed Google Scholar). Some drugs are oxidized by P450 2A6 (7.Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450 Structure, Mechanism, and Biochemistry. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2004: 377-531Google Scholar, 22.Nunoya K.I. Yokoi T. Kimura K. Kainuma T. Satoh K. Kinoshita M. Kamataki T. J. Pharmacol. Exp. Ther. 1999; 289: 437-442PubMed Google Scholar). P450 2A6 also catalyzes the oxidation of indoles (23.Gillam E.M.J. Aguinaldo A.M. Notley L.M. Kim D. Mundkowski R.G. Volkov A. Arnold F.H. Soucek P. DeVoss J.J. Guengerich F.P. Biochem. Biophys. Res. Commun. 1999; 265: 469-472Crossref PubMed Scopus (128) Google Scholar, 24.Gillam E.M.J. Notley L.M. Cai H. DeVoss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (247) Google Scholar), and we have used P450 2A6 mutants to synthesize new indirubins with activity as protein kinase inhibitors (25.Guengerich F.P. Sorrells J.L. Schmitt S. Krauser J.A. Aryal P. Meijer L. J. Med. Chem. 2004; 43: 3236-3241Crossref Scopus (73) Google Scholar). Recently x-ray crystal structures have been reported for P450 2A6, including forms with the substrates coumarin and nicotine bound (26.Yano J.K. Griffin K. Stout C.D. Johnson E.F. Abstracts of the 15th International Symposium on Microsomes and Drug Oxidations, Mainz, Germany, July 4–9, 2004. University of Mainz, Mainz, Germany2004: 109Google Scholar). These structures more than any of the other mammalian P450s solved to date have a small binding site akin to that of bacterial P450 101A1 (27.Poulos T.L. Finzel B.C. Gunsalus I.C. Wagner G.C. Kraut J. J. Biol. Chem. 1985; 260: 16122-16130Abstract Full Text PDF PubMed Google Scholar). The space for the substrate coumarin is very restricted, and the coumarin-bound structure has the C-7 atom located near the heme iron (26.Yano J.K. Griffin K. Stout C.D. Johnson E.F. Abstracts of the 15th International Symposium on Microsomes and Drug Oxidations, Mainz, Germany, July 4–9, 2004. University of Mainz, Mainz, Germany2004: 109Google Scholar). A major conformational change is required to open and close the enzyme and allow the substrate (coumarin) to enter and leave (26.Yano J.K. Griffin K. Stout C.D. Johnson E.F. Abstracts of the 15th International Symposium on Microsomes and Drug Oxidations, Mainz, Germany, July 4–9, 2004. University of Mainz, Mainz, Germany2004: 109Google Scholar). We have been studying aspects of catalysis of mammalian P450s, including the rate-limiting steps in reactions (28.Yun C.-H. Miller G.P. Guengerich F.P. Biochemistry. 2001; 40: 4521-4530Crossref PubMed Scopus (37) Google Scholar, 29.Guengerich F.P. Miller G.P. Hanna I.H. Sato H. Martin M.V. J. Biol. Chem. 2002; 277: 33711-33719Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 30.Guengerich F.P. Biol. Chem. 2002; 383: 1553-1564Crossref PubMed Scopus (65) Google Scholar, 31.Guengerich F.P. Krauser J.A. Johnson W.W. Biochemistry. 2004; 43: 10775-10788Crossref PubMed Scopus (79) Google Scholar). P450 2A6 was of interest because of the recently reported structure, the useful fluorescence properties and common use of coumarins as P450 substrates, and our inherent interest in the catalytic properties of P450 2A6 (8.Yun C.-H. Shimada T. Guengerich F.P. Mol. Pharmacol. 1991; 40: 679-685PubMed Google Scholar, 23.Gillam E.M.J. Aguinaldo A.M. Notley L.M. Kim D. Mundkowski R.G. Volkov A. Arnold F.H. Soucek P. DeVoss J.J. Guengerich F.P. Biochem. Biophys. Res. Commun. 1999; 265: 469-472Crossref PubMed Scopus (128) Google Scholar, 24.Gillam E.M.J. Notley L.M. Cai H. DeVoss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (247) Google Scholar, 25.Guengerich F.P. Sorrells J.L. Schmitt S. Krauser J.A. Aryal P. Meijer L. J. Med. Chem. 2004; 43: 3236-3241Crossref Scopus (73) Google Scholar, 32.Nakamura K. Martin M.V. Guengerich F.P. Arch. Biochem. Biophys. 2001; 395: 25-31Crossref PubMed Scopus (79) Google Scholar). Rates of several steps were measured (Scheme 1). Studies of O-dealkylation of 7-OR coumarins showed high kinetic hydrogen isotope effects and demonstrate the kinetic difficulty of C–H bond breaking. The 7-OR coumarins showed extensive formation of 3-OH products as well as 7-OH coumarin. Together the results provide a picture of a very dynamic catalytic cycle with considerably more flexibility than apparent with the more efficient bacterial P450s, providing some potential insight into rate differences. Chemicals—Coumarin, 7-OH coumarin, 7-OMe coumarin, and 7-OEt coumarin were purchased from Aldrich and recrystallized from C2H5OH/H2OorCH3OH/H2O before use. 5-Deazaflavin was a gift of the late V. Massey (University of Michigan, Ann Arbor, MI) (36.Massey V. Hemmerich P. Biochemistry. 1978; 17: 9-16Crossref PubMed Scopus (292) Google Scholar). Deuterated 7-OMe coumarins were prepared by reaction of 7-OH coumarin with deuterated methyl iodides (Cambridge Isotopes, Cambridge, MA) in the usual manner (29.Guengerich F.P. Miller G.P. Hanna I.H. Sato H. Martin M.V. J. Biol. Chem. 2002; 277: 33711-33719Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 37.Furniss B.S. Hannaford A.J. Rogers V. Smith P.W.G. Tatchell A.R. Vogel's Textbook of Practical Organic Chemistry. John Wiley & Sons, New York1978: 753Google Scholar, 38.Zubia E. Rodriguez L.F. Massanet G.M. Collado I.G. Tetrahedron. 1992; 48: 4239-4246Crossref Scopus (61) Google Scholar) and recrystallized from CH3OH/H2O. [1-Ethyl-d2]-7-OEt coumarin was prepared in the same way from [1-d2]-ethyl iodide (Cambridge). In the synthesis of [1-ethyl-d1]-7-OEt coumarin, CH3CHO was reduced with LiAlD4 in diethylene glycol diethyl ether to prepare [1-d1]-C2H5OH (39.Gellman A.J. Buelow M.T. Street S.C. Morton T.H. J. Phys. Chem. A. 2000; 104: 2476-2485Crossref Scopus (31) Google Scholar), and the distilled product was reacted with tosyl chloride in dry pyridine to form the tosylate (72% yield, m.p. 27–30 °C (literature (40.Tipson R.S. J. Org. Chem. 1944; 9: 235-241Crossref Scopus (271) Google Scholar), 21 °C). The tosylate was then reacted with 7-OH coumarin in acetone/K2CO3 (under reflux) in the same manner as used with the alkyl iodides to yield [1-ethyl-d1]-7-OEt coumarin, which was recrystallized from CH3OH, H2O (39% yield, m.p. 85–87 °C, literature (41.Ishifuku K. Sakurai H. Okamoto H. Sato S. Yakugaku Zasshi. 1953; 73: 332-334Crossref Google Scholar), 88–90 °C); UV (CH3OH) ϵ323 1.23 × 104 m–1 cm–1; electrospray MS, m/z 191.1 (MH+); NMR (CDCl3) δ 1.37 (dd, 3H, CH3), 4.03 (m, 1H, CHD), 6.22 (d, 1H, H-3), 6.69–6.82 (m, 2H, H-6, H-8), 7.33 (d, 1H, H-5), 7.60 (d, 1H, H-4). All deuterated coumarin derivatives were >98% isotopically enriched at the site of modification as judged by MS and NMR spectroscopy. The synthesis of 3-OH coumarins was done using the general procedure of Neubauer and Flatow (42.Neubauer O. Flatow L. Hoppe-Seyler's Z. Physiol. Chem. 1907; 52: 375-398Crossref Scopus (4) Google Scholar), which involves condensation of salicylaldehyde or a 4-substituted salicylaldehyde with hippuric acid (N-benzoylglycine) to form the N-benzoyl enamine, which was hydrolyzed in 10 n NaOH (100 °C, 45 min) to give the desired coumarin. 4-OEt salicylaldehyde was prepared by BCl3 treatment of 2,4-(OEt)2 salicylaldehyde (Aldrich) in CH2Cl2 (80% yield) (43.Hinterding K. Knebel A. Herrlich P. Waldmann H. Bioorg. Med. Chem. 1998; 6: 1153-1162Crossref PubMed Scopus (49) Google Scholar). The identities of the 3-OH coumarins were confirmed by their m.p. values and spectroscopy: 3-OH coumarin, m.p. 151–154 °C (literature (44.Rajyalakshmi K. Srinivasan V.R. Ind. J. Chem. B. 1978; 16: 156Google Scholar), 154 °C), UV (CH3OH) ϵ307 1.22 × 104 m–1 cm–1, ϵ294 1.19 × 104 m–1 cm–1, ϵ235 4.91 × 103 m–1 cm–1, fluorescence (CH3OH) λexcitation 310 nm, λemission 395 nm, electrospray MS, m/z 163.1 (MH+), NMR (CDCl3) δ 6.48 (bs, 1H, H-4), 7.46–7.67 (m, 4H, H-5,6,7,9); 3-OH,7-OMe coumarin, m.p. 179–182 °C (literature (45.Indahl S.R. Scheline R.R. Xenobiotica. 1971; 1: 13-24Crossref PubMed Scopus (18) Google Scholar), 175.5–177.5 °C), UV (CH3OH) ϵ322 1.4 × 104 m–1 cm–1,ϵ235 5.6 × 103 m–1, ϵ215 8.4 × 10 m–1 cm–1, electrospray MS, m/z 193.1 (MH+), NMR (CDCl3) δ 3.79 (s, 3H, CH3), 6.88 (dd, 1H, H-6), 6.94 (d, 1H, H-8), 7.08 (s, 1H, H-4), 7.43 (d, 1H, H-5); 3-OH,7-OEt coumarin, m.p. 155–157 °C, UV (CH3OH) ϵ322 1.34 × 104 m–1 cm–1, ϵ235 5.6 × 103 m–1 cm–1, ϵ215 8.4 × 10 m–1 cm–1, electrospray MS, m/z 206.9 (MH+), NMR (CDCl3) δ 1.32 (t, 3H, CH3), 4.05 (q, 2H, CH2), 6.87 (dd, 1H, H-6), 6.89 (d, 1H, H-8), 7.08 (s, 1H, H-4), 7.42 (d, 1H, H-5). Enzymes—P450 2A6 was expressed from a plasmid (originally obtained from P. Soucek, National Institute of Public Health, Prague) in Escherichia coli, except that a His5 tag was attached to the C terminus (24.Gillam E.M.J. Notley L.M. Cai H. DeVoss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (247) Google Scholar, 46.Soucek P. Arch. Biochem. Biophys. 1999; 370: 190-200Crossref PubMed Scopus (53) Google Scholar). Rat NADPH-P450 reductase was expressed in E. coli and purified as described (47.Hanna I.H. Teiber J.F. Kokones K.L. Hollenberg P.F. Arch. Biochem. Biophys. 1998; 350: 324-332Crossref PubMed Scopus (182) Google Scholar). Recombinant human b5 was expressed in E. coli JM109 cells from a plasmid (pSE420 (Amp)) kindly provided by Satoru Asahi (Takeda Pharmaceutical, Osaka, Japan). The protein was solubilized and purified to electrophoretic homogeneity using modifications of the DEAE-cellulose and hydroxylapatite chromatography methods described elsewhere (48.Shimada T. Misono K.S. Guengerich F.P. J. Biol. Chem. 1986; 261: 909-921Abstract Full Text PDF PubMed Google Scholar). Spectroscopy—NMR spectra were recorded using Bruker 300 and 400 MHz instruments in the Vanderbilt facility. UV-visible spectra were generally acquired using an OLIS/Cary 14 or a OLIS/Aminco DW2a instrument (OLIS, Bogart, GA). Mass spectra were recorded using HPLC-MS methods (octadecylsilane columns, positive ion-electrospray, or atmospheric pressure chemical ionization) in the Vanderbilt facility using a Thermo-Finnigan TSQ-7000 instrument (Thermo-Finnigan, Sunnyvale, CA). Fluorescence measurements were made using either an SPEX Fluoromax-3 instrument (SPEC/Jobin Yvon, Edison, NJ) or an OLIS RSM-1000 instrument (OLIS), operating in the stopped-flow mode. Stopped-flow kinetic UV-visible measurements were made using an OLIS RSM-1000 instrument (slit width 1.24–3.16 nm for absorbance beam). Some kinetic traces were obtained in the single wavelength mode; the rapid scanning mode was used with a 16 × 1-mm scanning disk to obtain spectra (16–1000 scans s–1). In some cases the acquired spectra were used to derive kinetic traces at individual wavelengths, and fitting was done with the manufacturer's software. Assays—Typical steady-state coumarin oxidation reactions included 50 pmol P450 2A6, 100 pmol of NADPH-P450 reductase, 50 pmol of b5 (when indicated), and 30 μg of di-12:0 GPC in 0.50 ml of 50 mm potassium phosphate buffer (pH 7.4) along with a specified amount of the coumarin substrate. An aliquot of an NADPH-generating system was used to start reactions (final concentrations, 10 mm glucose 6-phosphate, 0.5 mm NADP+, and 1 international unit of yeast glucose 6-phosphate ml–1 (49.Guengerich F.P. Hayes A.W. Principles and Methods of Toxicology. 4th Ed. Taylor & Francis, Philadelphia2001: 1625-1687Google Scholar)). Stock coumarin solutions (5 mm) were made in H2O. 7-OMe and 7-OEt coumarin stocks (50 mm) were made in CH3CN and diluted into enzyme reactions, with final organic solvent concentrations <1% (v/v). Incubations were generally done for 10 min at 37 °C, terminated with 0.10 ml of 17% HClO4, and centrifuged (103× g, 10 min). CH2Cl2 (1.0 ml) was added to the supernatant to extract the products followed by centrifugation at 103× g (process repeated one more time). The organic layers were combined, and the CH2Cl2 was removed under a N2 stream. The products were analyzed by HPLC using a Toso ODS-80TM octadecylsilane (C18) column (4.6 × 150 mm, 5 μm) with the mobile phase H2O:CH3CN (55:45, v/v) containing 10 mm HClO4, a flow rate of 1.0 ml min–1, and monitoring at A330. Kinetic parameters (Km and kcat) were determined using nonlinear regression analysis with Graph-Pad Prism software (Graph-Pad, San Diego, CA). In some cases (stopped-flow), 7-OH coumarin was monitored directly (F390/460). Assays involving competitive deuterium isotope effects were done by HPLC-MS analysis of formaldehyde or acetaldehyde derived from [methyl-d3]-7-OMe coumarin or [1-ethyl-d2]-7-OEt coumarin, respectively, using the general approach described elsewhere (51.Okazaki O. Guengerich F.P. J. Biol. Chem. 1993; 268: 1546-1552Abstract Full Text PDF PubMed Google Scholar, 52.Guengerich F.P. Yun C.-H. Macdonald T.L. J. Biol. Chem. 1996; 271: 27321-27329Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). With [1-ethyl-d1]-7-OEt and [1-ethyl-d2]-7-OEt coumarin as substrates, the mass spectra were complicated due to the presence of contaminants in some of the reagents, particularly the solvents and glycerol. To minimize complications arising from residual aldehyde contamination in reagents and solvents, the following changes were made to the procedure. Reconstituted enzyme solutions (P450 2A6, NADPH-P450 reductase, and b5) were dialyzed against glycerol-free 50 mm potassium phosphate buffer (pH 7.4) containing 0.2 mm EDTA and 0.1 mm dithiothreitol (two changes over 12 h at 4 °C) before the addition of di-12:0 GPC. Dissolution of 7-OMe and 7-OEt coumarin in H2O was achieved by sonication (Branson sonicator, microtip probe, 70% full power) (Branson, Danbury, CT) to avoid the introduction of organic solvents. Aqueous stock solutions (∼0.5 mm) were then filtered and quantitated spectrophotometrically (see above). Hexanes and CH3CN were heated with and distilled from 2,4-dinitrophenylhydrazine. 2,4-Dinitrophenylhydrazine (for use as a derivatization reagent) was recrystallized twice from CH3OH/H2O, dried in vacuo, dissolved in 6 n HCl (0.1%, w/v), and washed multiple times with a hexane, CH2Cl2 mixture (7:3, v/v) to remove hydrazone impurities before use for derivatization. Deuterium incorporation was determined using HPLC/negative ion atmospheric pressure chemical ionization MS of the resulting 2,4-dinitrophenylhydrazone derivatives (source temperature 550 °C; heated capillary voltage 20 V; heated capillary temperature 180 °C; ionization current 5 μA; sheath gas (N2) pressure 70 p.s.i.; auxiliary gas (N2) pressure 10 p.s.i.) (53.Kolliker S. Oehme M. Dye C. Anal. Chem. 1998; 70: 1979-1985Crossref PubMed Scopus (135) Google Scholar). Anaerobic experiments involved the use of glass anaerobic cuvettes and tonometers with a gas train connected to a manifold, alternating between vacuum and Ar. The basic systems are described elsewhere (54.Foust G.P. Burleigh Jr., B.D. Mayhew S.G. Williams Jr., C.H. Massey V. Anal. Biochem. 1969; 27: 530-535Crossref PubMed Scopus (60) Google Scholar, 55.Burleigh Jr., B.D. Foust G.P. Williams Jr., C.H. Anal. Biochem. 1969; 27: 536-544Crossref PubMed Scopus (63) Google Scholar, 56.Palfey B.A. Johnson K.A. Kinetic Analysis of Macromolecules. Oxford University Press, Oxford2003: 203-228Google Scholar), and recent further modifications have been described (31.Guengerich F.P. Krauser J.A. Johnson W.W. Biochemistry. 2004; 43: 10775-10788Crossref PubMed Scopus (79) Google Scholar). The OLIS RSM-1000 stopped-flow spectrophotometer has stainless steel lines leading up to the observation cell instead of Teflon, reducing the diffusion of O2. As described earlier (31.Guengerich F.P. Krauser J.A. Johnson W.W. Biochemistry. 2004; 43: 10775-10788Crossref PubMed Scopus (79) Google Scholar), the drive syringes were filled with anaerobic 0.10 m Na2S2O4 (in 0.2 m potassium phosphate buffer, pH 7.4) overnight before use to scrub O2 (31.Guengerich F.P. Krauser J.A. Johnson W.W. Biochemistry. 2004; 43: 10775-10788Crossref PubMed Scopus (79) Google Scholar, 56.Palfey B.A. Johnson K.A. Kinetic Analysis of Macromolecules. Oxford University Press, Oxford2003: 203-228Google Scholar). The drive syringes were then loaded with the contents of a tonometer containing 0.25 mm safranin T and 0.5 mm methyl viologen (photo-reduced) in anaerobic 0.10 m Tris·HCl buffer (pH 7.7) containing 10 mm EDTA. The lack of O2 in the system is indicated by blue color (methyl viologen radical cation), as opposed to red (oxidized safranin). Thus, the final displacement of the dye by the enzyme solution provides a reasonable check on the anaerobic nature of the system. Oxidations Catalyzed by P450 2A6—Earlier work led to the characterization of P450 2A6 as the major coumarin 7-hydroxylase (8.Yun C.-H. Shimada T. Guengerich F.P. Mol. Pharmacol. 1991; 40: 679-685PubMed Google Scholar, 14.Yamano S. Tatsuno J. Gonzalez F.J. Biochemistry. 1990; 29: 1322-1329Crossref PubMed Scopus (417) Google Scholar, 15.Miles J.S. McLaren A.W. Forrester L.M. Glancey M.J. Lang M.A. Wolf C.R. Biochem. J. 1990; 267: 365-371Crossref PubMed Scopus (204) Google Scholar). Recently the structure of a P450 2A6 crystal has been reported (26.Yano J.K. Griffin K. Stout C.D. Johnson E.F. Abstracts of the 15th International Symposium on Microsomes and Drug Oxidations, Mainz, Germany, July 4–9, 2004. University of Mainz, Mainz, Germany2004: 109Google Scholar), with coumarin bound and positioned with the C-7 atom near the iron atom. Assays of P450 2A6-catalyzed coumarin oxidation commonly utilize a sensitive fluorescence assay that reports 7-hydroxylation (57.Ullrich V. Weber P. Hoppe-Seyler's Z. Physiol. Chem. 1972; 353: 1171-1177Crossref PubMed Scopus (638) Google Scholar, 58.Soucek P. J. Chromatogr. B. Biomed. Appl. 1999; 734: 23-29Crossref PubMed Scopus (27) Google Scholar). HPLC-UV assays indicated the formation of the single product 7-OH coumarin by chromatographic and spectral comparison to standard material (Table I, also see Supplemental Fig. 1). 7-OMe and 7-OEt coumarins were O-dealkylated to form 7-OH coumarin, but both of these substrates also formed the 3-hydroxy products, as judged by comparisons with synthetic materials. The identification of 3-OH, 7-OEt coumarin as a product of oxidation of 7-OEt coumarin has been reported previously with human liver microsomes (59.Jung B. Graf H. Ullrich V. Biol. Chem. Hoppe-Seyler. 1985; 366: 23-31Crossref PubMed Scopus (17) Google Scholar, 60.Fisher M.B. Jackson D. Kaerner A. Wrighton S.A. Borel A.G. Drug Metab. Dispos. 2002; 30: 270-275Crossref PubMed Scopus (16) Google Scholar). The 3-hydroxylation of coumarin has been reported with human liver microsomes (61.van Iersel M.L. Henderson C.J. Walters D.G. Price R.J. Wolf C.R. Lake B.G. Xenobiotica. 1994; 24: 795-803Crossref PubMed Scopus (43) Google Scholar, 62.Born S.L. Caudill D. Fliter K.L. Purdon M.P. Drug Metab. Dispos. 2002; 30: 483-487Crossref PubMed Scopus (93) Google Scholar). Neither we nor Born et al. (62.Born S.L. Caudill D. Fliter K.L. Purdon M.P. Drug Metab. Dispos. 2002; 30: 483-487Crossref PubMed Scopus (93) Google Scholar) detected conversion of coumarin to 3-OH coumarin by P450 2A6 systems. 23-Hydroxycoumarin was prepared (see "Experimental Procedures") and used as a standard for HPLC assays. A baculovirus-infected insect cell microsomal system was used in the earlier study (62.Born S.L. Caudill D. Fliter K.L. Purdon M.P. Drug Metab. Dispos. 2002; 30: 483-487Crossref PubMed Scopus (93) Google Scholar), but no positive control for 7-hydroxylation was involved.Table ISteady-state parameters for coumarin oxidation catalyzed by P450 2A6SubstrateWithout b5With b57-OH3-OH7-OH3-OHk catKmk catKmk catKmk catKms−1μms−1μms−1μms−1μmCoumarin0.075 ± 0.0081.8 ± 0.70.148 ± 0.0171.4 ± 0.67-OMe coumarin0.040 ± 0.00213 ± 20.016 ± 0.00112 ± 30.037 ± 0.00210 ± 20.018 ± 0.00211 ± 27-OEt coumarin0.045 ± 0.00339 ± 70.133 ± 0.023120 ± 300.033 ± 0.00310 ± 40.167 ± 0.01715 ± 5 Open table in a new tab Subsequent analysis of steady-state kinetic parameters indicated that 3-hydroxylation was observed to a greater extent for 7-OEt coumarin than 7-OMe coumarin (Table I). 3Higher rates of coumarin hydroxylation were observed when organic solvents were omitted from the reaction. Although microsomal coumarin 7-hydroxyation has been reported not to be very sensitive to CH3OH or dimethylsulfoxide (50.Chauret N. Gauthier A. Nicoll-Griffith D.A. Drug Metab. Dispos. 1998; 26: 1-4PubMed Google Scholar), we found inhibitory effects of CH3OH and C2H5OH in our work with the reconstituted enzyme system. Coumarin stocks can be prepared at 5 mm in H2O and stored at 4 °C without any difficulty. The addition of b5 stimulated the formation of 7-hydroxylation of coumarin 2-fold, as reported by others (46.Soucek P. Arch. Biochem. Biophys. 1999; 370: 190-200Crossref PubMed Scopus (53) Google Scholar, 63.Tan Y. Patten C.J. Smith T. Yang C.S. Arch. Biochem. Biophys. 1997; 342: 82-91Crossref PubMed Scopus (50)

Referência(s)