Oxidation of N-nitrosoalkylamines by human cytochrome p450 2a6: Sequential oxidation to aldehydes and carboxylic acids and analysis of reaction steps

Article abstract of DOI:10.1074/jbc.M109.088039

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 (^Dk_app ～ 10), which was highly expressed in a variety of competitive and non-competitive experiments. The ^Dk_app 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 considering the k_cat and K_m 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. Substoichiometric 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).

Full text of DOI:10.1074/jbc.M109.088039

Enzymology:
Oxidation of N-Nitrosoalkylamines by
Human Cytochrome P450 2A6:
SEQUENTIAL OXIDATION TO
ALDEHYDES AND CARBOXYLIC
ACIDS AND ANALYSIS OF REACTION
STEPS
Goutam Chowdhury, M. Wade Calcutt and F.
Peter Guengerich
J. Biol. Chem. 2010, 285:8031-8044.
doi: 10.1074/jbc.M109.088039 originally published online January 8, 2010
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 11, pp. 8031–8044, March 12, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Oxidation of N-Nitrosoalkylamines by Human Cytochrome
P450 2A6
SEQUENTIAL OXIDATION TO ALDEHYDES AND CARBOXYLIC ACIDS AND ANALYSIS OF
□
S
REACTION STEPS*
Received for publication, November 25, 2009, and in revised form, December 26, 2009 Published, JBC Papers in Press, January 8, 2010, DOI 10.1074/jbc.M109.088039
Goutam Chowdhury1, M. Wade Calcutt1, and F. Peter Guengerich2
From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-0146
Cytochrome P450 (P450) 2A6 activates nitrosamines, includ-
ing N,N-dimethylnitrosamine (DMN) and N,N-diethylnitros-
amine (DEN), to alkyl diazohydroxides (which are DNA-alkylat-
ing 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 ϳ 10), which
was highly expressed in a variety of competitive and non-com-
tions (4). The mammalian P450s are of considerable interest
because of their roles in the metabolism of steroids, eico-
sanoids, 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, the addition of a second electron, pro-
tonation, and rearrangement to generate a reactive iron-ox-
ygen complex poised near the substrate (6, 7). The active
complex can be described as a formal FeO3ϩ entity, with sim-
ilarity to Compound I of peroxidases, which can be used to
rationalize most reactions (7–9), although some alternate pos-
sibilities can also be considered. A generally accepted mecha-
nism for many P450 oxidations involves the abstraction of a
hydrogen atom by the FeO3ϩ entity, followed by “oxygen
rebound” to yield a hydroxylated product (10).
D
petitive experiments. The kapp for DEN was ϳ3 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 consid-
ering the kcat and Km parameters measured for oxidation of
DMN and DEN to the aldehydes and for oxidation of the alde-
hydes 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 (unla-
beled) aldehydes in the oxidations of DMN and DEN to carbox-
ylic acids. Substoichiometric 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 oxida-
tion (i.e. of DMN or DEN to an aldehyde).
Among the chemical carcinogens activated by mammalian
P450s are N,N-dialkylnitrosamines (also called N-nitrosodial-
kylamines) (11), including those found in tobacco products and
also the simple nitrosamines N,N-dimethylnitrosamine (DMN)
and N,N-diethylnitrosamine (DEN) (12). The mechanism of
activation is agreed to involve ␣-hydroxylation of the nitros-
amine in most cases (Fig. 1). The process is generally accepted
Ϫ
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 nitro-
sation. Rearrangement of an ␣-hydroxynitrosamine results in
the formation of an aldehyde and an alkyl diazohydroxide, the
latter of which can alkylate DNA (possibly via an alkyl nitre-
P4503 enzymes are found throughout nature and catalyze nium ion or carbocation) (Fig. 1) (11). This alkylation of DNA is
many reactions, most of which are mixed function oxida- generally accepted to be the basis of the tumor-initiating ability
of these nitrosamines. In 1973, Keefer et al. (15) reported that
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grants R37 CA090426 and T32 ES007028 (to M. W. C.), F32 ES012123 (to
perdeuterated DMN caused considerably fewer liver tumors in
rats than protiated DMN. The deuterated material also yielded
M. W. C.), and P30 ES000267 (to G. C.). This work was also supported by a
fewer methylated DNA adducts (16). Subsequent in vitro exper-
Merck fellowship (to G. C.).
iments with rat liver microsomes indicated that the conversion
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S13, including 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.
of DMN to HCHO was characterized by an increased Km but
little change in Vmax (17–19).
1 Both authors contributed equally to this work.
methylamine); DEN, N,N-diethylnitrosamine (N-nitrosodiethylamine);
HPLC, high performance liquid chromatography; LC, liquid chromatogra-
phy (including both HPLC and UPLC); MS, mass spectrometry; UPLC, ultra-
performance liquid chromatography. The conventions Dk ϭ intrinsic
kinetic deuterium isotope effect, DV ϭ Hkcat/Dkcat, and D(V/K) ϭ H(kcat/Km)/
D(kcat/Km)ofNorthrop(2, 3)areusedinthedesignationofkinetichydrogen
isotope effects (H, protium; D, deuterium).
2 To whom correspondence should be addressed: Dept. of Biochemistry and
Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638
RobinsonResearchBldg.,2200PierceAve.,Nashville,TN37232-0146.Tel.:615-
322-2261; Fax: 615-322-3141; E-mail: f.guengerich@vanderbilt.edu.
3 The abbreviations used are: P450, cytochrome P450 (also termed “heme-
thiolate protein P450” (1)); DMN, N,N-dimethylnitrosamine (N-nitrosodi-
MARCH 12, 2010•VOLUME 285•NUMBER 11
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Sequential Nitrosamine Oxidation by P450 2A6
isothiocyanate, LiAlD4, NaBD4, oxalic
acidꢀ(H2O)2, and 4Ј-nitrophenacyl
bromide (95%) were purchased
from Sigma. Di-[2,2Ј-d6]ethylamine
was purchased from CDN Isotopes
(Pointe-Claire, Quebec, Canada).
NaNO2 and potassium oxalate were
obtained from Fisher; sodium di-
formylimide was a product of TCI
America (Portland, OR). Tetra-
hexylammoniumꢀHSO4 (99%) was
purchased from Fluka (Buchs, Swit-
zerland). [14C]Formaldehyde (17.5
mM in H2O, 57 mCi mmolϪ1) and
[14C]sodium formate (18.9 mM in
FIGURE 1. Oxidation of DMN and DEN by P450 2A6 and generation of reactive alkyl diazohydroxides and
aldehydes. A, DMN; B, DEN.
Ϫ1
C2H5OH/H2O, 53 mCi mmol
)
were obtained from American
Radiolabeled Chemicals (St. Louis, MO). [14C]DMN (1.75 mM
in H2O, 57 mCi/mmol) was purchased from Moravek Bio-
chemicals (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 Inc.) (27).
One of the enzymes involved in the oxidation of DMN is
P450 2E1 (20, 21). This enzyme also oxidizes ethanol to acetal-
dehyde (22, 23), characterized by a non-competitive intermo-
lecular 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, acet-
aldehyde, 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).
Synthesis—Unlabeled formaldehyde was synthesized from
paraformaldehyde (Eastman Kodak Co.) to avoid complica-
tions resulting from organic solvents contained in commer-
cially available formaldehyde solutions. A round-bottom
flask containing 10 g of 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 form-
aldehyde, was passed through a Bakerbond quaternary amine
3-ml SPE column, and the formaldehyde was quantified follow-
ing derivatization with 2,4-dinitrophenylhydrazone and HPLC
(see below).
We extended the previous work with ethanol to DMN
because the original carcinogenesis studies on ethanol 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 deute-
rium isotope effects.
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 work up
of materials after chromatography), limiting the scale of the
synthetic work to Ն0.5 g. The identity and purity of all sub-
strates were established by NMR and MS (supplemental Figs.
S3 and 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) (supple-
mental Figs. S3 and S4). All deuterated nitrosamine derivatives
(see below) were obtained as mixtures of E- and Z-isomers
and were Ͼ97% isotopically enriched at the site(s) of modifica-
tion, as judged by MS and NMR spectroscopy (33). Detailed
syntheses and analytical data for all nitrosamines are included
in the supplemental material, including N-nitrosodi-[1,1Ј-
d6]methylamine (d3d3-DMN), N-nitrosodi-[1,1Ј-13C]methyl-
amine, N-nitrosodi-[1-d3]methylamine, N-nitrosodi-[1-d2,1Ј-
Our results show kinetic deuterium isotope effects on both
k
cat 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 inter-
molecular experiments. As in the overall oxidation of ethanol to
acetic acid by P450 2E1 (27), a fraction of the aldehyde (or
␣-hydroxynitrosamine) is not released from the enzyme prior
to further oxidation to the carboxylic acid. These results have
implications in consideration of the activation of carcinogenic
N-nitrosodialkylamines by P450 2A6 but also more generally in
multistep reactions catalyzed by P450 enzymes.
EXPERIMENTAL PROCEDURES
Chemicals—DMN, DEN, diacetamide, N-ethylacetamide, d2]methylamine, N-nitrosodi-[1-d2]ethylamine (d2d2-DMN),
acetaldoxime, acetaldehyde, [d6]dimethylamineꢀHCl, methyl N-nitrosodi-[1,1Ј-d2]ethylamine (d2d0-DEN), N-nitrosodi-
8032 JOURNAL OF BIOLOGICAL CHEMISTRY
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Sequential Nitrosamine Oxidation by P450 2A6
[2,2Ј-d3]ethylamine (d1d1-DEN), and N-nitroso-2-ethylamino- line), to minimize complications arising from residual alde-
ethanol (2-hydroxy-DEN).
hydes present in glycerol. A 60-␮l aliquot of an NADPH-gen-
Enzymes—P450 2A6 was expressed from a pCW plasmid erating system was used to start reactions (final concentrations
ϩ
in Escherichia coli DH5␣ (31, 34, 35). The yield of whole cell of 10 mM glucose 6-phosphate, 0.5 mM NADP , and 1 IU/ml
P450 hemoprotein expression was ϳ200 nmol/liter, as de- yeast glucose 6-phosphate dehydrogenase (40)). Incubations
termined by Fe2ϩ-CO versus Fe2ϩ difference spectra (36). were generally done for 15 min in a shaking water bath at 37 °C,
P450 2A6 was purified to electrophoretic homogeneity from terminated by the sequential addition of 100 ␮l of 10% (w/v)
solubilized 2A6 membrane fractions as described previously ZnSO4ꢀ7H2O and 100 ␮l of saturated aqueous Ba(OH)2ꢀ8H2O
(31, 34, 35) using a combination of ion exchange (DEAE- and centrifuged (2 ϫ 103 ϫ g). H2O (0.5 ml) and 2,4-dinitro-
Sephacel) and Ni2ϩ-nitrilotriacetic acid chromatography. phenylhydrazine (0.1% (w/v) in 6 M HCl) were added to the
Recombinant rat NADPH-P450 reductase was expressed in supernatant; derivatized aldehydes were then extracted into
E. coli and purified as described elsewhere (37). Recombinant hexane (0.9 ml) with moderate shaking at room temperature for
human cytochrome b5 was expressed in E. coli JM109 cells from 60 min. The hexane layer was evaporated under a gentle stream
a plasmid (pSE420(Amp)) provided by Satoru Asaki (Takeda of N2 at room temperature, and the residue was reconstituted in
Pharmaceuticals, Osaka, Japan). The protein was purified to 100 ␮l of a mixture of H2O/CH3CN (1:1, v/v). Hydrazone prod-
electrophoretic homogeneity using modification of the DEAE- ucts were analyzed by HPLC using a Zorbax octadecylsilane
cellulose and other chromatography methods described else- (C18) column (6.2 mm ϫ 80 mm, 3 ␮m; MacModd, Chadds
where (31, 38).
Ford, PA), with isocratic elution (2 ml/min; H2O/CH3CN,
Spectroscopy—NMR spectra were recorded using a Bruker 45:55, v/v), and UV detection (355 nm). Before use as a derivat-
300-MHz spectrometer in the Vanderbilt facility. UV-visible ization reagent, 2,4-dinitrophenylhydrazine was twice recrys-
spectra were acquired using an OLIS/Aminco DW2a instru- tallized from CH3OH/H2O (3:1), dried in vacuo, dissolved in
ment (OLIS, Bogart, GA). Mass spectra of synthetic products 6 M HCl (0.1%, w/v), and washed multiple times with a hexane/
were recorded in the Vanderbilt facility using a Thermo-Finni- CH2Cl2 mixture (7:3, v/v) to minimize interference resulting
gan TSQ-7000 instrument (ThermoFinnigan, Sunnydale, CA). from residual hydrazone contamination. Hexanes and CH3CN
LC-MS work was done with a Thermo LTQ instrument or a were heated with and distilled from 2,4-dinitrophenylhydr-
Waters Synapt mass spectrometer using an Acquity UPLC azine to remove residual aldehydes. Assays involving competi-
(Waters, Milford, MA), except for the competitive experiments tive deuterium isotope effects were done by LC-MS analysis
(inter- and intramolecular), which were done by LC-MS using (atmospheric pressure chemical ionization, negative ion) of
the TSQ-7000 instrument.
derivatized formaldehyde or acetaldehyde (source tempera-
General Assays—Aldehydes were quantified by HPLC/UV ture, 550 °C; heated capillary voltage, 20 V; heated capillary
analysis of the 2,4-dinitrophenylhydrazones as described previ- temperature, 180 °C; ionization current, 5 ␮A; sheath gas (N2)
ously (24, 39), with some modification. The sensitivity of the pressure, 70 p.s.i.; auxiliary gas (N2) pressure, 10 p.s.i.).
assays was improved by purification of some of the reagents and
For the DMN time course experiments, 0.5 nmol of 2A6, 1.0
by removal of glycerol from enzymes (by dialysis immediately nmol of NADPH-P450 reductase, 0.5 nmol of cytochrome b5,
prior to use), because commercial glycerol was found to be con- 30 ␮M L-␣-dilauroyl-sn-glycero-3-phosphocholine, NADPH (1
taminated with formaldehyde. Dansyl hydrazones were used in mM), and DMN (17 mM) were incubated in 0.2 ml of potassium
some LC-MS analyses because of the low (electrospray ioniza- phosphate buffer (100 mM, pH 7.4) at 37 °C for varying amounts
tion and atmospheric pressure chemical ionization) ionization of time. Reactions were initiated by adding NADPH. Incuba-
efficiencies of HCHO- and CH3CHO-derived DNPH hydra- tions were terminated by adding 200 ␮l of cold CH3CN and
zone derivatives. Applying the analytical methods described centrifuged (2 ϫ 103 ϫ g) for 5 min. The supernatants were
here to the analysis of unlabeled substrates, we were able to transferred to amber vials, and 20 nmol of DCDO and 600 ␮l of
quantitate acetic acid in extracts of enzyme product mixtures, a freshly prepared solution of dansylhydrazine (0.5 mg mlϪ1) in
but not formic acid, at the level of sensitivity required for this CH3CN containing 0.3% CH3CO2H (v/v) was added. The reac-
work. Therefore, we used 14C-labeled substrates and measured tion mixture was incubated at room temperature for 30 min
[14C]formic acid utilizing ion exchange SPE columns.
and dried under nitrogen. Finally, the residue was reconstituted
Nitrosamine N-Dealkylation Assays—Typical steady-state in 100 ␮l of a mixture of H2O/CH3CN (3:1, v/v). Dansylated
dealkylation reactions included 400 pmol of 2A6, 800 pmol of products were analyzed by LC-MS on a Waters Acquity UPLC
NADPH-P450 reductase, 400 pmol of cytochrome b5, 20 ␮g of system connected to either an LTQ (Thermo Fisher, Santa
L-␣-dilauroyl-sn-glycero-3-phosphocholine, and varying con- Clara, CA) or a Waters Synapt mass spectrometer using an
centrations of the nitrosamine substrate in 0.34 ml of 50 mM Aquity UPLC BEH C18 octadecylsilane column (1.7 ␮m, 2.1
potassium phosphate buffer (pH 7.4). Reaction vials (clear glass, mm ϫ 100 mm). LC conditions were as follows. Buffer A con-
1 dram) were sealed with Teflon-lined rubber septa because of tained 10 mM NH4CH3CO2 and 2% CH3CN (v/v), and buffer B
the volatility of the substrates. Reconstituted enzyme solutions contained 10 mM NH4CH3CO2 and 95% CH3CN (v/v). The
(P450 2A6, NADPH-P450 reductase, and cytochrome b5) were following gradient program was used, with a flow rate of 300
dialyzed against glycerol-free 50 mM potassium phosphate ␮l/min: 0–6.5 min, linear gradient from 25% B to 100% B (v/v);
buffer (pH 7.4) containing 0.2 mM EDTA and 0.1 mM dithio- 6.5–7.5 min, hold at 100% B; 7.5–8 min, linear gradient to 75%
threitol (two changes over 12 h at 4 °C), before the addition of A (v/v); 8–10 min, hold at 75% A (v/v). The temperature of the
the phospholipid (L-␣-dilauroyl-sn-glycero-3-phosphocho- column was maintained at 25 °C. Samples (20 ␮l) were infused
MARCH 12, 2010•VOLUME 285•NUMBER 11
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Sequential Nitrosamine Oxidation by P450 2A6
with an autosampler. MS analyses were performed in the elec- 0.18 mCi mmolϪ1) were initiated with an acetate-free NADPH-
trospray positive ion mode. The results of steady-state kinetic generating system (dialyzed enzymes), incubated for a specified
experiments were fit to hyperbolic plots using GraphPad Prism time at 37 °C, terminated by the addition of 100 ␮l of 10%
(GraphPad, Dan Diego, CA), and parameters and S.E. values ZnSO4ꢀ7H2O (w/v), and centrifuged (2 ϫ 103 ϫ g). The super-
were obtained with this program using non-linear regression.
natants were loaded onto BakerbondTM quaternary amine 3-ml
Acetic Acid Assays—P450 2A6 was reconstituted with cyto- SPE columns that had been washed with 6 ml of CH3OH and
chrome b5, NADPH-P450 reductase, and phospholipid as de- equilibrated with 10 ml of H2O. After loading, the columns
scribed for nitrosamine N-dealkylation assays (see above). were washed with 10 ml of H2O to remove residual aldehyde or
Reactions containing acetaldehyde (0–3.5 mM) were initiated nitrosamine substrate. The [14C]formic acid was eluted with 1.5
with an acetate-free NADPH-generating system (dialyzed en- ml of 0.1 M HCl, and radioactivity was measured by liquid scin-
zymes), incubated for 15 min at 37 °C, and terminated by freez- tillation spectrometry. Recovery was calibrated using a sodium
ing in a dry ice-acetone bath. Following lyophilization to near [14C]formate standard.
dryness, acids were acylated in 300 ␮l of CH2Cl2 containing
For time course experiments, reactions containing [14C]DMN
4-nitrophenacyl bromide (0.5 mM) and tetrahexylammonium- (17 mM, 0.3 mCi/mmol) were initiated with an acetate-free
HSO4 (0.03 mM). After 30 min at 50 °C, the CH2Cl2 phase was NADPH-generating system (dialyzed enzymes) (40), incubated
transferred to a clean vial, evaporated to dryness, and reconsti- for various times, and quenched by heating at 80 °C for 5 min.
tuted in CH3CN/H2O (1:1, v/v). Derivatized carboxylic nitro- [14C]HCO2H was quantitated by HPLC using a liquid scintilla-
phenacyl esters were analyzed by HPLC using an ODS-AQ tion flow counter and an Ultrasil amino column (4.6 mm ϫ 250
octadecylsilane (C18) hydrophilic end-capped column (YMC, mm, 10 ␮m; Beckman) with the following LC conditions. Buffer
4.6 mm ϫ 150 mm, 5 ␮m) with isocratic elution (1.4 ml/min, A contained 20% CH3OH (v/v), and buffer B contained 80%
H2O/CH3CN, 55:45, v/v) and UV detection (260 nm). Before CH3OH (v/v) and 500 mM NH4CH3CO2. The following gradi-
use as a derivatization reagent, 4-nitrophenacyl bromide was ent program was used, with a flow rate of 1 ml/min: 0–10 min,
twice recrystallized from CH3OH/H2O (3:1) and dried in 100% A; 10–25 min, linear gradient to 80% A (v/v); 25–26 min,
vacuo. CH3CN and CH2Cl2 were passed multiple times through linear gradient to 100% A; 26–30 min, hold at 100% A.
columns (3 cm ϫ 20 cm) of basic alumina (60–325 mesh;
Brockman activity I, Fisher) to minimize interference resulting tion were performed in a quench-flow apparatus (model RFQ-
from residual carboxylic acid contamination. 3, KinTek Corp., Austin, TX). Experiments were done with
Pre-steady-state Kinetics—Pre-steady-state kinetics of oxida-
Time course reactions for DEN oxidation were performed as N-nitrosodi-[1,1Ј-13C]methylamine (170 mM final concentra-
described for nitrosamine N-dealkylation assays (see above) tion) in the case of DMN and with N-nitrosodi-[2,2Ј-
with the exception that reactions were terminated by the addi- d3]ethylamine (1.4 mM final concentration) in the case of DEN.
tion of 200 ␮l of cold CH2Cl2, To the reaction mixture 0.5 nmol Reactions were performed at 37 °C, and P450 2A6 (100 pmol)
of CD3CO2H was added, and the products were derivatized was reconstituted as described for the nitrosamine N-dealkyla-
with 4-nitrophenacyl bromide, as described above. The derivat- tion assays (see above). Reactions were initiated with 1 mM
ized products were analyzed by LC-MS on a Waters Acquity NADPH for a period of time ranging from 5 ms to 10 s and
UPLC system connected to an LTQ mass spectrometer quenched with CH3CN containing 1% CH3CO2H. Propional-
(Thermo Fisher, Santa Clara, CA) using an Aquity UPLC BEH dehyde was used as an internal standard. Aldehydes were deri-
C18 octadecylsilane column (1.7 ␮m, 1 mm ϫ 100 mm). LC vatized with dansylhydrazine and quantitated by LC-MS as
conditions were as follows: buffer A contained 2% CH3CN and described for the DMN N-demethylation time course experi-
0.01% HCO2H (v/v), and buffer B contained 95% CH3CN and ments (see above). In the case of [13C]HCHO, the yields were
0.01% HCO2H (v/v). The following gradient program was used, corrected for the isotopic abundance of [13C]HCHO, resulting
with a flow rate of 150 ␮l minϪ1: 0–4.0 min, linear gradient from background [12C]HCHO.
from 95% A to 50% A; 4.0–4.5 min, linear gradient to 100% B;
For measuring oxidation of acetaldehyde to acetic acid,
4.5–5.5 min, hold at 100% B; 5.5–6 min, linear gradient to 95% [14C]CH3CHO (10 mM final concentration, 5 mCi/mmol) was
A; 6–8 min, hold at 95% A. The temperature of the column was used. Reactions were performed as described for the [14C]for-
maintained at 50 °C; the injection volume was 20 ␮l. Samples mic acid assays with the exception that 17% ZnSO4ꢀ7H2O (w/v)
were infused with an autosampler. MS analyses were performed was used to quench the reaction. The product [14C]CH3CO2H
in the negative ion mode. Electrospray ionization conditions was purified using BakerbondTM quaternary amine SPE col-
were as follows: source voltage, 4 kV, source current, 100 ␮A; umns and detected by liquid scintillation spectrometry (see
auxiliary gas flow rate setting, 37; sheath gas flow setting, 16; above).
capillary voltage, Ϫ4 V; capillary temperature, 380 °C; tube lens
voltage, Ϫ22 V.
Stopped-flow Kinetics—Analyses were done using an OLIS
RSM-1000 stopped-flow spectrophotometer in the rapid scan-
[14C]Formic Acid Assays—A concentrated (80 mM, 0.2 mCi ning monochromator mode, using the general procedures
mmolϪ1) stock solution of [14C]formaldehyde was prepared by described earlier (31).
adding paraformaldehyde-derived (unlabeled) formaldehyde
Ligand binding assays were done at ambient temperature
(see above) to commercially obtained [14C]formaldehyde. P450 (23 °C). P450 2A6 (final concentration in mixing cell, 2.0 ␮M) was
2A6 was reconstituted as described for the nitrosamine N-deal- mixed with either 12 mM DMN or 1.1 mM DEN (final concentra-
kylation assays (see above). Reactions containing [14C]formal- tions) in 50 mM potassium phosphate buffer (pH 7.4). Kinetic anal-
dehyde (0–50 mM, 0.2 mCi mmolϪ1) or [14C]DMN (17.5 mM, ysis was done using the absorbance changes at 420 nm (decrease).
8034 JOURNAL OF BIOLOGICAL CHEMISTRY
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Sequential Nitrosamine Oxidation by P450 2A6
TABLE 1
Kinetic isotope effects for oxidation of DMN and DEN to aldehydes
Kinetic isotope effecta
Substrate
Formaldehyde
Acetaldehyde
Intramolecular
CD2H-N(NO)-CD2Hb,c
10.2 Ϯ 0.2
15 Ϯ 3
d
CH3CHD-N(NO)-CHDCH3
3.7 Ϯ 0.2
3.0 Ϯ 0.1
c
CH3-N(NO)-CD3
d
CH3CH2-N(NO)-CD2CH3
Intermoleculare
c
CH3-N(NO)-CH3/CD3-N(NO)-CD3
12.0 Ϯ 0.7
11 Ϯ 1
d
CH3CH2-N(NO)-CH2CH3/CH3CD2-N(NO)-CD2CH3
2.7 Ϯ 0.2
3.6 Ϯ 0.2
c
CH3-N(NO)-CH3/CD3-N(NO)-CH3
d
CH3CH2-N(NO)-CH2CH3/CH3CD2-N(NO)-CH2CH3
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 deuterium/protium ratio of 2.
c Substrate concentration 17 mM.
d Substrate concentration 1.0 mM.
e Substrate mixture (1:1).
Reduction assays were done with ferric P450 2A6 and a 2-fold the mean results were compared, with the extent of decrease
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 (undiluted)
ligand from a 500-␮l 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 ␮l 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.
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, respec-
tively, 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 Ϯ
Ϫ1
0.0015 sϪ1 in the absence of cytochrome b5 and 0.11 Ϯ 0.05 s
in its presence. For DEN (used at 140 ␮M), the rates of acetal-
dehyde formation in the absence and presence of cytochrome
b5 were 0.04 Ϯ 0.002 and 0.15 Ϯ 0.68 sϪ1, respectively. Accord-
ingly, all further assays included cytochrome b5. Initial param-
In the case of ligand binding reactions, the reaction time was
either 15 s or 150 ms. For reduction, reaction times were 3 or
30 s. In both cases, 4–7 individual mixing reactions were used
to derive rate constants (using the OLIS software, single expo-
nential fits) and averaged.
Ϫ1
eters of kcat ϭ 0.42 s and Km ϭ 15 mM were estimated for
DMN oxidation to formaldehyde, and kcat ϭ 0.13 sϪ1 and Km ϭ
0.15 mM for DEN oxidation to acetaldehyde. These results are
consistent with literature indicating the preferential catalytic
efficiency of P450 2A6 toward DEN (28, 32). With this prelim-
inary information, concentrations of 17 mM DMN and 1 mM
DEN were used for the competitive and intrinsic kinetic deute-
rium isotope effect studies (see below).
Pulse-Chase Experiments—Experiments were done with
[14C]DMN (17 mM, 0.5 mCi mmolϪ1) in the case of DMN and
with N-nitrosodi-[2,2Ј-d3]ethylamine (0.14 mM) in the case of
DEN. In both cases, the reaction was initiated (37 °C) with 2.5
␮M P450 2A6, 5.0 ␮M NADPH-P450 reductase, 2.5 ␮M cyto-
chrome b5, 30 ␮M L-␣-dilauroyl-sn-glycero-3-phosphocholine,
and an NADPH-generating system (see above). 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 (see above) with the exception that,
after sample loading, the column was washed with 20% CH3OH
(5 ml) 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-deu-
terated CH3CO2H in the reagents served as an internal stand-
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 contrib-
ute to rate limitation (3). In principle, comparisons of rates of
C–H and C–D bond cleavage at a carbon atom substituted with
both protium and deuterium should provide an estimate of Dk,
the intrinsic isotope effect. These values, estimated by MS of
the aldehyde products, were 10.2 Ϯ 0.2 and 3.7 Ϯ 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
ard (ϳ10-fold higher level). Assays were run in triplicate, and two deuteriums, the contribution could be as high as 1.4. Thus,
MARCH 12, 2010•VOLUME 285•NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8035

Sequential Nitrosamine Oxidation by P450 2A6
TABLE 2
Non-competitive intermolecular kinetic isotope effects for oxidation of DMN and DEN to aldehydes
Substrate
Producta
kcat
Km
k
cat/Km
DV
D(V/K)
Ϫ1
Ϫ1
s
mM
M
Ϫ1 s
d0-DMN
d3d3-DMN
d0-DEN
HCHO
HCDO
0.45 Ϯ 0.01
0.092 Ϯ 0.02b
0.14 Ϯ 0.01
0.11 Ϯ 0.01
17 Ϯ 1
26 Ϯ 2
2.1 Ϯ 0.9
1000 Ϯ 120
920 Ϯ 70
44 Ϯ 15
4.8 Ϯ 1.0b
1.2 Ϯ 0.1
13 Ϯ 5
CH3CHO
CH3CDO
0.14 Ϯ 0.02
0.12 Ϯ 0.01
d2d2-DEN
1.1 Ϯ 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).
the true Dk for DMN oxidation could be as low as 10/1.4 ϭ 7. not directly reflect the “off” rates. The high Kd values prevent
The contribution of a geminal secondary kinetic isotope effect estimation of the dissociation rate by extrapolation to zero sub-
should be less in the case of the methylene in DEN (-CHD-) strate concentration (31, 52). A dissociation rate was estimated
(i.e. Յ1.2), so the Dk should be Յ3.5 and possibly ϳ3.4
indirectly by mixing a preformed P450 2A6-DEN complex with
We also measured the apparent kinetic hydrogen isotope 4-phenylimidizole, which is presumed to occupy the same site
effects for unsymmetrically deuterated DMN and DEN (Table but yields a different spectral complex.5 The rapid release of
1). In principle, the comparison of these values with the esti- DMN and DEN was indicated by these experiments and the
mates of the intrinsic kinetic isotope effects (see above) can competitive intermolecular isotope effect results (see below).
provide an estimate of the ability of a substrate to turn within
k1
the active site of the enzyme (48–51). These values were as high
P450ꢀDEN 9|= P450 ϩ DEN
as the values obtained with N-nitrosodi-[1-d2,1Ј-d2]methyl-
amine and N-nitrosodi-[1,1Ј-d2]ethylamine (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 rotating within the active site.
The rates of binding of DMN and DEN to P450 2A6 were mea-
sured. Binding of either substrate to P450 2A6 yielded a “Type I”
difference spectrum, with an apparent shift of low spin iron
(␭max 418 nm) to high spin (␭max 390 nm). The apparent Kd was
12 mM for DMN and 1.1 mM for DEN (supplemental Figs. S5
and S6). This change could be observed in a stopped-flow spec-
trophotometer and occurred at (first-order) rates of 80 Ϯ 18
kϪ1
k2
P450 ϩ 4-phenylimidazole 9= P450ꢀ4-phenylimidazole
|
kϪ2
REACTIONS 1 and 2
Competitive Intermolecular Kinetic Deuterium Isotope
Effects—The competitive isotope effects for the mixtures of d0
and perdeuterated nitrosamines (Table 1) were within experi-
mental error of the intramolecular values, the pseudointrinsic
isotope effects. In addition, the values seen in the “mixed”
experiments were just as high, with d0 substrate plus asym-
metrically labeled nitrosamines (Table 1). If exchanges were
slow, these values should also be lower.5
s
Ϫ1 with 12 mM DMN and 73 Ϯ 22 sϪ1 with 1.1 mM DEN (at
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.
23 °C).5 These parameters are for the binding of ligands but do
4 A 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 hydro-
gen at the 4-methylene carbon by recombinant mouse P450 2a4 and 2a5.
The selective abstraction of an (R)- versus (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 compet-
itive 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 are nearly as large and within
experimental error.
Deuterium substitution of DMN affected both kcat and Km
(Table 2), with DV ϭ 4.8 and DKm ϭ 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
5 One 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 ϭ kon[S] ϩ 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 ϳ0.15 mM (Table 3) and the
spectrally estimated Kd (increase in absorbance at 390 nm, decrease at
420 nm) for binding DEN was ϳ1.1 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 preformed P450
2A6ꢀDEN complex was rapidly mixed (at 13 °C) with an excess of 4-pheny-
limidazole, and the rate of the change in the spectrum (from a ␭max 390 nm
complex to a ␭max 427 nm complex) was measured (0.86 Ϯ 0.08 sϪ1) and
compared with the rate of the formation of the latter complex in the
absence of DEN (0.86 Ϯ 0.12 sϪ1). Analysis of the data with the kinetic
model shown in Reactions 1 and 2, using Kd,DEN ϭ kϪ1/k1 ϭ 1100 ␮M (from
a spectral titration) and Kd,4-phenylimidazole ϭ 0.51 Ϯ 0.10 ␮M (from spectral
titration) and an experiment involving 5 mM DEN (5-fold Ͼ Ks) and 10 ␮M
4-phenylimidazole (20-fold Ͼ Ks) with the program DynaFit (assuming a
simple competitive model) indicated that k1 and k2 (at 13 °C) were ϳ5 ϫ
105
M
Ϫ1 sϪ1, andthuskϪ1 mustbeϾ500sϪ1, whichisconsistentwiththeKd
(Kd,DEN ϭ kϪ1/k1 ϭ 5 ϫ 102
M
Ϫ1 sϪ1/5 ϫ 105 Ϫ1 sϪ1 ϳ 1 mM) (see supple-
M
mental Fig. S7). From these results, we conclude that the exchange of DEN
and, by inference, DMN with ferric P450 2A6 is very rapid (ϳ100 sϪ1) and is
not an issue in the interpretation of the kinetic deuterium isotope effects.
8036 JOURNAL OF BIOLOGICAL CHEMISTRY
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Sequential Nitrosamine Oxidation by P450 2A6
FIGURE 3. Comparison of rates of formaldehyde formation from d0d3-
DMN with d0d0- and d3d3-DMN.
cate that P450 2A6 exchanges substrates rapidly, consonant
with the results of studies with P450 2A6 and coumarin (31).
One question is whether such exchange can occur at the stage
of the actual oxygen complex (putative FeO3ϩ).
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 previ-
ously with P450 1A2 (51). To address the question of whether
such exchange can occur at the stage of the actual oxygen com-
plex (putative FeO3ϩ) 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-break-
ing step is relatively more difficult, the enzyme could (if the step
were slow enough relative to exchange) dissociate the deuter-
ated substrate, bind the protiated substrate, and oxidize the
protiated substrate. The intermolecular non-competitive iso-
tope 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 FeO3ϩ complex, the rate
of product formation should reflect the substrate isotopic com-
FIGURE 2. Non-competitive intermolecular kinetic isotope effects for oxi-
dation of DMN and DEN to aldehydes. A, DMN. B, DEN. The points are
means Ϯ 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.
TABLE 3
Oxidation of aldehydes to carboxylic acids
Substrate Product
kcat
Km
k
cat/Km
DV
D(V/K)
Ϫ1
Ϫ1
s
mM
M
Ϫ1 s
HCHO HCO2H
0.010 Ϯ 0.001 2.1 Ϯ 1.4 4.8 Ϯ 3.3
CH3CHO CH3CO2H
CH3CDO CH3CO2H
0.12 Ϯ 0.01
0.12 Ϯ 0.01
1.3 Ϯ 0.3 92 Ϯ 21
2.7 Ϯ 0.5 44 Ϯ 10 0.97Ϯ 2.0 Ϯ 0.6
have less error based on visual observation of the tangents (Fig. position (i.e. the amount of product formed from the oxidation
2), was ϳ13, within experimental error of the approximated of d0d3-DMN (HCHO plus DCDO) should be intermediate
intrinsic Dk (Table 1). When DEN oxidation was measured, DV between the amounts formed from d0-DMN (HCHO) and
and D(V/K) were near unity (Table 3), within experimental d3d3-DMN (DCDO)). When the experiment was done, the lat-
error.
ter result was obtained (Fig. 3). A similar set of experiments
Limited Methyl (␤) Hydroxylation of DEN—One possibility using deuterated DEN analogues could not be done because the
to consider is that P450 2A6 can generate alternate products non-competitive intermolecular isotope effect was near unity
when a C–D bond replaces C–H, which could affect the inter- (Table 2).
pretation of the kinetic isotope effects. P450s can catalyze a
Oxidations of Aldehydes to Carboxylic Acids—Both HCHO
Ϫ
reaction generating NO2 (55), although this is a relatively and CH3CHO were substrates for P450 2A6 oxidation (Table
minor pathway. Another possibility is that deuterium substitu- 3). The oxidation of CH3CHO was much more efficient than
tion of the ␣-carbon of DEN could favor hydroxylation of the that of HCHO, by a factor of 20-fold. The efficiencies (kcat/Km)
methyl group. We synthesized 2-hydroxy-DEN, characterized for oxidations of both aldehydes were ϳ10-fold less than for the
it, and used it as an LC-MS standard to monitor its possible oxidations of the corresponding nitrosamines to the aldehydes
formation from DEN by P450 2A6. With d0-DEN, the rate was (Table 2).
Ͻ0.004 minϪ1 (with a DEN concentration of 1 mM). With d2d2-
A non-competitive intermolecular kinetic isotope effect of
DEN, the rate was ϳ0.011 minϪ1. We conclude that methyl 2.0 was observed for the P450 2A6-catalyzed acetaldehyde oxi-
hydroxylation is not a major pathway, even when the ␣-carbon dation D(V/K) parameter, but not DV; the isotope effect was
is deuterium-substituted.
only on Km. A similar observation for acetaldehyde oxidation
Comparisons of Product Formation with d0-, d3-, and was made with human P450 2E1 (27). The P450 2A6-catalyzed
d3d3-DMN—The lack of attenuation of kinetic isotope effects oxidation of formaldehyde was not analyzed for an isotope
in the intermolecular competitive experiments (Table 1) and effect because the assay of formic acid was not sufficiently sen-
the results of the 4-phenylimidazole binding experiments5 indi- sitive without the use of 14C-labeled material.
MARCH 12, 2010•VOLUME 285•NUMBER 11
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Sequential Nitrosamine Oxidation by P450 2A6
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 B. The products were measured by LC-MS analysis of the dansyl
hydrazones.
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).
FIGURE 4. Time courses of conversion of DMN and DEN to aldehydes and
carboxylicacids. A, conversionofDMNtoHCHOandHCO2H. B, conversionof
DEN to CH3CHO and CH3CO2H. C, conversion of DEN to CH3CO2H, scale
expanded for clarity. The substrate concentration was 17 mM and 140 ␮M in A
and B, respectively, and the P450 2A6 concentration was 2.5 ␮M.
NADPH Oxidation Rates and Coupling Efficiency—In the
absence of any added substrate, the reconstituted P450 2A6
system oxidized NADPH at a rate of 0.63 sϪ1 (i.e. 0.63 nmol
of NADPH/s/nmol of P450. With 17 mM DMN (Km concen-
tration), the rate was 1.5 sϪ1, and with 0.14 mM DEN (Km
concentration), the rate was 0.75 sϪ1. Under these condi-
tions, the rates of formation of the aldehydes with the same
reagents were 0.05 and 0.075 sϪ1, 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. Accord-
ingly, pulse-chase experiments were done, in which reac-
tions were initiated with labeled DMN or DEN (concentra-
tion ϳ 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 scintilla-
tion spectrometry or LC-MS. The final ratios of HCHO/
[14C]HCHO and of CH3CHO/CD3CHO were ϳ16 and ϳ7,
respectively, based on the total amount of aldehydes formed
in 20 min. If all of the intermediate aldehyde was in equilib-
rium (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
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. 4, B and C). 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 repro-
ducible bursts in both cases, with 2–4% product formed in the
rapid phase (Fig. 5). A small burst was also detected in the oxi-
dation 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 (see above),
with the lower UV region obscured by the absorbance of the
ligand (supplemental Figs. S5 and S6). The estimated Kd val-
ues 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 and S10), with apparent Kd values of
ϳ200 mM.
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). Pre-
liminary anaerobic studies showed that all of the Na2S2O4-re-
ducible P450 2A6, when DEN was present, was reduced within acetic acid was labeled (Fig. 6).
8038 JOURNAL OF BIOLOGICAL CHEMISTRY
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Sequential Nitrosamine Oxidation by P450 2A6
Kinetic Modeling—An overall scheme of the major events substrate to product (Fig. 4). A minimal model was devel-
of the reactions is shown in Fig. 7, including known rates of oped that is consistent with these data (Fig. 9), with S denot-
some of the non-enzymatic reactions (56–59). If the alde- ing the nitrosamine, P the aldehyde, and Q the carboxylic
hyde product dissociates from P450 2A6, then the rates of acid. Critical features included fitting the time courses of
formation of the carboxylic acids (measured in Fig. 4) can be formation of the aldehyde and acid simultaneously, with a
described by a model of a coupled reaction with separate kcat lack of a lag for the acid, as well as showing a partial kinetic
and Km values for the two steps (Tables 2 and 3). The pre- burst and an isotope effect for DMN. The rate constants for
dicted time courses of formation of the aldehydes and car- ligand binding were set at 107 Ϫ1 sϪ1 (consistent with other
M
boxylic acids (see supplemental Fig. S12) were compared work with P450 2A6 (31) and accepted diffusion-limited
with the experimental values (from Fig. 4) in Fig. 8. Two rates of interaction of enzymes and ligands (44), with rate
striking features were clear: (i) the theoretical plots under- constants for ligand release balanced to fit the spectrally esti-
predict carboxylic acid formation, and (ii) the theoretical mated Kd values (supplemental Figs. S5, S6, S9, and S10). The
model predicts lag phases for carboxylic acid formation, rate constants for steps 1–4 were set not to be faster than the
which is intuitive due to the relatively high Km values for rapid formation of product by P450 2A6 (Fig. 5).
oxidation of the two aldehydes (Table 3).
Fits are shown for DMN and DEN oxidations to aldehydes
A comprehensive but minimal kinetic model was devel- and acids in Fig. 10, using the rate constants shown in Table 4
oped utilizing KinTek Explorerꢁ software. The model is nec- for the steps in Fig. 9 (see also supplemental Fig. S13). The time
essarily complex because it must include multiple phenom- courses of product formation fit the experimental data well, and
ena, including (i) normal events known to occur in P450 partial bursts are predicted (as shown with the inset for DMN in
reactions, including separate substrate binding and oxygen Fig. 10B). Applying an intrinsic Dk of 12 for DMN N-dealkyla-
activation steps, (ii) the irreversible loss of reduced oxygen tion (Table 1) reduced the rate of HCHO production (v) by
species from the activated complex (see above) (60), (iii) the 3.5-fold, and applying an intrinsic Dk of 3 for DEN N-dealkyla-
partial burst kinetics (Fig. 5), (iv) the lack of affinity of the tion (Table 1) reduced the rate of CH3CHO formation by only
aldehydes for the enzyme (supplemental Figs. S9 and S10), 1.2-fold (cf. Table 2).
(v) the use of the product as a substrate in the second reac-
DISCUSSION
tion (Table 3), (vi) the non-competitive kinetic deuterium
The original impetus for this work was an in vivo study
showing a strong deuterium isotope effect on the hepatocar-
cinogenicity of DMN (15). At that
time, none of the mammalian P450s
had been characterized. Subse-
quently, P450s 2A6 and 2E1 were
identified as the major catalysts
involved in the oxidation and bio-
activation of short-chain N-nitros-
amines, especially DMN and DEN
(28, 32, 61). Nitrosamines should
not be regarded only as xenobiot-
ics, in that the in vivo nitrosation
of secondary amines is a well
established phenomenon (62–64)
and considered to be of relevance
isotope effect seen with DMN but not DEN (Table 2), and
(vii) the maximum rates possible for converting part of the
FIGURE 6. Pulse-chase experiments. See “Experimental Procedures” for details. Reactions were started with
labeled DMN (A) or DEN (B) (concentration ϳKm; 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).
in understanding human cancer
etiology (65). Previous work on the
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 and S6). E, enzyme (P450 2A6); RCHO, aldehyde; RCH(OH)2,
hydrated aldehyde; RCO2H, carboxylic acid.
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Sequential Nitrosamine Oxidation by P450 2A6
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 and 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 with a methyl (e.g. 94.5 ver-
sus 98 kcal/mol) (66).6 Several lines of investigation argue
that these substrates can tumble and/or exchange rapidly
(Table 1) but not after the P450 is in the activated state and
poised for C–H bond cleavage (Figs. 3 and 11).
One interesting aspect of this study is the observed pro-
cessivity of oxidation. P450 2A6 oxidizes the nitrosamines to
aldehydes (Table 2) and oxidizes both aldehydes (HCHO and
CH3CHO) to carboxylic acids (Table 3). However, the alde-
hyde oxidations are not particularly efficient (Table 3), and
combining the kcat and Km values for the individual oxida-
tions yields predictions that have two major discrepancies
with the experimental results (Figs. 4 and 8): (i) with DEN
and particularly DMN, the calculations based on the individ-
ual kcat and Km values underestimate carboxylic acid forma-
tion, 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 alde-
hydes were found to have only low affinity for (ferric) P450
2A6 (supplemental Figs. S9 and S10).
A kinetic model was developed that is consistent with
most of the observed results. In order to account for the lack
of dissociation of the aldehyde(s), a solution used in our ear-
lier studies on P450 2E1 (27) was invoked, namely that the
P450 undergoes a conformational change upon binding sub-
strate (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
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. A, general mechanism; B, fit of DMN data; C, fit of DEN data.
stepwise oxidation of ethanol to acetic acid by human P450 complex is left in a conformational state leading to catalysis,
2E1 identified an intrinsic kinetic deuterium isotope effect avoiding the need to release, rebind, and then undergo the
(expressed primarily in Km), burst kinetics, and a processive activating conformational change. Thus, the rate constants
pathway (24, 27). In the oxidation of DMN and DEN by P450 for events leading to catalysis in the second cycle compete
2A6, some similar phenomena were seen, and some of the with others in the “first” cycle (Fig. 9A). Another required
same mechanisms seem applicable, although several features feature is an equilibrium of an activated P450 2A6-substrate
are more complex.
complex with an unproductive complex, added to explain
The best estimates for the intrinsic kinetic deuterium iso- the (pre-steady-state) partial bursts observed for production
tope effects for the oxidations of DMN and DEN by P450 2A6 of aldehydes (70–72). The abortive generation of reduced
are ϳ10 and ϳ3, respectively, as estimated from the non- oxygen species (Fig. 9) was included in light of the low effi-
competitive intramolecular studies (Table 1, lines 1 and 2), ciency of NADPH coupling (see above). Any model must also
with caveats about the contribution of secondary isotope have a C–H bond-breaking step that can account for the high
effects. Similar kinetic isotope effects were measured in a kinetic deuterium isotope effects (for DMN) (Table 2).
variety of competitive experiments with labeled DMN and
DEN (Table 1), arguing that exchange of the nitrosamine
6 The values in Ref. 66 are general, but the difference in dissociation energy
between a methyl versus methylene C–H bond is similar in other literature
summaries. We did not find specific bond energy values for DMN and DEN
substrates is very rapid, a conclusion supported by direct
measurements of on-rates (70–80 sϪ1 with 12 mM DMN or
in our searches. Bond energies have been measured for the hydrogen in
1.1 mM DEN at 23 °C) and by an experiment in which the
methylamine and ethylamine (67), with a difference of 3.8 kcal molϪ1 that
dissociation rate of a P450 2A6-DEN complex was estimated
by displacement of another ligand (4-phenylimidazole)5
(supplemental Fig. S7).
is very similar to the general difference of 3.5 kcal molϪ1 cited in Ref. 66. Of
course, thebondenergiesareperturbedinthefreeaminesrelativetoDMN
and DEN due to the tendency of these amines to lose electrons.
8040 JOURNAL OF BIOLOGICAL CHEMISTRY
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Sequential Nitrosamine Oxidation by P450 2A6
Literature rates of non-enzymatic
steps are shown in Fig. 7, including
the rates of rearrangement of ␣-
hydroxynitrosamines 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, par-
ticularly if they are reasonable
approximations of rate constants in
the enzyme active site. One issue is
the time needed for the breakdown
of the ␣-hydroxynitrosamine 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 proc-
ess, before hydrogen atom abstrac-
tion (Fig. 7). One means of circum-
venting this kinetic problem is to
use the alternative peroxide mecha-
nism (27, 76, 77) (Fig. 11B), avoiding
the need for the slow hydration of
CH3CHO (Fig. 7) (57, 58). (A third
Ϫ
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 path-
ways (Fig. 11) is operative (a small
kinetic deuterium isotope effect was
observed in a non-competitive ex-
periment (Table 4), but this, by
itself, does not have a single mecha-
nistic interpretation).
FIGURE 9. Scheme used for kinetic modeling of nitrosamine oxidations. A, scheme with P450 conforma-
A model (Fig. 9) was constructed
based upon the seven points listed
under “Kinetic Modeling.” Several
strengths of the model are as fol-
lows: (i) the kinetic courses of alde-
hyde and carboxylic acid formation
tions and valence states. S, nitrosamine; P, aldehyde; Q, carboxylic acid. B, simplified scheme used in analysis
(Table4andsupplementalFig. S13). E, P4502A6;S, nitrosamine;F, conformationalstateofEutilizedincatalysis;
J, non-productive conformation of E; G, F with active Fe-O entity poised for oxidation; P, aldehyde; Q, carboxylic
acid;O, reducedoxygenspecies(H2O2 orH2O);H, sumofEandFformsofP4502A6(combinedforsimplification
because the conversion of F to E is not critical to this series of events).
Finally, the model incorporates the spectrally measured Kd
match the experimental results, with no lags observed for car-
boxylic 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 and S6); (iii) the model yields substoichiometric 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 (P450Ј
Fe3ϩ-S), the unproductive complex in Fig. 9, yielded only a full
kinetic burst (1 product/enzyme), not the partial burst. JP
(P450Ј Fe3ϩ-P) mirrors JS (i.e. if there is an unproductive com-
plex with the nitrosamine, one might be expected with the alde-
hyde as substrate), and if JP is eliminated (Fig. 9), the reaction is
values for the nitrosamines and aldehydes.7
7 The crystal structures of P450 2A6 show only space for a single substrate (30,
75), althoughnoneoftheligandsisassmallasDMN. Harrelson etal. (73, 74)
have proposed models of P450 2A6 (and P450 2E1) with two ligands pres-
ent (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-Hofstee plots of steady-state kinetic results.
However, the data points of Fig. 5 (also supplemental Table S3 of Ref. 74)
could be readily fit to standard hyperbolic plots (kcat ϭ 0.51 Ϯ 0.02 minϪ1
K
,
m ϭ 107 Ϯ 15 ␮M; kcat 2.5 Ϯ 0.1 minϪ1, Km ϭ 81 Ϯ 12 ␮M; r2 ϭ 0.98 in both
cases) without the need to apply a Hill equation, and it should be noted
that the only abnormal points in the Eadie-Hofstee plots were obtained at
the lowest enzyme velocities (Fig. 5 of Ref. 74). Accordingly we developed
our models with only a single ligand in the active site in light of limited
evidence for more ligands.
MARCH 12, 2010•VOLUME 285•NUMBER 11
JOURNAL OF BIOLOGICAL CHEMISTRY 8041

Sequential Nitrosamine Oxidation by P450 2A6
too fast, and rate constants must be attenuated. If the F form
(P450* Fe3ϩ) 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 substoichiometric. The step GS 3 E ϩ O
(P450 FeO3ϩ 3 H2O2 ϩ H2O) is logical, in light of the observed
uncoupling (see above); if these steps (GS, GP 3 E ϩ O) are
dropped, the model still fits, but some rate constants must be
attenuated. Nevertheless, the current model does have some
deficiencies, including the following: (i) the predicted burst
is substoichiometric but does not completely fit the experi-
mental results, and (ii) the predicted expressed non-compet-
itive kinetic isotope effect (Table 2) is larger (D(V/K) 13 Ϯ 5)
than predicted by the model (3.2; see above). 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 estro-
gens with the accumulation of low concentrations of inter-
mediates (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 oxida-
tion of pregnenolone to dehydroepiandrosterone by P450
17A1, about 20% of the intermediate 17␣-hydroxypreg-
nenolone 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 pro-
FIGURE 10. Fitting of data points for time courses of nitrosamine oxida-
tion to kinetic model of Fig. 9. A, DMN to HCHO and HCO2H. B, expansion of
first 30 s of A. C, DEN to CH3CHO and CH3CO2H. D, amplitude expansion of C.
The values of the rate constants used are shown in Table 4, with the numbers cessivity (83). Even less information is available about P450s
of the reaction steps (Fig. 9) corresponding to each rate constant.
that do not normally have defined physiological roles in ste-
roid metabolism. Rat P450 2C11 oxidizes testosterone to
TABLE 4
16␣-hydroxyandrostenedione via androstenedione, and
Kinetic modeling of oxidations of DMN and DEN by P450 2A6
All units are in M and sϪ1 (bimolecular reactions in MϪ1 sϪ1, for forward steps 1, 8,
Sugiyama et al. (84) estimated that ϳ15% of the andro-
stenedione 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 sug-
gested that ϳ90% of the acetaldehyde did not dissociate in
the process (see above). The processivity of P450 2E1 in
nitrosamine oxidations has not been reported in the litera-
ture to date.
and 11). Rate constants are limited to two significant digits.
DMN
DEN
Reaction step
(Fig. 9)
Reaction (Fig. 9B)
kforward kreverse kforward kreverse
1
E ϩ S % ES
ES % FS
107
150
6.0
1.0
1.2
3.0
1.5
107
2.2
0.40
107
57
1.5 ϫ 105
107
150
5.2
4.1
1.3
7.0
1.0
107
2.0
0.36
107
57
104
1.2
2
1.0
3
FS 3 GS
4
GS % FP
0.3
10
0.8
10
5
FP % EP
6
FS % JS
1.2
1.0
7
GS 3 E ϩ O
E ϩ P % EP
FP % GP
8
2 ϫ 106
1.3
2 ϫ 106
1.0
9
Acknowledgment—We thank K. Trisler for assistance in preparation
of the manuscript.
10
11
12
13
GP % HQ
E ϩ Q % HQ
FP % JP
1.0
1.0
107
107
2.5
2.4
GP 3 E ϩ O
1.0
1.0
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