Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6

Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6 SEQUENTIAL OXIDATION TO ALDEHYDES AND CARBOXYLIC ACIDS AND

ANALYSIS OF REACTION STEPS* S Goutam Chowdhury1, M. Wade Calcutt1, and F. Peter Guengerich*

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Running title: Sequential Nitrosamine Oxidation by P450 2A6 Address correspondence to: Prof. F. Peter Guengerich, Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 2200 Pierce Avenue, Nashville, Tennessee 37232-0146, Telephone: (615) 322-2261, FAX: (615) 322-3141, E-mail: [email protected]

Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA alkylating agents) and also aldehydes (HCHO from DMN and CH3CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect (Dkapp ~ 10), which was highly expressed in a variety of competitive and non-competitive experiments. The Dkapp for DEN was ~ 3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO2H and CH3CO2H, respectively. In neither case was a lag observed, which was unexpected in consideration of the kcat and Km parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Sub-stoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate

binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e., of DMN or DEN to an aldehyde). P4502 enzymes are found throughout nature and catalyze many reactions, most of which are mixed-function oxidations (4). The mammalian P450s are of considerable interest because of their roles in the metabolism of steroids, eicosanoids, drugs, chemical carcinogens, and other important molecules (5). The general mechanistic features of P450 reactions include substrate binding, reduction to the ferrous state, binding of O2, addition of a second electron, protonation, and rearrangement to generate a reactive iron-oxygen complex poised near the substrate (6, 7). The active complex can be described as a formal FeO3+ entity, with similarity to Compound I of peroxidases, which can be used to rationalize most reactions (7-9), although some alternate possibilities can also be considered. A generally accepted mechanism for many P450 oxidations involves the abstraction of a hydrogen atom by the FeO3+ entity, followed by “oxygen rebound” to yield a hydroxylated product (10). Among the chemical carcinogens activated by mammalian P450s are N,N-

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.088039The latest version is at JBC Papers in Press. Published on January 8, 2010 as Manuscript M109.088039

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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dialkylnitrosamines (also called N-nitrosodialkylamines) (11), including those found in tobacco products and also the simple nitrosamines N,N-dimethylnitrosamine (DMN)2 and N,N-diethylnitrosamine (DEN)2 (12). The mechanism of activation is agreed to involve α-hydroxylation of the nitrosamine in most cases (Fig. 1). The process is generally accepted to involve hydrogen atom transfer instead of the alternate 1e¯ oxidation implicated for some amines (7, 8, 13, 14) because of the high oxidation potential of the nitrogen atom due to nitrosation. Rearrangement of an α-hydroxy nitrosamine results in the formation of an aldehyde and an alkyl diazohydroxide, the latter of which can alkylate DNA (possibly via an alkyl nitrenium ion or carbocation) (Fig. 1) (11). This alkylation of DNA is generally accepted to be the basis of the tumor-initiating ability of these nitrosamines. In 1973 Keefer et al. (15) reported that perdeuterated DMN caused considerably fewer liver tumors in rats than protiated DMN. The deuterated material also yielded fewer methylated DNA adducts (16). Subsequent in vitro experiments with rat liver microsomes indicated that the conversion of DMN to HCHO was characterized by an increased Km but little change in Vmax (17-19). One of the enzymes involved in the oxidation of DMN is P450 2E1 (20, 21). This enzyme also oxidizes ethanol to acetaldehyde (22, 23), characterized by a non-competitive intermolecular deuterium isotope effect of 5 on the Km but none on kcat with human P450 2E1 (24). These results were explained by the presence of a rate-limiting step following the formation of the product acetaldehyde, as clearly documented by the observed pre-steady-state kinetic burst of product formation. P450 2E1 indicated that the enzyme also oxidizes the first product, acetaldehyde, to acetic acid (25-27). Pulse chase experiments and fitting to kinetic models indicated that although P450 2E1 does not have a high intrinsic activity for oxidizing

acetaldehyde, the kinetic course of ethanol oxidation yielded limited exchange of the intermediate acetaldehyde with the medium (27). We extended the previous work with ethanol to DMN because the original carcinogenesis studies on had been done with this compound (15, 16). P450 2A6 was examined because of several general catalytic and other similarities with P450 2E1 (5, 28, 29) and the considerable amount of structural (30) and kinetic (31) information about this enzyme. P450 2A6 oxidizes both DMN and DEN, as does P450 2E1; the catalytic efficiency of P450 2A6 is higher than that of P450 2E1 for DEN oxidation (28, 32). Several aspects of the design of the earlier P450 2E1 experiments with ethanol (24, 27) were used as a framework for the present study, including the kinetic deuterium isotope effects. Our results show kinetic deuterium isotope effects on both kcat and Km for the oxidation of DMN to formaldehyde, but the intrinsic isotope effect for DEN oxidation to acetaldehyde is considerably lower and is attenuated in non-competitive intermolecular experiments. As in the overall oxidation of ethanol to acetic acid by P450 2E1 (27), a fraction of the aldehyde (or α-hydroxy nitrosamine) is not released from the enzyme prior to further oxidation to the carboxylic acid. These results have implications in considerations of the activation of carcinogenic N-nitrosodialkylamines by P450 2A6 but also more generally in multi-step reactions catalyzed by P450 enzymes.

Experimental Procedures

Chemicals—DMN, DEN, diacetamide, N-ethylacetamide, acetaldoxime, acetaldehyde, [d6]-dimethylamine·HCl, methyl isothiocyanate, LiAlD4, NaBD4, oxalic acid·(H2O)2, and 4´-nitrophenacyl bromide (95%) were purchased from Sigma-Aldrich


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Chemical Co. (St. Louis, MO). Di-[2,2´-d6]-ethylamine was purchased from CDN isotopes (Pointe-Claire, Quebec, Canada). NaNO2 and potassium oxalate were obtained from Fisher Scientific Co. (Pittsburgh, PA); sodium diformylimide was a product of TCI America (Portland, OR). Tetrahexylammonium·HSO4 (99%) was purchased from Fluka (Buchs, Switzerland). [14C]-Formaldehyde (17.5 mM in H2O, 57 mCi mmol-1) and [14C]-sodium formate (18.9 mM in C2H5OH/H2O, 53 mCi mmol-1) were obtained from American Radiolabeled Chemicals (St. Louis, MO). [14C]-DMN (1.75 mM in H2O, 57 mCi mmol-1) was purchased from Moravek Biochemicals (Brea, CA). Aqueous solutions of [14C]-HCHO and [14C]-DMN were purified (from acid contaminants) prior to use by passing them through BakerbondTM disposable quaternary amine 3-ml SPE columns (J.T. Baker, Phillipsburg, NJ) (27).

Synthesis—Unlabeled formaldehyde was synthesized from paraformaldehyde (Eastman, Rochester, NY) to avoid complications resulting from organic solvents contained in commercially available formaldehyde solutions. A round-bottom flask containing 10 g paraformaldehyde was heated to 125 °C and purged gently with dry N2 gas; the exiting vapors were passed through a bubbler containing 100 ml of H2O. The aqueous trap solution, containing absorbed monomeric formaldehyde, was passed through a Bakerbond quaternary amine 3-ml SPE column and the formaldehyde was quantified following derivatization with 2,4-dinitrophenylhydrazone and HPLC (vide infra).

The general approach to synthesis of deuterated nitrosamine substrates involved LiAlD4 reduction of various precursors to amines, followed by nitrosation (supplemental Figs. S1 and S2). The water solubility and volatility of the intermediates and products were issues in limiting the scale of the

reactions. Also, final purity of the labeled DMN and DEN was not acceptable without distillation (because of these limitations in the workup of materials after chromatography), limiting the scale of the synthetic work to ≥ 0.5 g. The identity and purity of all substrates was established by NMR and MS (supplemental Figs. S3, S4). Several of the compounds show complex NMR proton splitting due to the E/Z character imposed by the nitroso group, consistent with earlier literature (33) (supplemental Figs. S3, S4). All deuterated nitrosamine derivatives (vide infra) were obtained as mixtures of E- and Z- isomers and were >97% isotopically enriched at the site(s) of modification as judged by MS and NMR spectroscopy (33). Detailed syntheses and analytical data for all nitrosamines are included in the Supplementary Data section, including N-nitrosodi-[1,1´-d6]-methylamine (d3d3-DMN), N-nitrosodi-[1,1´-13C]-methylamine ([13C]-DMN), N-nitrosodi-[1-d3]-methylamine (d3d0-DMN), N-nitrosodi-[1-d2,1´-d2]-methylamine (d2d2-DMN), N-nitrosodi-[1-d2]-ethylamine (d2d0-DEN), N-nitrosodi-[1,1´-d2]-ethylamine (d1d1-DEN), N-nitrosodi-[2-,2’-d3]-ethylamine (d3d3-DEN), and N-nitroso-2-ethylaminoethanol (2-hydroxy DEN).

Enzymes—P450 2A6 was expressed from a pCW plasmid in Escherichia coli DH5α (31, 34, 35) . The yield of whole cell P450 hemoprotein expression was ~200 nmol l-1, as determined by Fe2+-CO vs. Fe2+ difference spectra (36). P450 2A6 was purified to electrophoretic homogeneity from solubilized 2A6 membrane fractions as described previously (31, 34, 35) using a combination of ion-exchange (DEAE-Sephacel) and Ni2+-nitrilotriacetate chromatography. Recombinant rat NADPH-P450 reductase was expressed in E. coli and purified as described elsewhere (37). Recombinant human cytochrome b5 was expressed in E. coli JM109 cells from a


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plasmid (pSE420(Amp)) provided by Satoru Asaki (Takeda Pharmaceuticals, Osaka, Japan). The protein was purified to electrophoretic homogeneity using modification of the DEAE-cellulose and other chromatography methods described elsewhere (31, 38). Spectroscopy—NMR spectra were recorded using a Bruker 300 MHz spectrometer in the Vanderbilt facility. UV-visible spectra were acquired using an OLIS/Aminco DW2a instrument (OLIS, Bogart, GA). Mass spectra of synthetic products were recorded in the Vanderbilt facility using a Thermo-Finnigan TSQ-7000 instrument (ThermoFinnigan, Sunnydale, CA). LC-MS work was done with a Thermo LTQ instrument or a Waters Synapt mass spectrometer using an Acquity UPLC (Waters, Milford, MA), except for the competitive experiments (inter- and intra-molecular), which were done by LC-MS using the TSQ-7000 instrument.

Assays: General—Aldehydes were quantified by HPLC/UV analysis of the 2,4-dinitrophenylhydrazones as described previously (24, 39), with some modification. The sensitivity of the assays was improved by purification of some of the reagents and by removal of glycerol from enzymes (by dialysis immediately prior to use), because commercial glycerol was found to be contaminated with formaldehyde. Dansyl hydrazones were used in some LC-MS analyses because of the low (ESI and APCI) ionization efficiencies of HCHO- and CH3CHO-derived DNPH hydrazone derivatives. Applying the analytical methods described here to the analysis of unlabeled substrates, we were able to quantitate acetic acid in extracts of enzyme product mixtures, but not formic acid, at the level of sensitivity required for this work. Therefore we used 14C-

labeled substrates and measured [14C]-formic acid utilizing ion-exchange SPE columns. Nitrosamine N-Dealkylation Assays—Typical steady-state dealkylation reactions included 400 pmol 2A6, 800 pmol NADPH-P450 reductase, 400 pmol cytochrome b5, 20 µg DLPC, and varying concentrations of the nitrosamine substrate in 0.34 ml of 50 mM potassium phosphate buffer (pH 7.4). Reaction vials (clear glass, 1-dram) were sealed with Teflon-lined rubber septa because of the volatility of the substrates. Reconstituted enzyme solutions (P450 2A6, NADPH-P450 reductase, and cytochrome 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 the phospholipid (DLPC), to minimize complications arising from residual aldehydes present in glycerol. A 60-µl aliquot of an NADPH-generating system was used to start reactions (final concentrations of 10 mM glucose 6-phosphate, 0.5 mM NADP+, and 1 IU of yeast glucose 6-phosphate dehydrogenase ml-1 (40)). Incubations were generally done for 15 min in a shaking water bath at 37 °C, terminated by the sequential addition of 100 µl of 10% (w/v) ZnSO4·7H2O and 100 µl of saturated aqueous Ba(OH)2·8H2O, and centrifuged (2 × 103 × g). H2O (0.5 ml) and 2,4-dinitrophenylhydrazine (0.1%, w/v, in 6 M HCl) were added to the supernatant; derivatized aldehydes were then extracted into hexane (0.9 ml) with moderate shaking at room temperature for 60 min. The hexane layer was evaporated under a gentle stream of N2 at room temperature, and the residue was reconstituted in 100 µl of a mixture of H2O:CH3CN (1:1, v/v). Hydrazone products were analyzed by HPLC using a Zorbax octadecylsilane (C18) column (6.2 mm × 80 mm, 3 µm, MacModd, Chadds Ford, PA), with isocratic elution (2 ml min-1; H2O:CH3CN, 45:55, v/v), and UV detection


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(355 nm). Before use as a derivatization reagent, 2,4-dinitrophenylhydrazine was twice recrystallized from CH3OH/H2O (3:1), dried in vacuo, dissolved in 6 M HCl (0.1%, w/v), and washed multiple times with a hexane-CH2Cl2 mixture (7:3, v/v) to minimize interference resulting from residual hydrazone contamination. Hexanes and CH3CN were heated with and distilled from 2,4-dinitrophenylhydrazine to remove residual aldehydes. Assays involving competitive deuterium isotope effects were done by LC-MS analysis (APCI, negative ion) of derivatized formaldehyde or acetaldehyde (source temperature 550 °C; heated capillary voltage 20 V; heated capillary temperature 180 °C; ionization current 5 µA; sheath gas (N2) pressure 70 psi; auxiliary gas (N2) pressure 10 psi). For the DMN time course experiments, 0.5 nmol 2A6, 1.0 nmol NADPH-P450 reductase, 0.5 nmol cytochrome b5, 30 µM DLPC, NADPH (1 mM), and DMN (17 mM) were incubated in 0.2 ml of potassium phosphate buffer (100 mM, pH 7.4) at 37 °C for varying amounts of time. Reactions were initiated by adding NADPH. Incubations were terminated by adding 200 µl of cold CH3CN and centrifuged (2 × 103 × g) for 5 min. The supernatants were transferred to amber vials and 20 nmol of DCDO and 600 µl of a freshly prepared solution of dansylhydrazine (0.5 mg ml-1) in CH3CN containing 0.3% CH3CO2H (v/v) was added. The reaction mixture was incubated at room temperature for 30 min and dried under nitrogen. Finally, the residue was reconstituted in 100 µl of a mixture of H2O:CH3CN (3:1, v/v). Dansylated products were analyzed by LC-MS on a Waters Acquity UPLC system connected to either an LTQ (Thermo Fisher, Santa Clara, CA) or a Waters Synapt mass spectrometer using an Aquity UPLC BEH C18 octadecylsilane column (1.7 µm, 2.1 mm × 100 mm). LC conditions were as follows: buffer A

contained 10 mM NH4CH3CO2 and 2% CH3CN (v/v), and buffer B contained 10 mM NH4CH3CO2 and 95% CH3CN (v/v). The following gradient program was used, with a flow rate of 300 µl min-1: 0-6.5 min, linear gradient from 25% B to 100% B (v/v); 6.5-7.5 min, hold at 100% B; 7.5-8 min, linear gradient to 75% A (v/v); 8-10 min, hold at 75% A (v/v). The temperature of the column was maintained at 25 °C. Samples (20 µl) were infused with an auto-sampler. MS analyses were performed in the ES positive ion mode.

The results of steady-state kinetic experiments were fit to hyperbolic plots using GraphPad Prism (GraphPad, Dan Diego, CA) and parameters and standard errors were obtained with this program using non-linear regression. Acetic Acid Assays—P450 2A6 was reconstituted with cytochrome b5, NADPH-P450 reductase, and phospholipid as described for nitrosamine N-dealkylation assays (vide supra). Reactions containing acetaldehyde (0-3.5 mM) were initiated with an acetate-free NADPH-generating system (dialyzed enzymes), incubated for 15 min at 37 °C, and terminated by freezing in a dry ice-acetone bath. Following lyophilization to near dryness, acids were acylated in 300 µl of CH2Cl2 containing 4-nitrophenacyl bromide (0.5 mM) and tetrahexylammonium-HSO4 (0.03 mM). After 30 min at 50° C, the CH2Cl2 phase was transferred to a clean vial, evaporated to dryness, and reconstituted in CH3CN/H2O (1:1, v/v). Derivatized carboxylic nitrophenacyl esters were analyzed by HPLC using an ODS-AQ octadecylsilane (C18) hydrophilic end-capped column (YMC, 4.6 mm × 150 mm, 5 µm) with isocratic elution (1.4 ml min-1, H2O:CH3CN, 55:45, v/v) and UV detection (260 nm). Before use as a derivatization reagent, 4-nitrophenacyl bromide was twice recrystallized from CH3OH/H2O (3:1) and dried in vacuo. CH3CN


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and CH2Cl2 were passed multiple times through columns (3 cm × 20 cm) of basic alumina (60-325 mesh, Brockman activity I, Fisher) to minimize interference resulting from residual carboxylic acid contamination. Time course reactions for DEN oxidation were performed as described for nitrosamine N-dealkylation assays (vide supra) with the exception that reactions were terminated by the addition of 200 µl of cold CH2Cl2, To the reaction mixture 0.5 nmol of CD3CO2H was added and the products were derivatized with 4-nitrophenacyl bromide, as described above. The derivatized products were analyzed by LC-MS on a Waters Acquity UPLC system connected to an LTQ mass spectrometer (Thermo Fisher, Santa Clara, CA) using an Aquity UPLC BEH C18 octadecylsilane column (1.7 µm, 1 mm × 100 mm). LC conditions were as follows: buffer A contained 2% CH3CN and 0.01% HCO2H (v/v), and buffer B contained 95% CH3CN and 0.01% HCO2H (v/v). The following gradient program was used, with a flow rate of 150 µl min-1: 0-4.0 min, linear gradient from 95% A to 50% A; 4.0-4.5 min, linear gradient to 100% B; 4.5-5.5 min, hold at 100% B; 5.5-6 min, linear gradient to 95% A; 6-8 min, hold at 95% A. The temperature of the column was maintained at 50 °C; the injection volume was 20 µl. Samples were infused with an auto-sampler. MS analyses were performed in the negative ion mode. ESI conditions were as follows: source voltage 4 kV, source current 100 µA, auxiliary gas flow rate setting 37, sheath gas flow setting 16, capillary voltage -4 V, capillary temperature 380 °C, tube lens voltage -22 V.

[14C]-Formic Acid Assays—A concentrated (80 mM, 0.2 mCi mmol-1) stock solution of [14C]-formaldehyde was prepared by adding paraformaldehyde-derived (unlabeled) formaldehyde (vide supra) to commercially obtained [14C]-formaldehyde. P450 2A6 was reconstituted as described for

the nitrosamine N-dealkylation assays (vide supra). Reactions containing [14C]-formaldehyde (0-50 mM, 0.2 mCi mmol-1) or [14C]-DMN (17.5 mM, 0.18 mCi mmol-1) were initiated with an acetate-free NADPH-generating system (dialyzed enzymes), incubated for a specified time at 37 °C, terminated by the addition of 100 µl of 10% ZnSO4·7H2O (w/v), and centrifuged (2 × 103 × g). The supernatants were loaded onto BakerbondTM quaternary amine 3-ml SPE columns that had been washed with 6 ml of CH3OH and equilibrated with 10 ml of H2O. After loading, the columns were washed with 10 ml H2O to remove residual aldehyde or nitrosamine substrate. The [14C]-formic acid was eluted with 1.5 ml of 0.1 M HCl and radioactivity was measured by liquid scintillation spectrometry. Recovery was calibrated using a sodium [14C]-formate standard.

For time course experiments, reactions containing [14C]-DMN (17 mM, 0.3 mCi mmol-1) were initiated with an acetate-free NADPH-generating system (dialyzed enzymes) (40), incubated for various time and were quenched by heating at 80 °C for 5 min. [14C]-HCO2H was quantitated by HPLC using a liquid scintiallation flow counter and an Ultrasil amino column (Beckman, San Rafael, CA, 4.6 mm × 250 mm, 10 µm) with the following LC conditions: buffer A contained 20% CH3OH (v/v), and buffer B contained 80% CH3OH (v/v) and 500 mM NH4CH3CO2. The following gradient program was used, with a flow rate of 1 ml min-1: 0-10 min, 100% A; 10-25 min, linear gradient to 80% A (v/v); 25-26 min, linear gradient to 100% A; 26-30 min, hold at 100% A.

Pre-steady-state Kinetics—Pre-steady-

state kinetics of oxidation were performed in a quench-flow apparatus (model RFQ-3, KinTek Corp., Austin, TX). Experiments were done with [13C]-DMN (170 mM final concentration) in the case of DMN and with


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d3d3-DEN (1.4 mM final concentration) in the case of DEN. Reactions were performed at 37 °C and P450 2A6 (100 pmol) was reconstituted as described for the nitrosamine N-dealkylation assays (vide supra). Reactions were initiated with 1 mM NADPH for a period of time ranging from 5 ms to 10 s and quenched with CH3CN containing 1% CH3CO2H. Propionaldehyde was used as an internal standard. Aldehydes were derivatized with dansylhydrazine and quantitated by LC-MS as described for the DMN N-demethylation time course experiments (vide supra). In the case of [13C]-HCHO, the yields were corrected for the isotopic abundance of [13C]-HCHO resulting from background [12C]-HCHO.

For measuring oxidation of acetaldehyde to acetic acid, [14C]-CH3CHO (10 mM final concentration, 5 mCi mmol-1) was used. Reactions were performed as described for the [14C]-formic acid assays with the exception that 17% ZnSO4·7H2O (w/v) was used to quench the reaction. The product [14C]-CH3CO2H was purified using BakerbondTM quaternary amine SPE columns and detected by liquid scintillation spectrometry (vide supra).

Stopped-flow Kinetics—Analyses were

done using an OLIS RSM-1000 stopped-flow spectrophotometer in the rapid scanning monochromator mode, using the general procedures described earlier (31). Ligand binding assays were done at ambient temperature (23 °C). P450 2A6 (final concentration in mixing cell 2.0 µM) was mixed with either 12 mM DMN or 1.1 mM DEN (final concentrations) in 50 mM potassium phosphate buffer (pH 7.4). Kinetic analysis was done using the absorbance changes at 420 nm (decrease). Reduction assays were done with ferric P450 2A6 and a 2-fold molar excess of NADPH-P450 reductase under an anaerobic CO atmosphere using procedures described

earlier (31, 41). The rate of the increase in absorbance at 450 nm was used in measurements of rates. The ligands used (DMN, DEN, HCHO, and CH3CHO) are all volatile and therefore were added to the tonometers containing ferric P450 2A6 after degassing and equilibration (with CO) was finished by opening a tonometer port (under positive CO pressure) and adding the (neat) ligand from a 500-µl syringe (Gastight number 1750, Hamilton, Reno, NV), fitted with a ground joint for sealing to a port on the tonometer, which had been fitted with a screw-type driver and calibrated to deliver 5.7 µl per turn. A fitted glass cap was then placed back on the tonometer (under positive CO pressure), and the device was used to load one of the drive syringes of the stopped-flow instrument. In the case of ligand binding reactions, the reaction time was either 15 s or 150 ms. For reduction, reaction times were 3 s or 30 s. In both cases, 4 to 7 individual mixing reactions were used to derive rate constants (using the OLIS software, single exponential fits) and averaged. Pulse-chase Experiments—Experiments were done with [14C]-DMN (17 mM, 0.5 mCi mmol-1) in the case of DMN and with d3d3-DEN (0.14 mM) in the case of DEN. In both cases the reaction was initiated (37 °C) with 2.5 µM P450 2A6, 5.0 µM NADPH-P450 reductase, 2.5 µM cytochrome b5, 30 µM di-12 GLPC, and an NADPH-generating system (vide supra). After 1 or 2 min either (unlabeled) HCHO (2 mM, in the case of DMN) or CH3CHO (1.5 mM, in the case of the substrate DEN) was added and the reaction was allowed to proceed another 18 or 19 min (at 37 °C) (to 20 min total reaction time). In the case of DMN, the product [14C]-HCO2H was measured as described for the [14C]-formic acid assays (vide supra) with the exception that, after sample loading, the column was washed with 20% CH3OH (5 ml)


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and H2O (10 ml) to completely elute the residual [14C]-DMN substrate. The product [14C]-HCO2H was then eluted with 1 M HCl. In the case of DEN, the product CD3CO2H was derivatized with 4-nitrophenacyl bromide and analyzed by LC-MS, using the procedure described earlier. The non-deuterated CH3CO2H in the reagents served as an internal standard (~ 10-fold higher level). Assays were run in triplicate and the mean results were compared, with the extent of decrease due to the presence of the added aldehyde being indicative of the fraction of the unlabeled aldehyde (or its equivalent) that exchanged.

Results Preliminary Assays—P450 2A6-

catalyzed rates of conversion of DMN and DEN to formaldehyde and acetaldehyde, respectively, were constant up to at least 15 min, and this reaction time was used in most subsequent assays. Both reactions were highly dependent upon the presence of cytochrome b5: with DMN (used at 17 mM) the rate of HCHO production was 0.0030 (± 0.0015) s-1 in the absence of cytochrome b5 and 0.11 (± 0.05) s-1 in its presence. For DEN (used at 140 µM) the rates of acetaldehyde formation in the absence and presence of cytochrome b5 were 0.04 (± 0.002) and 0.15 (± 0.68) s-1, respectively. Accordingly all further assays included cytochrome b5. Initial parameters of kcat = 0.42 s-1 and Km = 15 mM were estimated for DMN oxidation to formaldehyde and kcat = 0.13 s-1 and Km = 0.15 mM for DEN oxidation to acetaldehyde. These results are consistent with literature indicating the preferential catalytic efficiency of P450 2A6 towards DEN (28, 32). With this preliminary information, concentrations of 17 mM DMN and 1 mM DEN were used for the competitive and intrinsic kinetic deuterium isotope effect studies (vide infra).

Intramolecular Kinetic Deuterium Isotope Effects—The Dk values (Table 1) form a basis for comparison with other isotope effects, which should show attenuation if specific steps contribute to rate limitation (3). In principle, comparisons of rates of C-H and C-D bond cleavage at a carbon atom substituted with both H and D should provide an estimate of Dk, the intrinsic isotope effect. These values, estimated by MS of the aldehyde products, were 10.2 (± 0.2) and 3.7 (± 0.2) for DMN and DEN, respectively.

Both of the values have caveats. The Dk value of 10 for the -CHD2 group(s) of DMN has the potential contribution of a geminal secondary isotope effect on the C-H value (42). In the general literature these are usually 1.0-1.2 (42-44), including the few cases in which they have been estimated for P450s (45, 46). Such secondary isotope effects are multiplicative, so with two deuteriums the contribution could be as high as 1.4. Thus, the true Dk for DMN oxidation could be as low as 10/1.4 = 7. The contribution of a geminal secondary kinetic isotope effect should be less in the case of the methylene in DEN (-CHD-), i.e. ≤ 1.2, so the Dk should be ≤ 3.5 and possibly ~ 3.3

We also measured the apparent kinetic hydrogen isotope effects for unsymmetrically deuterated DMN and DEN (Table 1). In principle, the comparison of these values with the estimates of the intrinsic kinetic isotope effects (vide supra) can provide an estimate of the ability of a substrate to turn within the active site of the enzyme (48-51). These values were as high as the values obtained with d2d2-DMN and d1d1-DEN (Table 1), within experimental error, and do not provide evidence for restricted motion in the active site, which would have the expected effect of attenuating these values. However, even if effects are not seen in such experiments they must be considered in light of the alternate interpretation that the substrates rapidly exchange with the medium, as opposed to


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rotating within the active site. The rates of binding of DMN and DEN to P450 2A6 were measured. Binding of either substrate to P450 2A6 yielded a “Type I” difference spectrum, with an apparent shift of low-spin iron (λmax 418 nm) to high-spin (λmax 390 nm). The apparent Kd was 12 mM for DMN and 1.1 mM for DEN (supplemental Figs. S5, S6). This change could be observed in a stopped-flow spectrophotometer and occurred at (first-order) rates of 80 (± 18) s-1 with 12 mM DMN and 73 (± 22) s-1 with 1.1 mM DEN (at 23 °C).4 These parameters are for the binding of ligands but do not directly reflect the “off” rates. The high Kd values prevent estimation of the dissociation rate by extrapolation to zero substrate concentration (31, 52). A dissociation rate was estimated indirectly by mixing a pre-formed P450 2A6-DEN complex with 4-phenylimidizole, which is presumed to occupy the same site but yields a different spectral complex.4 The rapid release of DMN and DEN was indicated by these experiments and the competitive intermolecular isotope effect results (vide infra). Competitive Intermolecular Kinetic Deuterium Isotope Effects—The competitive isotope effects for the mixtures of d0 and perdeuterated nitrosamines (Table 1) were within experimental error of the intramoleular values, the pseudo-intrinsic isotope effects. In addition, the values seen in the “mixed” experiments were just as high, with d0 substrate plus asymmetrically labeled nitrosamines (Table 1). If exchanges were slow, these values should also be lower.4

Non-competitive Intermolecular Kinetic Deuterium Isotope Effects—Comparison of the non-competitive intermolecular deuterium isotope effects with estimates of the intrinsic isotope effect (Dk) is a generally accepted approach to assess the extent to which the C-H bond-breaking step contributes to limiting an overall catalytic

reaction of an enzyme (2, 3, 54), particularly if physical steps such as substrate binding and release are not slow, as shown earlier for DMN and DEN. Deuterium substitution of DMN affected both kcat and Km (Table 2), with DV = 4.8 and DKm = 0.38. Both the DV and DK values have inherent error due to the difficulty in the estimation of kcat with d3d3-DMN; the D(V/K) value, which is considered to have less error based on visual observation of the tangents (Fig. 2), was ~13, within experimental error of the approximated intrinsic Dk (Table 1). When DEN oxidation was measured, DV and D(V/K) were near unity (Table 3), within experimental error. Limited Methyl (β) Hydroxylation of DEN—One possibility to consider is that P450 2A6 can generate alternate products when a C-D bond replaces C-H, which could affect the interpretation of the kinetic isotope effects. P450s can catalyze a reaction generating NO2¯ (55), although this is a relatively minor pathway. Another possibility is that deuterium substitution of the α-carbon of DEN could favor hydroxylation of the methyl group. We synthesized 2-hydroxy DEN, characterized it, and used it as an LC-MS standard to monitor its possible formation from DEN by P450 2A6. With d0 DEN the rate was < 0.004 min-1 (with a DEN concentration of 1 mM). With d2d2 DEN the rate was ~ 0.011 min-1. We conclude that methyl hydroxylation is not a major pathway, even when the α-carbon is deuterium substituted.

Comparisons of Product Formation with d0-, d3-, and d3d3-DMN—The lack of attenuation of kinetic isotope effects in the intermolecular competitive experiments (Table 1) and the results of the 4-phenylimidazole binding experiments4 indicate that P450 2A6 exchanges substrates rapidly, consonant with the results of studies with P450 2A6 and coumarin (31). One


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question is whether such exchange can occur at the stage of the actual oxygen complex (putative FeO3+).

The presence of a high isotope effect in the non-competitive intermolecular experiments (Table 1) permits the application of another type of experiment, which we have applied previously with P450 1A2 (51). To address the question of whether such exchange can occur at the stage of the actual oxygen complex (putative FeO3+) formation, we applied another type of experiment using DMN and two DMN analogues (DMN-d3 and DMN-d3d3). The presence of deuterium in the substrate is not sensed in an enzyme catalytic cycle until the chemistry of C-H(D) bond breaking begins. Because the C-D bond breaking step is relatively more difficult, the enzyme could (if the step were slow enough relative to exchange) dissociate the deuterated substrate, bind the protiated substrate, and oxidize the protiated substrate. The intermolecular non-competitive isotope effect, and the overall rate of product formation, would not be attenuated. However, if no exchange of the substrate can occur after complete activation of the FeO3+ complex, the rate of product formation should reflect the substrate isotopic composition, i.e. the amount of product formed from the oxidation of d0d3-DMN (HCHO plus DCDO) should be intermediate between the amounts formed from d0-DMN (HCHO) and d3d3-DMN (DCDO). When the experiment was done, the latter result was obtained (Fig. 3). A similar set of experiments using deuterated DEN analogues could not be done because the non-competitive intermolecular isotope effect was near unity (Table 2).

Oxidations of Aldehydes to Carboxylic Acids—Both HCHO and CH3CHO were substrates for P450 2A6 oxidation (Table 3). The oxidation of CH3CHO was much more efficient than that of HCHO, by a factor of 20-fold. The efficiencies (kcat/Km) for oxidations

of both aldehydes were ~ 10-fold less than for the oxidations of the corresponding nitrosamines to the aldehydes (Table 2). A non-competitive intermolecular kinetic isotope effect of 2.0 was observed for the P450 2A6-catalyzed acetaldehyde oxidation D(V/K) parameter, but not DV; the isotope effect was only on Km. A similar observation for acetaldehyde oxidation was made with human P450 2E1 (27). The P450 2A6-catalyzed oxidation of formaldehyde was not analyzed for an isotope effect because the assay of formic acid was not sufficiently sensitive without the use of 14C-labeled material.

Time Courses of Aldehyde and Carboxylic Acid Formation from DMN and DEN—Careful assays showed that DMN was oxidized to both HCHO and HCO2H in a linear course, over a period of 10 min (Fig. 4A). Similarly, oxidations of DEN to both CH3CHO and CH3CO2H were apparently linear (at least 15 min) (Fig. 4B, 4C). Most notably, no lag was observed in the formation of the carboxylic acid in either case.

Pre-steady-state kinetic analysis of the conversion of DMN and DEN to the respective aldehydes showed small but reproducible bursts in both cases, with 2-4% product formed in the rapid phase (Fig. 5). A small burst was also detected in the oxidation of CH3CHO (supplemental Fig. S8).

Spectrally-determined Binding of

Ligands to P450 2A6—The addition of DMN or DEN to (ferric) P450 2A6 produced a classic Type I heme Soret difference spectrum (vide supra), with the lower UV region obscured by the absorbance of the ligand (supplemental Figs. S5, S6). The estimated Kd values for DMN and DEN were 12 and 1.1 mM, respectively. The Type I spectral changes for the aldehydes were even weaker (supplemental Figs. S9, S10), with apparent Kd values of ~ 200 mM.


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Rates of P450 2A6 Reduction—Rates

of reduction of P450 2A6 have been shown to be slow in the absence of substrate and enhanced by the presence of the substrate coumarin (31). Preliminary anaerobic studies showed that all of the Na2S2O4-reducible P450 2A6, when DEN was present, was reduced within 60 s. Stopped-flow kinetics assays established that the rate of reduction of P450 2A6 was enhanced by the presence of DMN or DEN but not formaldehyde or acetaldehyde (supplemental Fig. S11).

NADPH Oxidation Rates and

Coupling Efficiency—In the absence of any added substrate, the reconstituted P450 2A6 system oxidized NADPH at the rate of 0.63 s-1

(i.e., 0.63 nmol NADPH s-1 (nmol P450)-1). With 17 mM DMN (Km concentration) the rate was 1.5 s-1 and with 0.14 mM DEN (Km concentration) the rate was 0.75 s-1. Under these conditions the rates of formation of the aldehydes with the same reagents were 0.05 and 0.075 s-1, respectively (Fig. 4). Thus, with DMN and DEN the efficiency of coupling to generate nitrosamine reaction products was 3 and 10%, respectively.

Pulse Chase Experiments—The lack

of lag phases in the production of carboxylic acids (Fig. 4) suggested that the aldehyde products of the nitrosamines might be retained by P450 2A6 and not in exchange with the medium. Accordingly pulse chase experiments were done, in which reactions were initiated with labeled DMN or DEN (concentration ~ Km). A large excess of the appropriate unlabeled aldehyde was added after 1-2 min, the reaction was quenched after 20 min, and the isotopic incorporation of the carboxylic acid products was measured by liquid scintillation spectrometry or LC-MS. The final ratios of HCHO/14C-HCHO and of CH3CHO/CD3CHO were ~ 16 and ~ 7, respectively, based on the total amount of

aldehydes formed in 20 min. If all of the intermediate aldehyde was in equilibrium (in each case), this approach should have eliminated all but 5-10% of the label in the recovered carboxylic acid. However, 80-90% of the formic acid and 40-60% of the acetic acid was labeled (Fig. 6).

Kinetic Modeling—An overall scheme

of the major events of the reactions is shown in Fig. 7, including known rates of some of the non-enzymatic reactions (56-59). If the aldehyde product dissociates from P450 2A6, then the rates of formation of the carboxylic acids (measured in Fig. 4) can be described by a model of a coupled reaction with separate kcat and Km values for the two steps (Tables 2, 3). The predicted time courses of formation of the aldehydes and carboxylic acids (see supplemental Fig. S12) were compared to the experimental values (from Fig. 4) in Fig. 8. Two striking features were clear: (i) the theoretical plots under-predict carboxylic acid formation, and (ii) the theoretical model predicts lag phases for carboxylic acid formation, which is intuitive due to the relatively high Km values for oxidation of the two aldehydes (Table 3).

A comprehensive but minimal kinetic model was developed utilizing KinTek Explorer® software. The model is necessarily complex because it must include multiple phenomena, including (i) normal events known to occur in P450 reactions, including separate substrate binding and oxygen activation steps, (ii) the irreversible loss of reduced oxygen species from the activated complex (vide supra) (60), (iii) the partial burst kinetics (Fig. 5), (iv) the lack of affinity of the aldehydes for the enzyme (supplemental Figs. S9, S10), (v) the use of the product as a substrate in the second reaction (Table 3), (vi) the non-competitive kinetic deuterium isotope effect seen with DMN but not DEN (Table 2), and (vii) the maximum rates possible for converting part of the substrate to product


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(Fig. 4). A minimal model was developed that is consistent with these data (Fig. 9), with S denoting the nitrosamine, P the aldehyde, and Q the carboxylic acid. Critical features included fitting the time courses of formation of the aldehyde and acid simultaneously, with a lack of a lag for the acid, as well as showing a partial kinetic burst and an isotope effect for DMN. The rate constants for ligand binding were set at 107 M-1 s-1 (consistent with other work with P450 2A6 (31) and accepted diffusion-limited rates of interaction of enzymes and ligands (44), with rate constants for ligand release balanced to fit the spectrally estimated Kd values (supplemental Figs. S5, S6, S9, S10). The rate constants for steps 1-4 were set not to be faster than the rapid formation of product by P450 2A6 (Fig. 5).

Fits are shown for DMN and DEN oxidations to aldehydes and acids in Fig. 10, using the rate constants shown in Table 4 for the steps in Fig. 9 (see also supplemental Fig. S13). The time courses of product formation fit the experimental data well, and partial bursts are predicted (as shown with the inset for DMN in Fig. 10B). Applying an intrinsic Dk of 12 for DMN N-dealkylation (Table 1) reduced the rate of HCHO production (v) by 3.5-fold, and applying an intrinsic Dk of 3 for DEN N-dealkylation (Table 1) reduced the rate of CH3CHO formation by only 1.2-fold (cf. Table 2).

Discussion The original impetus for this work was an in vivo study showing a strong deuterium isotope effect on the hepatocarcinogenicity of DMN (15). At that time none of the mammalian P450s had been characterized. Subsequently P450s 2A6 and 2E1 were identified as the major catalysts involved in the oxidation and bioactivation of short-chain N-nitrosamines, especially DMN and DEN (28, 32, 61). Nitrosamines should not be regarded only as xenobiotics, in that the in

vivo nitrosation of secondary amines is a well-established phenomenon (62-64) and considered to be of relevance in understanding human cancer etiology (65). Previous work on the stepwise oxidation of ethanol to acetic acid by human P450 2E1 identified an intrinsic kinetic deuterium isotope effect (expressed primarily in Km), burst kinetics, and a processive pathway (24, 27). In the oxidation of DMN and DEN by P450 2A6, some similar phenomena were seen and some of the same mechanisms seem applicable, although several features are more complex.

The best estimates for the intrinsic kinetic deuterium isotope effects for the oxidations of DMN and DEN by P450 2A6 are ~ 10 and ~ 3, respectively, as estimated from the non-competitive intramolecular studies (Table 1, lines 1 and 2), with caveats about the contribution of secondary isotope effects. Similar kinetic isotope effects were measured in a variety of competitive experiments with labeled DMN and DEN (Table 1), arguing that exchange of the nitrosamine substrates is very rapid, a conclusion supported by direct measurements of on-rates (70 to 80 s-1 with 12 mM DMN or 1.1 mM DEN at 23 °C) and by an experiment in which the dissociation rate of a P450 2A6-DEN complex was estimated by displacement of another ligand (4-phenylimidazole)4 (supplemental Fig. S7).

Major conclusions from the kinetic isotope effect work are that the isotope effect is much higher for oxidation of the methyl group (DMN) than the methylene group (DEN). Another major conclusion is that hydrogen atom abstraction is a rate-limiting step in the case of DMN but not DEN (Table 3, Fig. 2). A simple explanation for the difference may be the inductive effect and the inherent energy of breaking a methylene C-H bond compared to a methyl (e.g., 94.5 vs. 98 kcal mol-1 (66)).5 Several lines of investigation argue that these substrates can tumble and/or exchange rapidly (Table 1) but not after the


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P450 is in the activated state and poised for C-H bond cleavage (Figs. 3, 11).

One interesting aspect of this study is the observed processivity of oxidation. P450 2A6 oxidizes the nitrosamines to aldehydes (Table 2) and oxidizes both aldehydes (HCHO, CH3CHO) to carboxylic acids (Table 3). However, the aldehyde oxidations are not particularly efficient (Table 3), and combining the kcat and Km values for the individual oxidations yields predictions that have two major discrepancies with the experimental results (Figs. 4, 8): (i) with DEN and particularly DMN, the calculations based on the individual kcat and Km values underestimate carboxylic acid formation, and (ii) the predicted lag phases for carboxylic acid formation are not observed experimentally. The results of the pulse chase experiments with both aldehydes (Fig. 6) indicate that a dissociative model cannot explain the results on formation of carboxylic acids (Fig. 8). However, the aldehydes were found to have only low affinity for (ferric) P450 2A6 (supplemental Figs. S9, S10).

A kinetic model was developed that is consistent with most of the observed results. In order to deal with the lack of dissociation of the aldehyde(s), a solution used in our earlier studies on P450 2E1 (27) was invoked, namely that the P450 undergoes a conformational change upon binding substrate (see reviews of P450 crystal structures for physical evidence (68,69) and then relaxes its conformation after forming product. We postulate that the P450 2A6-aldehyde complex is left in a conformational state leading to catalysis, avoiding the need to release, re-bind, and then undergo the activating conformational change. Thus, the rate constants for events leading to catalysis in the second cycle compete with others in the “first” cycle (Fig. 9A). Another required feature is an equilibrium of an activated P450 2A6-substrate complex with an unproductive complex, added to explain the (pre-steady-

state) partial bursts observed for production of aldehydes (70-72). The abortive generation of reduced oxygen species (Fig. 9) was included in light of the low efficiency of NADPH coupling (vide supra). Any model must also have a C-H bond-breaking step that can account for the high kinetic deuterium isotope effects (for DMN) (Table 2). Finally, the model incorporates the spectrally-measured Kd values for the nitrosamines and aldehydes.6

Literature rates of non-enzymatic steps are shown in Fig. 7, including the rates of rearrangement of α-hydroxy nitrosamines to aldehydes and the hydration of aldehydes/dehydration of hydrated aldehydes. One initial consideration was that these phenomena might help explain the kinetic isotope effect results, but they do not help with the partial bursts and processivity, particularly if they are reasonable approximations of rate constants in the enzyme active site. One issue is the time needed for the breakdown of the α-hydroxy nitrosamine to the aldehyde (Fig. 7). Another issue, if one uses a classic hydrogen atom abstraction mechanism for P450 2A6 (Fig. 11A), is that the aldehyde would need to hydrate, a slow process, before hydrogen atom abstraction (Fig. 7). One means of circumventing this kinetic problem is to use the alternative peroxide mechanism (27, 76, 77) (Fig. 11B), avoiding the need for the slow hydration of CH3CHO (Fig. 7) (57, 58). (A third option could be 1e- oxidation of one of the hydroxyl groups (13, 78).) At this time we do not have definite evidence as to which of these pathways (Fig. 11) is operative (a small kinetic deuterium isotope effect was observed in a non-competitive experiment (Table 4) but this, by itself, does not have a single mechanistic interpretation).

A model (Fig. 9) was constructed based upon the seven points listed under Results, Kinetic Modeling. Several strengths of the model are: (i) the kinetic courses of aldehyde and carboxylic acid formation match


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the experimental results, with no lags observed for carboxylic acid formation in either case (Fig. 10); (ii) the same rate constants can be utilized for DMN and DEN (Table 4), modified only to match the apparent nitrosamine affinity (supplemental Figs. S5, S6); (iii) the model yields sub-stoichiometric bursts of product formation (Fig. 10) to match the observed results (Fig. 5); and (iv) a kinetic deuterium isotope effect was expressed in the case of DMN but not DEN (cf. Table 3). Several points should be made about the model. Eliminating JS (P450’ Fe3+-S), the unproductive complex in Fig. 9, yielded only a full kinetic burst (1 product/enzyme), not the partial burst. JP (P450’ Fe3+-P) mirrors JS (i.e., if there is an unproductive complex with the nitrosamine, one might be expected with the aldehyde as substrate), and if JP is eliminated (Fig. 9) the reaction is too fast and rate constants must be attenuated. If the F form (P450* Fe3+) of the enzyme (Fig. 9) is eliminated (F regenerated from a productive conformational change following substrate binding) then carboxylic acid formation is too slow, and the burst phase is not sub-stoichiometric. The step GS E + O (P450 FeO3+ H2O2 + H2O) is logical, in light of the observed uncoupling (vide supra); if these steps (GS, GP E + O) are dropped the model still fits but some rate constants must be attenuated. Nevertheless, the current model does have some deficiencies, including: (i) the predicted burst is sub-stoichiometric but does not completely fit the experimental results, and (ii) the predicted expressed non-competitive kinetic isotope effect (Table 2) is larger (D(V/K) 13 ± 5) than predicted by the model (3.2, vide supra). Further improvement may be in order, although the mechanism is

already fairly complex and we hesitate to include additional steps without justification.

Several P450s catalyze sequential oxidations in steroid metabolism (5, 7). P450 19A1 oxidizes androgens to estrogens with the accumulation of low concentrations of intermediates (79). P450 11B2 converts deoxycorticosterone to aldosterone without the dissociation of intermediates from the enzyme, as judged by pulse and quench experiments (80). Similar approaches led to the conclusion that in the oxidation of pregnenolone to dehydroepiandrosterone by P450 17A1 about 20% of the intermediate 17α-hydroxypregnenolone did not dissociate from the enzyme (81), although a study with P450 17A1 transfected into HEK-293 cells yielded a different conclusion (82). The literature with P450 19A1, the steroid aromatase, is controversial regarding processivity (83). Even less information is available about P450s that do not normally have defined physiological roles in steroid metabolism. Rat P450 2C11 oxidizes testosterone to 16α-hydroxyandrostenedione via androstenedione, and Sugiyama et al. (84) estimated that ~15% of the androstenedione intermediate does not dissociate in the process. The only other previous work in this area comes from our own laboratory with P450 2E1 and the conversion of ethanol to acetic acid via acetaldehyde (27); pulse experiments suggested that ~90% of the acetaldehyde did not dissociate in the process (vide supra). The processivity of P450 2E1 in nitrosamine oxidations has not been reported in the literature to date.

Acknowledgment–We thank K. Trisler for assistance in preparation of the manuscript.

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49, 1239-1249 64. Loeppky, R. N., Tomasik, W., Outram, J. R., and Feicht, A. (1982) IARC Sci. Publ.,

Monograph 41, Int. Agency Res. Cancer, Lyon, pp 41-56 65. Bartsch, H., Ohshima, H., Shuker, D. E. G., Pignatelli, B., and Calmels, S. (1990) Mut.

Res. 238, 255-267 66. Carey, F. A., and Sundberg, R. J. (1990) Advanced Organic Chemistry, Plenum Press,

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Chem. Soc. 119, 8925-8932 68. Poulos, T. L., and Johnson, E. F. (2005) in Cytochrome P450: Structure Mechanism, and

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69. Johnson, E. F., and Stout, C. D. (2005) Biochem. Biophys. Res. Commun. 338, 331-336 70. Spence, R. A., Anderson, K. S., and Johnson, K. A. (1996) Biochemistry 35, 1054-1063 71. Suo, Z., and Johnson, K. A. (1997) Biochemistry 36, 12459-12467 72. Furge, L. L., and Guengerich, F. P. (1999) Biochemistry 38, 4818-4825 73. Harrelson, J. P., Henne, K. R., Alonso, D. O., and Nelson, S. D. (2007) Biochem.

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Sci. U. S. A. 93, 4644-4648 78. Guengerich, F. P., Willard, R. J., Shea, J. P., Richards, L. E., and Macdonald, T. L.

(1984) J. Am. Chem. Soc. 106, 6446-6447 79. Grogan, J., Shou, M., Zhou, D., Chen, S., and Korzekwa, K. R. (1993) Biochemistry 32,

12007-12012 80. Imai, T., Yamazaki, T., and Kominami, S. (1998) Biochemistry 37, 8097-8104 81. Yamazaki, T., Ohno, T., Sakaki, T., Akiyoshi-Shibata, M., Yabusaki, Y., Imai, T., and

Kominami, S. (1998) Biochemistry 37, 2800-2806 82. Soucy, P., and Luu-The, V. (2000) Eur. J. Biochem. 267, 3243-3247 83. Fishman, J. (1982) Cancer Res. 42, 3277s-3280s 84. Sugiyama, K., Nagata, K., Gillette, J. R., and Darbyshire, J. F. (1994) Drug Metab.

Dispos. 22, 584-591

FOOTNOTES

* This work was supported by US National Institutes of Health grants R37 CA090426,

T32 ES007028 (M.W.C.), F32 ES012123 (M.W.C.), and P30 ES000267 and a Merck fellowship


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(G.C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

S The on-line version of this article (available at http://ww.jbc.org) contains summaries of synthetic schemes, details of synthesis of nitrosamine, 1H NMR spectra of DMN and DEN and isotopically labeled derivatives, spectral changes in P450 2A6 induced by ligands, rates of P450 2A6 reduction, and DynaFit and Explorer kinetic models and scripts.

‡To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 2200 Pierce Avenue, Nashville, TN 37232-0146. Tel: 615-322-2261; Fax: 615-322-3141; E-mail: [email protected]. 1Both authors contributed equally to this work.

2The abbreviations used are: APCI, atmospheric pressure chemical ionization; DMN, N,N-dimethylnitrosamine (N-nitrosodimethylamine); DEN, N,N-diethylnitrosamine (N-nitrosodiethylamine); DLPC, L-α-dilauroyl-sn-glycero-3-phosphocholine; ES, electrospray; HPLC, high performance liquid chromatography; HR, high resolution; LC, liquid chromatography (including both HPLC and UPLC); MS, mass spectrometry; P450, cytochrome P450 (also termed “heme-thiolate protein P450 (1)); UPLC, ultraperformance liquid chromatography. The conventions Dk = intrinsic kinetic deuterium isotope effect, DV = Hkcat/Dkcat, and D(V/K) = H(kcat/Km)/D(kcat/Km) of Northrop (2, 3) are used in the designation of kinetic hydrogen isotope effects (H: protium, D: deuterium).

3A more fundamental consideration is the prochirality of the methylene hydrogens. Work

on the oxidation of the more complex tobacco-specific nitrosamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (“NNK”) by Jalas et al. (47) has shown the stereoselective removal of the pro-R hydrogen at the 4-methylene carbon by recombinant mouse P450 2a4 and 2a5. The selective abstraction of an R vs. S methylene hydrogen atom would have the overall effect of changing the DEN value reported in line 2 of Table 1 from a non-competitive to a competitive experiment. If there is a significant attenuation of the kinetic isotope effect in going to a competitive experiment, then the interpretation is that the rate of exchange of the substrate (DEN) is a factor. However, the values for the competitive isotope effects measured subsequently in Table 1 aare nearly as large and within experimental error.

4One approach to the rate of exchange of ligands with ferric P450 2A6 involved a more direct kinetic analysis. For substrates with high affinity, the equation kobs = kon [S] + koff can be used for a simple two-state system (52) and has been applied to the binding of coumarin to P450 2A6 (31). However, the Km for DEN was ~ 0.15 mM (Table 3) and the spectrally estimated Kd (increase in absorbance at 390 nm, decrease at 420 nm) for binding DEN was ~ 1.1 mM (supplemental Fig. S6), so the kobs values would be expected to be too fast to measure by stopped-flow methods. We employed an alternative approach, with the assumption that DEN and the “Type II” (53) ligand 4-phenylimidazole occupy the same space, a concept supported by the available crystal structures of P450 2A6 bound to coumarin and 8-methoxypsoralen (30). A pre-


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formed P450 2A6·DEN complex was rapidly mixed (at 13 °C) with an excess of 4-phenylimidazole and the rate of the change in the spectrum (from a λmax 390 nm complex to a λmax 427 nm complex) was measured (0.86 ± 0.08 s-1) and compared to the rate of the formation of the latter complex in the absence of DEN (0.86 ± 0.12 s-1). Analysis of the data with the kinetic model k1

P450•DEN P450 + DEN k-1 k2

P450 + 4-phenylimidazole P450•4-phenylimidazole k-2 using Kd,DEN = k-1/k1 = 1100 µM (from a spectral titration) and Kd,4-phenylimidazole = 0.51 (± 0.10) µM (from spectral titration) and an experiment involving 5 mM DEN (5-fold > Ks) and 10 µM 4-phenylimidazole (20-fold > Ks) with the program DynaFit—assuming a simple competitive model—indicated that k1 and k2 (at 13 °C) were ~ 5 × 105 M-1 s-1 and thus k-1 must be > 500 s-1, which is consistent with the Kd (Kd,DEN = k-1/k1 = 5 × 102 M-1 s-1/5 × 105 M-1 s-1 ~ 1 mM) (see supplemental Fig. S7). From these results we conclude that the exchange of DEN and, by inference, DMN with ferric P450 2A6 is very rapid (~ 100 s-1) and is not an issue in the interpretation of the kinetic deuterium isotope effects.

5The values in reference (66) are general but the difference in dissociation energy between a methyl vs. methylene C-H bond is similar in toher literature summaries. We did not find specific bond energy values for DMN and DEN in our searches. Bond energies have been measured for the hydrogen in methylamine and ethylamine (67), with a difference of 3.8 kcal mol-1 that is very similar to the general difference of 3.5 kcal mol-1 cited in ref. (66). Of course, the bond energies are perturbed in the free amines relative to DMN and DEN due to the tendency of these amines to lose electrons.

6The crystal structures of P450 2A6 show only space for a single substrate (30,75), although none of the ligands is as small as DMN. Harrelson et al. (73, 74) have proposed models of P450 2A6 (and P450 2E1) with two ligands present (simultaneously) in the active site. These proposals are based upon some patterns of kinetic deuterium isotope effects seen with xylenes and also on abnormalities in Eadie-Hostee plots of steady-state kinetic results. However, the data points of Fig. 5 (also Supporting Information Table S3) of ref. (74) could be readily fit to standard hyperbolic plots (kcat 0.51 ± 0.02 min-1, Km 107 ± 15 µM; kcat 2.5 ± 0.1 min-1, Km 81 ± 12 µM; r2 0.98 in both cases) without the need to apply a Hill equation, and it should be noted that the only abnormal point in the Eadie-Hofstee plots were obtained at the lowest enzyme velocities (Fig. 5 of (74)). Accordingly we developed our models with only a single ligand in the active sit in light of limited evidence for more ligands.


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Table 1

Kinetic isotope effects for oxidation of DMN and DEN to aldehydes

Kinetic isotope effecta

Product

Substrate Formaldehyde Acetaldehyde

Intramolecular

CD2H-N(NO)-CD2Hb, c 10.2 ± 0.2

CH3CHD-N(NO)-CHDCH3d 3.7 ± 0.2

CH3-N(NO)-CD3c 15 ± 3

CH3CH2-N(NO)-CD2CH3 d 3.0 ± 0.1

Intermolecular e

CH3-N(NO)-CH3/CD3-N(NO)-CD3c 12.0 ± 0.7

CH3CH2-N(NO)-CH2CH3/CH3CD2-N(NO)-CD2CH3d 2.7 ± 0.2

CH3-N(NO)-CH3/CD3-N(NO)-CH3 c 11 ± 1

CH3CH2-N(NO)-CH2CH3/CH3CD2-N(NO)-CH2CH3d 3.6 ± 0.2

a Measured from MS by comparison of the expected peaks for individual isotopic products, with

statistical correction for 13C contributions.

b With statistical correction for a D/H ratio of 2.

c Substrate concentration 17 mM.

d Substrate concentration 1.0 mM.

e Substrate mixture (1:1).


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Table 2

Non-competitive intermolecular kinetic isotope effects for oxidation of DMN and DEN to

aldehydes

Substrate

Producta

kcat

(s-1)

Km

(mM)

kcat/Km

(M-1 s-1)

DV

D(V/K)

d0-DMN HCHO 0.45 ± 0.01 17 ± 1 26 ± 2

d3d3-DMN HCDO 0.092 ± 0.02b 44 ± 15 2.1 ± 0.9 4.8 ± 1.0b 13 ± 5

d0-DEN CH3CHO 0.14 ± 0.01 0.14 ± 0.02 1000 ± 120

d2d2-DEN CH3CDO 0.11 ± 0.01 0.12 ± 0.01 920 ± 70 1.2 ± 0.1 1.1 ± 0.2

a Measured by HPLC of 2,4-dinitrophenylhydrazones of the products, with UV detection.

b These values contain error because of the inability to saturate the enzyme, and the D(V/K) value

is a better (upper) estimate (i.e., ratios of tangents in Fig. 2).


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Table 3

Oxidation of aldehydes to carboxylic acids

Substrate

Product

kcat

(s-1)

Km

(mM)

kcat/Km

(M-1 s-1)

DV

D(V/K)

HCHO HCO2H 0.010 ± 0.001 2.1 ± 1.4 4.8 ± 3.3

CH3CHO CH3CO2H 0.12 ± 0.01 1.3 ± 0.3 92 ± 21

CH3CDO CH3CO2H 0.12 ± 0.01 2.7 ± 0.5 44 ± 10 0.97 ± 2.0 ± 0.6


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Table 4

Kinetic modeling of oxidations of DMN and DEN by P450 2A6

DMN DEN

Reaction step

(Fig. 9)

Reaction (Fig. 9B)

kforward kreverse kforward kreverse

1 E + S ES 107 1.5 × 105 107 104

2 ES FS 150 1.0 150 1.2

3 FS GS 6.0 _ 5.2 _

4 GS FP 1.0 0.3 4.1 0.8

5 FP EP 1.2 10 1.3 10

6 FS JS 3.0 1.2 7.0 1.0

7 GS E + O 1.5 — 1.0 —

8 E + P EP 107 2 × 106 107 2 × 106

9 FP GP 2.2 1.3 2.0 1.0

10 GP HQ 0.40 1.0 0.36 1.0

11 E + Q HQ 107 107 107 107

12 FP JP 57 2.5 57 2.4

13 GP E + O 1.0 — 1.0 —

All units are in M and s-1 (bimolecular reactions in M-1 s-1, for forward steps 1, 8, and 11). Rate constants are limited to two significant digits.


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FIGURE LEGENDS FIGURE 1. Oxidation of DMN and DEN by P450 2A6 and generation of reactive alkyl diazohydroxides and aldehydes. FIGURE 2. Noncompetitive intermolecular kinetic isotope effects for oxidation of DMN and DEN to aldehydes. A, DMN. B, DEN. The points are means (± range) for duplicate assays at each concentration indicated. In some cases the range was within the size of the point and is not shown. See Table 3 for estimated parameters. FIGURE 3. Comparison of rates of formaldehyde formation from d0d3-DMN with d0d0- and d3d3-DMN. FIGURE 4. Time courses of conversion of DMN and DEN to aldehydes and carboxylic acids. A, Conversion of DMN to HCHO and HCO2H. B, Conversion of DEN to CH3CHO and CH3CO2H. C, conversion of DEN to CH3CO2H, scale expanded for clarity The substrate concentration was 17 mM and 140 µM in Parts A and B, respectively, and the P450 2A6 concentration was 2.5 µM. FIGURE 5. Burst kinetics for the oxidations of nitrosamines to aldehydes by P450 2A6. A, DMN to HCHO. B, DEN (d3d3) to CH3CHO. C, expanded plot from part B. The products were measured by LC-MS analysis of the dansyl hydrazones. FIGURE 6. Pulse chase experiments. See Experimental Procedures for details. Reactions were started with labeled DMN or DEN (concentration ~ Km, Table 4) and, after 1 or 2 min, the appropriate unlabeled aldehyde was added in large excess over the calculated yield of labeled aldehyde (Fig 4). FIGURE 7. Scheme of enzymatic and non-enzymatic events in nitrosamine oxidation. Rates of the non-enzymatic reactions are from the literature (56-59). The Kd values for DMN and DEN were estimated using spectral titrations (supplemental Figs. S5, S6). E: enzyme (P450 2A6); RCHO: aldehyde; RCH(OH)2: hydrated aldehyde; RCO2H: carboxylic acid. FIGURE 8. Comparison of experimental time courses of product formation with predictions based on kcat and Km values for individual reactions. See supplemental Fig. S12 for DynaFit script and differential equations used in the modeling. FIGURE 9. Scheme used for kinetic modeling of nitrosamine oxidations. A, scheme with P450 conformations and valence states. S: nitrosamine, P: aldehyde, Q: carboxylic acid. B, simplified scheme used in analysis (Table 4, supplemental Fig. S13). E: P450 2A6; S: nitrosamine; F: conformational state of E utilized in catalysis; J: non-productive conformation of E; G: F with active Fe-O entity poised for oxidation; P: aldehyde; Q: carboxylic acid; O, reduced oxygen species (H2O2 or H2O); H: sum of E and F forms of P450 2A6 (combined for simplification because the conversion of F to E is not critical to this series of events).


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FIGURE 10. Fitting of data points for time courses of nitrosamine oxidation to kinetic model of Fig. 9. A, DMN to HCHO and HCO2H. B, DEN to CH3CHO and CH3CO2H. The values of the rate constants used are shown in Table 4, with the numbers of the reaction steps (Fig. 9) corresponding to each rate constant. FIGURE 11. Alternate oxidation mechanisms for aldehydes (27, 77).


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NNH3C CH3

OP450

2A6

NNH3C CH2

O

O—H HCHO + CH3-N=N-OH

P4502A6

HCO2H [CH3+] + N2 + OH-

A

B NNH3CH2C CH2CH3

OP450

2A6

NNH3CH2C CHCH3

O

CH3CHO + CH3CH2-N=N-OH

P4502A6

CH3CO2H [ CH3CH2+] + N2 + OH-

O—H

Fig. 1


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Fig. 2


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Fig. 3

sd0d0

d0d3

d3d3


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Fig. 4

C


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Fig. 5

C by guest on April 5, 2018

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Fig. 6

Relative yield acid (%)compared to noaddition of carrier aldehyde

DMN DEN


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E +

E•

E + RCHO

E•RCHO

Fig. 7

E + RCO2H

E•RCO2H

0.1 s-1 (HCHO)10 s-1 (CH3CHO)

10 s-1 (HCHO)0.005 s-1 (CH3CHO)

R' NN O

R

OH

R' NN O

R

OH E + RCH(OH)2

E•RCH(OH)2

0.0033 s-1(HCHO)0.005 s-1 (CH3CHO)

E +

E•R' N

N O

R

R' NN O

RKdDMN 12 mM

DEN 1.1 mM[O] [O]


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Time, min

B

C

experimental

theoretical

theoretical

experimental

DMN HCO2H

DEN CH3CO2H

Fig. 8

A


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P450 Fe3+

1

2

1034

6

9

5

8

13

7

12

11

Fig. 9

P450 Fe3+•S

P450* Fe3+•S

P450' Fe3+•S

P450: ground stateP450*: active conformationP450': an inactive conformation

P450* FeO3+•S

P450 Fe3+

+ H2O2 + H2O

P450 Fe3+

+ H2O2 + H2O

P450* Fe3+•P

P450 Fe3+•P

P450' Fe3+•P

P450* FeO3+•P

P450 Fe3+•Q

1st

cycle

2nd cycle

A

BE

ES

GS

EPGP

HQ

E + O

JP JS

1

2

1034

6

9

5

8

13

7

12

11

FSFP

E + O


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Fig. 10

Time, s


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AHCOH

OH

FeV=O FeIV—OH

COH

OH•

FeIV—OH FeIII

C OHOH

CO

OH

BC

O

H

FeIII—O-O-

CH

OHO O FeIII

CO

OH

FeIII—OH

CO

OH

FeIII + OH-

OH

Fig. 11


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Goutam Chowdhury, M. Wade Calcutt and F. Peter Guengerichoxidation to aldehydes and carboxylic acids and analysis of reaction steps

Oxidation of N-nitrosoalkylamines by human cytochrome p450 2A6. Sequential

published online January 8, 2010J. Biol. Chem.

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