Regular ArticleRandom Mutagenesis of Human Cytochrome P450 2A6 and Screening with Indole Oxidation Products1☆,☆☆,★,★★,☆☆☆,★★★
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1This study was supported in part by United States Public Health Service Grants R35 CA44353, R01 CA90426, and P30 ES00267 (F.P.G.).
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1This study was supported in part by United States Public Health Service Grants R35 CA44353, R01 CA90426, and P30 ES00267 (F.P.G.).
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Current address: Department of Pharmacy, Gunma University School of Medicine, Gunma, Japan.
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We did not extend our analysis of the kinetics of pigment formation to more concentrations. Sensitivity is not the issue (Fig. 3), but the estimation of Km values for indole 3-hydroxylation is complicated by the instability of the initial enzymatic prpoduct (3-hydroxyindole) in the presence of oxygen (17) and the lag observed in the nonenzymatic oxidation of 3-hydroxyindole to indigo (Fig. 3A). Another complexity in attempting to do steady-state kinetics of indigo formation is the varying λmax of the product (Fig. 3B), presumably a reflection of the formation of multiple products with overlapping spectra. It is possible to estimate kinetic parameters for some of the stable initial oxidation products, e.g., 2-hydroxyindole (17) (Fig. 4), but these are not related to pigment formation.
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Similar results (rates and spectra) were obtained in a set of duplicate experiments in which the reactions were stopped after 20 min by the addition of 1% sodium dodecyl sulfate (to 1%, w/v).
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We have found the color patterns produced by the mutants with some of the substituted indoles to be very reproducible, in repeated assays (P. Aryal and F. P. Guengerich, unpublished observations).
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To whom correspondence may be addressed at Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building (Medical Research Building I), 23rd and Pierce Avenues, Nashville, TN 37232-0146. Fax: (615) 322-3141. E-mail: [email protected].
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Abbreviations used: P450, cytochrome P450 [also termed heme-thiolate P450 (1)]; WT, wild-type (P450 2A6); NPR, NADPH-P450 reductase; PCR, polymerase chain reaction; SRS, substrate recognition sequence; StEP, staggered extension process; IPTG, isopropyl-β--thiogalactoside; LB, Luria broth.