From the Biomedical Research Centre, University of
Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United
Kingdom the ¶ Departments of Biochemistry and Chemistry,
University of Leicester, University Road, Leicester LE1 7RH, United
Kingdom, and the
Biological NMR Centre and Department of
Biochemistry, University of Leicester, PO Box 138, University Road,
Leicester LE1 9HN, United Kingdom
Received for publication, September 17, 2002, and in revised form, November 21, 2002
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ABSTRACT |
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Cytochrome P450 2D6 (CYP2D6)
metabolizes a wide range of therapeutic drugs. CYP2D6 substrates
typically contain a basic nitrogen atom, and the active-site residue
Asp-301 has been implicated in substrate recognition through
electrostatic interactions. Our recent computational models point to a
predominantly structural role for Asp-301 in loop positioning (Kirton,
S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S.,
and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and
suggest a second acidic residue, Glu-216, as a key determinant in the
binding of basic substrates. We have evaluated the role of Glu-216 in
substrate recognition, along with Asp-301, by site-directed
mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with
neutral residues (Gln, Phe, or Leu) greatly decreased the affinity
(Km values increased 10-100-fold) for the
classical basic nitrogen-containing substrates bufuralol and
dextromethorphan. Altered binding was also manifested in significant
differences in regiospecificity with respect to dextromethorphan,
producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of
Asp-301 to Gln and Asn had similarly profound effects on substrate
binding and regioselectivity. Intriguingly, removal of the negative
charge from either 216 or 301 produced enzymes (E216A, E216K, and
D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a
basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (Km 108 µM,
kcat 5 min Cytochromes P450 are a superfamily of heme-containing enzymes
responsible for the oxidative metabolism of an extremely wide variety
of substrates. Human cytochrome P450 2D6
(CYP2D6)1 is one of the most
important members of this family due to its central role in the
metabolism of many drugs in common clinical use (1), such as opioids,
antidepressants, neuroleptics, and various cardiac medications. CYP2D6
is polymorphic, giving rise to wide interindividual and ethnic
differences in drug metabolism (2, 3). Inheritance of the defective
gene results in the "poor metabolizer" phenotype that results in
impaired drug oxidation reactions (4) and may be linked to altered
disease susceptibility (5, 6). P450-drug and drug-drug interactions
involving CYP2D6 ligands are a prime consideration in the development
of new drugs, emphasizing the importance of a detailed understanding of
the factors that govern the substrate specificity of this enzyme.
CYP2D6 substrates are structurally diverse, but several key structural
features have been identified (reviewed in Ref. 7). These include a
basic nitrogen atom 5-10 Å from the site of metabolism that is
present in the majority of CYP2D6 substrates, although it is far from
being a universal requirement. For example, CYP2D6 can metabolizes
progesterone (8) and pregnenolone (9), and Guengerich et al.
have recently described a spirosulphonamide as a high affinity
substrate of CYP2D6, which lacks a basic nitrogen (10). Furthermore,
the enzyme can catalyze N-dealkylation reactions of
substrates such as deprenyl (11), amitriptyline (12), methamphetamine (13), the neurotoxin
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (14), and
dextromethorphan (15), a commonly used antitussive.
An ionic interaction between a negatively charged carboxylate group in
the active site of the enzyme and the basic nitrogen atom of substrates
has been proposed to be the key determinant of the specificity of
CYP2D6 reactions (16). A number of structural models have pointed to
aspartate 301 in the I helix being the specific residue involved (4,
16-18) (see Fig. 1). This is supported by mutagenesis studies that
showed that substitution of Asp-301 by neutral residues leads to a
marked reduction in catalytic activity against "classical" CYP2D6
substrates (17, 19, 20), although it has little effect on activity
against a substrate lacking a basic nitrogen (10). However, it is not
clear if the carboxylate of this residue interacts directly with the
basic nitrogen of the substrate within the active site as generally
assumed, or whether it has a structural role, perhaps in the
positioning of the B'-C loop in the active site (21), and is therefore
an indirect determinant of substrate specificity (20).
Our recent modeling studies (21), which incorporate information from
the recently determined crystal structure of rabbit 2C5 (22), indicate
that a second acidic residue, Glu-216, is in a position where it may
play a role in the binding of the basic nitrogen of CYP2D6 substrates
(Fig. 1). We now report site directed mutagenesis studies of Glu-216 and Asp-301 aimed at clarifying their
respective roles in CYP2D6 substrate specificity and
regiospecificity.
Chemicals
Terrific Broth (TB; 12g/liter bacto-tryptone, 24g/liter
bacto-yeast extract, and 4ml/liter glycerol), chloramphenicol,
Mutagenesis
The isolation of the cDNAs and construction of expression
plasmids ompA 2D6(His7) (pB81) and pJR7 (human P450
reductase) have been described elsewhere (23-25). Site-directed
mutagenesis was performed using the single-stranded DNA template method
(26) using pB81 as a template, the dut-
ung-E. coli strain CJ236, and the mutating
oligonucleotide. A list of the oligonucleotides used with the mutated
nucleotides underlined is as follows: E216Q, 3'-cag aaa gcc cga
ctg ctc ctt cag tcc; E216D, 3'-cag aaa gcc cga
gtc ctc ctt cag tcc; E216F, 3'-cag aaa gcc cga
gaa ctc ctt cag tcc; E216A, 3'-cag aaa gcc cga
cgc ctc ctt cag tcc; E216K, 3'-cag aaa gcc cga
ctt ctc ctt cag tcc; D301E, 3'-ggc aga gaa cag
ctc agc cac cac tat; D301Q, 3'-ggc aga gaa cag
ctg agc cac cac tat; D301N' 3'-ggc aga gaa cag
gtt agc cac cac tat and D301A, 3'-ggc aga gaa cag
ggc agc cac cac tat. Note, all oligonucleotides are reverse
complement sequences and written in the direction 3' to 5'. The
presence of the desired mutations was confirmed by automated DNA sequencing.
Coexpression of the P450s and P450 Reductase in E. coli
Expression was carried out essentially as previously described
(25). Briefly, pB81 and pB81-mutant plasmids were co-transfected with
pJR7 into the E. coli strain JM109. Cultures were grown in TB at 30 °C until the A600 reached
>0.8, whereupon the heme precursor Enzyme Assays
All reactions were carried out in triplicate at 37 °C with
shaking. HPLC analysis was carried out using a Hewlett Packard 1100 HPLC and Chemstation software.
Bufuralol 1'-Hydroxylation--
Reactions were carried out in 50 mM potassium phosphate, pH 7.4, 0-800 µM
bufuralol, 10 pmol P450, and an NADPH-generating system (5 mM glucose 6-phosphate, 1 unit of glucose 6-phosphate dehydrogenase, 1 mM NADP+) in a total volume of
300 µl. After 3 min of pre-incubation at 37 °C, reactions were
initiated with the addition of the NADPH-generating system and
incubated for a further 6 min before stopping with 15 µl of 60%
perchloric acid. Samples were incubated on ice for 10 min before
centrifugation at 16,100 × g for 10 min to remove particulate material. Routinely, 100-µl aliquots of the reaction supernatant were injected onto HPLC. Metabolites were separated using a
Hypersil ODS column (5 µm; 125 × 4.0 mm), flow rate, 1.0 ml/min. A step gradient was applied using 100 mM ammonium
acetate, pH 5 (Buffer A) and acetonitrile (Buffer B). The gradient
profile was 0 min = 73% A:27% B; 11.3 min = 60% A:40% B;
12.3min = 49% A:51% B; and 13.3 min = 73% A:27% B (all
v/v). The fluorescent metabolite 1'-hydroxy bufuralol was detected
using Dextromethorphan O- and N-Demethylation--
Assays were carried
out in 50 mM potassium phosphate, pH 7.4, 0-2000
µM dextromethorphan (for Km
determination), or 100 µM dextromethorphan (for the
regioselectivity studies), 10 pmol P450, and an NADPH-generating system
in a total volume of 200 µl. After a 3-min pre-incubation at
37 °C, reactions were initiated by the addition of the
NADPH-generating system and were incubated a further 6 min before
stopping with 100 µl of ice-cold methanol and 5 µl of 60%
perchloric acid. Samples were incubated on ice for 10 min before
centrifugation to remove particulate material. Metabolites were
separated by HPLC using a Hypersil C18 BDS column (5 µm; 250 × 4.6 mm) with a flow rate of 1 ml/min. Isocratic separation was used
with mobile phases of 100 mM ammonium acetate, pH 5, and
acetonitrile mixed at ratios of 78%:22% (v/v) respectively for the
separation of dextrorphan and 68%:32% (v/v) for 3-methoxymorphinan.
Metabolites were detected by fluorescence ( Testosterone 6 Diclofenac 4'-Hydroxylation--
Assays were carried out in 100 mM Tris-HCl, pH 7.4, 500 µM diclofenac (for
the specific activity determination) or 0-750 µM diclofenac (for kinetic analysis of the double mutant), 10 pmol P450,
and an NADPH-generating system in a total volume of 200 µl. Following
a 3-min pre-incubation at 37 °C, reactions were initiated by the
addition of the NADPH-generating system and incubated for 90 min (for
specific activities) or 4 min (for kinetics) before termination by the
addition of 200 µl of ice-cold methanol. Samples were incubated on
ice for 10 min before centrifugation to remove particulate material.
Metabolites were separated by HPLC using a Hypersil ODS column (5 µm;
250 × 4.6 mm) run isocratically at 1 ml/min with mobile phase of
20 mM potassium phosphate, pH 7, and acetonitrile
(77%:23% (v/v), respectively) and detected at 280 nm. 4'-Hydroxy
diclofenac formation was quantified using an authentic standard. A
minor peak corresponding to a 5'-hydroxy metabolite was seen in some
cases but not quantified.
Tolbutamide 4-Methylhydroxylation--
Assays were carried out
in 50 mM potassium phosphate, pH 7.4, 0-2000
µM tolbutamide, 10 pmol P450, and an NADPH-generating system in a total volume of 250 µl. Following a 3-min pre-incubation at 37 °C, reactions were initiated by the addition of the
NADPH-generating system and incubated for 6 min before termination by
the addition of 50 µl of 10% trichloroacetic acid. Samples were
incubated on ice for 10 min before centrifugation to remove particulate
material. Metabolites were separated by HPLC using a Hypersil ODS
column (5 µm; 250 × 4.6 mm). Step gradient separation using
acetonitrile (Buffer A), 10 mM sodium acetate, pH 4.3, (Buffer B) was used. The gradient profile was: 0 min = 32% A:68%
B; 11.5 min = 49% A:51% B; 12.5 min = 32% A:68% B (all
v/v), and the metabolite was detected at 230 nm. Tolbutamide
4-methylhydroxylation formation was quantitated using an authentic standard.
Nifedipine N-Oxidation--
Reactions were carried out in 100 mM potassium phosphate, pH 7.85, 0-1000 µM
nifedipine, 10 pmol P450, and an NADPH-generating system in a total
volume of 200 µl. After a 3-min pre-incubation at 37 °C, reactions
were started by the addition of the NADPH-generating system, and
samples were incubated for 10 min before stopping with 100 µl of
ice-cold methanol. Samples were incubated on ice for 10 min before
centrifugation at 16,100 × g for 10 min to remove particulate material. Aliquots of the supernatant were injected onto
HPLC. Metabolites were separated using a Hypersil ODS column (5 µm;
125 × 4.0 mm) and run isocratically at 1.0 ml/min using a mobile
phase of water:methanol:acetonitrile (45:25:30) (v/v/v). The oxidized
nifedipine metabolite was detected at 254 nm and quantitated with
reference to an authentic standard.
Modeling
The models of CYP2D6 were produced, and the dockings of
substrates performed as described previously (21). In brief, the models
of CYP2D6 were produced using comparative modeling (Modeler; Ref. 28)
with five structural templates Protein Expression--
A series of CYP2D6 mutants were
constructed in which the acidic amino acid residues Glu-216 or Asp-301
were replaced with acidic, basic, and uncharged residues. Additionally
both charged residues were neutralized in the double mutant
E216Q/D310Q. Levels of expression of the mutant P450s ranged from
150-645 nmol/liter E. coli culture compared with 300 nmol/liter for wild type CYP2D6. P450 reductase levels ranged between
289 and 467 nmol of cytochrome c reduced/min/mg of E. coli membrane. The D301A mutant failed to form spectroscopically
detectable P450, and the D301N mutant was found to be less stable than
the other mutant isoforms in the presence of sodium dithionite during
P450(Fe2+)-CO versus P450(Fe2+)
difference spectroscopy. Thus, mutations to the Asp-301 residue generally had a destabilizing effect on the enzyme, in agreement with
previous findings (20).
Metabolism of Typical CYP2D6 Substrates--
Kinetic analysis was
carried out on the mutant P450s co-expressed in E. coli with
human cytochrome P450 reductase using bufuralol and dextromethorphan,
two prototypical CYP2D6 substrates that each contain a basic nitrogen
(Fig. 2). The kinetic parameters for
bufuralol 1'-hydroxylation and dextromethorphan
O-demethylation are shown in Table
I. Conservative replacements of Glu-216
or Asp-301 with Asp and Glu, respectively, produced small (2-6-fold) increases in Km values and had negligible effects on kcat for both substrates. However, replacement
of either of the negatively charged side chains with a neutral group
had much larger effects, particularly on the Km
values.
Substitution of Asp-301 by asparagine or glutamine led to a 130- to
145-fold increase in Km values for bufuralol; for
dextromethorphan, the increase was 80-fold with the D301Q mutant but as
much as 1400-fold for D301N. For both substrates, the effects on
kcat were modest, ranging from a 30% decrease
to a 70% increase.
The role of Glu-216 has not hitherto been established experimentally.
Substitution of this residue by glutamine, alanine, or phenylalanine
had rather similar effects to the Asp-301 substitution as far as
bufuralol 1'-hydroxylation is concerned; Km was
increased by 100- to 170-fold, and kcat
increased by 40-70%, there being little effect of the bulk of the
neutral side chain at this position. For dextromethorphan
O-demethylation, the effects on Km were
smaller (10-25-fold increase), but kcat was decreased by as much as 5-fold in the E216Q mutant.
Substitution of both acidic residues by glutamine in E216Q/D301Q led to
larger increases in Km values for both bufuralol and
dextromethorphan than observed for the individual substitutions. However, the effects on the kcat values were
quite different. Both the individual E216Q and D301Q substitutions led
to an ~50% increase in kcat for bufuralol,
but the double mutant showed a 40% decrease in
kcat relative to the wild type (less than half the rate of either individual mutant). An analogous effect but in the
opposite direction was seen for dextromethorphan, where the individual
substitutions lead to a decrease in kcat by as much as 5-fold, while the double mutant shows an ~4-fold increase.
A clear difference between the two substrates is also seen in the
effects of replacing the negative charge at position 216 by a positive
charge in the E216K mutant. Such a mutation might be expected to show
the largest reduction in binding affinity for nitrogenous substrates
through repulsion of the basic nitrogen atom by the positive side
chain. For bufuralol, this mutant shows kinetic constants
indistinguishable from those of the neutral E216Q mutant. For
dextromethorphan, by contrast, there is a much greater increase in
Km values (120-fold as opposed to 11-fold) and a
greater decrease in kcat values (10-fold
versus 5-fold). Dextromethorphan, the more rigid substrate
experiences a larger differential effect.
Regiospecificity of Dextromethorphan Dealkylation--
To gain
further information on the effect of mutations on the positioning of
substrate in the active site, changes in the ratios of dextromethorphan
metabolites were analyzed. The major pathway of dextromethorphan
metabolism by CYP2D6 is O-demethylation, with the
formation of dextrorphan. However, CYP2D6 can also catalyze demethylation at the basic nitrogen to produce 3-methoxymorphinan (3MM)
in a concentration-dependent manner (15). To probe any changes in regioselectivity produced by the mutations, the ratio of the
two activities was measured at 0.1 mM dextromethorphan. The
wild type enzyme and the conservative mutants E216D and D301E produced
similar dextrorphan/3MM ratios of ~8:1, indicating a strong
preference for the O-demethylation pathway (Fig.
3). However, this ratio was substantially
decreased following removal of either of the negative charges,
consistent with a decreased preference for binding the basic nitrogen
close to Glu-216 and Asp-301, distant from the heme iron.
Dextrorphan:3MM ratios as low as 1:1 were observed, for the E216F,
E216K, D301N, D301Q, and E216Q/D301Q mutants.
Metabolism of Novel Substrates--
If the interaction of the two
acidic residues of CYP2D6 with the substrate is a key determinant of
the substrate specificity of the enzyme, specifically of its preference
for substrates containing a basic nitrogen, the mutants in which one or
both of these residues have been mutated might be expected to show a
broader substrate specificity. As an initial test of this, we measured
the ability of the mutants to oxidize two atypical CYP2D6 substrates
lacking a basic nitrogen. The compounds used were testosterone
(uncharged; a CYP3A4 substrate) and diclofenac (negatively charged; a
CYP2C9 substrate) (Fig. 2). Measured rates of formation of
6
The wild type enzyme did catalyze the hydroxylation of these two
atypical substrates, though at rates
Removal of the carboxyl groups from residues 216 and 301 had larger
effects on the diclofenac 4'-hydroxylase activity. The wild type,
E216D, and D301E enzymes produced only barely detectable quantities of
4'-hydroxy diclofenac (Table II), but a number of other mutants showed
significant increases in the rate of product formation. Mutants E216Q,
E216F, and D301N produced rates ~5-, 10-, and 22-fold higher than the
wild type enzyme, respectively, while the turnover rates of the E216A,
E216K, and D301Q derivatives were increased 50- to 75-fold. The double
mutant E216Q/D301Q produced the highest diclofenac 4'-hydroxylase
activity of 3.84 min
Since neutralization of both Glu-216 and Asp-301 in the double mutant
produced the highest levels of diclofenac activity, the kinetic
parameters of this mutant were determined for diclofenac, tolbutamide,
a CYP2C9 substrate which lacks a carboxylate group, and nifedipine, a
non-steroidal CYP3A4 substrate (see Fig. 2 for structures). As shown in
Table III, the
kcat values for each of these three compounds,
which are not normally metabolized to a significant extent by CYP2D6,
ranged between 1-5 min CYP2D6 shows a clear, though not exclusive, preference for the
metabolism of basic substrates that contain a nitrogen atom protonated
at physiological pH. A common hypothesis has been that this may be due
to electrostatic interactions of the protonated atom with the
carboxylate group of Asp-301 (4, 16-20). Several studies have pointed
to a possible role for a second carboxylate group, that of Glu-216, as
a binding determinant, principally to explain the metabolism of the
larger substrates with a basic nitrogen atom Comparative analysis of the alignment of P450 sequences has shown that
Asp-301 is present in a number of P450s that do not metabolizes basic
substrates, such as members of the 2C family that are known to
hydroxylate steroids (21). Glu-216 is also not sufficient by itself to
confer a preference for basic substrates as it is present without
Asp-301 in P450s that do not metabolizes basic substrates, such as the
CYP73A family that preferentially metabolizes cinnamic acid (21).
However, basic substrates are metabolized by all four known P450s that
contain a residue equivalent to both Glu-216 and Asp-301; CYP2D6,
CYP2D14 (bovine), CYP2D4 (rat), and CYP2J1 (rabbit). Thus indicating
strongly that if both Asp-301 and Glu-216 are present, basic substrates
appear to be preferred and both residues are important for the
metabolism of these substrates.
It is clear from the results presented here, that removal of the
negative charge at either position 216 or position 301 significantly increases the Km values for the
classical CYP2D6 substrates bufuralol and dextromethorphan (Table I).
This shows clearly that the role of Glu-216 is thus not limited to
substrates in which the basic nitrogen is relatively remote from the
site of oxidation. It is apparent that the relative importance of these two residues is different for the two substrates studied; the Km for bufuralol is similarly affected by the
neutralization of either Glu-216 or Asp-301, whereas for
dextromethorphan the substitution of Asp-301 clearly has a larger
effect than that of Glu-216. The models of the CYP2D6-bufuralol and
CYP2D6-dextromethorphan complexes (Fig. 1) give insight into a possible
explanation for this. The basic nitrogen of both bufuralol and
dextromethorphan is predicted (Fig. 1) to lie close to the negatively
charged carboxylate group of Glu-216, but relatively distant from that
of Asp-301. This in turn suggests the existence of an ionic interaction
between Glu-216 and the basic nitrogen The models suggest that the role of Asp-301 is also electrostatic in
nature but that this interaction is weaker than that with Glu-216.
Despite this weaker interaction, the effect on Km of
mutating Asp-301 is at least as great as when Glu-216 is mutated. The
models provide a possible explanation of this apparent paradox; they
suggest that Asp-301 is responsible for maintaining the integrity of
the active site via a hydrogen bond with the backbone amides of two residues in the B-C loop, Val-119 and Phe-120. Given this scenario, it might be expected that the mutation D301N would behave similarly to wild type; the modeling (consistent with the experimental Km) suggests that this might not be the case because the Glu-216 and Asp-301 also play an important role in the regiospecificity
of CYP2D6 toward dextromethorphan. The wild type enzyme shows a strong
(8-fold) preference for O-demethylation over
N-demethylation, but this is essentially abolished by
mutation of either or both of these residues to neutral side chains.
These observations are consistent with the idea that the negatively
charged "patch" in the active site created by these two residues
(notwithstanding the structural rearrangement we suggest could result
from removal of the negative charge on Asp-301) is the principal
determinant of the regiospecificity of CYP2D6 toward dextromethorphan
and probably also toward other substrates that contain a basic
nitrogen. While there are thus marked effects of both these negatively
charged residues on the binding of basic substrates, it is also clear that for some neutral substrates, such as the spirosulphonamide studied
by Guengerich et al. (10), the position of the bound substrate and/or its binding interactions with other residues in the
active site are such that mutation of Asp-301 has little effect on binding.
A striking demonstration of a different aspect of the importance of
Glu-216 and Asp-301 in determining the substrate specificity of CYP2D6
is afforded by the activity of the E216Q/D301Q mutant toward a number
of compounds that are not normally metabolized to a significant extent
by this enzyme. The double mutation increased the rate of
4'-hydroxylation of diclofenac 1000-fold to a
kcat value within a factor of five of that of
CYP2C9, which plays the dominant role in diclofenac metabolism in man
(38). Similar comparisons showed that CYP2D6 E216Q/D301Q metabolizes
tolbutamide almost as well as CYP2C9 and nifedipine almost as well as
CYP3A4 (although its rate of metabolism of testosterone fell well short of that of CYP3A4). These observations indicate that the size and shape
of the CYP2D6 active site is such as to allow it to bind a wide range
of compounds. (This range does not apparently extend to molecules the
size of testosterone; we have earlier shown that testosterone binding
is facilitated by the substitution of Phe-483 by a smaller residue
(23)). The binding site of CYP2D6 is thus intrinsically rather
promiscuous in nature, and the two residues Asp-301 and Glu-216, while
they are obviously not the only factor, do play a crucial role in
defining the CYP2D6 substrate specificity not simply by favoring the
binding of basic substrates but also by discriminating against acidic
substrates. It will be important to take into account this dual role of
these two key residues in further development of predictive models of
the specificity of CYP2D6.
1) along with
nifedipine (Km 28 µM,
kcat 2 min
1) and tolbutamide
(Km 315 µM,
kcat 1 min
1), which are not
normally substrates for CYP2D6, were within an order of magnitude of
those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and
Asp-301 thus effectively alters substrate recognition illustrating the
central role of the negative charges provided by both residues in
defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The active site region of our models
of CYP2D6. A, the hydrogen bonds between the carboxyl
of Asp-301 and the backbone amides of Val-119 and Phe-120.
B, the docked position of dextromethorphan. C,
the docked position of bufuralol.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-aminolevulinic acid, dithiothreitol, glucose 6-phosphate,
NADP+, phenylmethylsulfonyl fluoride, sodium dithionite,
cytochrome c, dextromethorphan, diclofenac, tolbutamide,
nifedipine, and testosterone were all purchased from Sigma.
Ampicillin was obtained from Beecham Research (Welwyn Garden City, UK),
and isopropyl
-D-thiogalactopyranoside from Melford
Laboratories (Ipswich, UK). Glucose 6-phosphate dehydrogenase (type
VII) was from Roche Molecular Biochemicals. HPLC grade solvents were
purchased from Rathburn Chemicals (Walkerburn, UK), and HPLC columns
from Agilent (Crawford Scientific, UK). DNA modifying enzymes were
obtained from Invitrogen and Promega. Dextrorphan, 3-methoxymorphinan, 6
-hydroxy-testosterone, bufuralol, 1'-hydroxy bufuralol, nifedipine N-oxide, hydroxy-tolbutamide, and 4'-hydroxydiclofenac were
purchased from Ultra Fine Chemicals (Manchester, UK). All other
chemicals were from BDH (Poole, UK). Library-efficient competent
Escherichia coli JM109 were purchased from Promega.
-aminolevulinic acid was
added to a final concentration of 1 mM. Induction was
initiated with the addition of
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 1 mM. Cultures were grown until the appearance of P450 in the CO reduced spectra of whole cells (usually 24 h), at which point cells were harvested. Sphaeroplasts were prepared and sonicated, and the membrane fraction pelleted by ultracentrifugation at 100,000 × g. Membranes were
resuspended in TSE buffer (50 mM Tris, pH 7.6, 250 mM sucrose, 10% glycerol), and the P450 content determined
by P450(Fe2+)-CO versus P450(Fe2+)
difference spectra. P450 reductase activity was estimated by NADPH-dependent cytochrome c reduction (27).
Membranes were stored at
70 °C until required.
ex = 252;
em = 302 and quantitated
with reference to an authentic standard.
ex = 270;
em = 312) and quantitated with reference to authentic standards.
-Hydroxylation--
Assays were carried out in
50 mM Hepes, pH 7.4, 400 µM testosterone, 30 pmol P450, and an NADPH-generating system in a total volume of 200 µl. After a 3-min pre-incubation at 37 °C, reactions were
initiated by the addition of the NADPH-generating system and incubated
for 3 h before stopping with 100 µl of ice-cold methanol and 5 µl of 60% perchloric acid. Samples were incubated on ice for 10 min
before centrifugation to remove particulate material. Metabolites were
separated by HPLC using a Hypersil ODS (5 µm; 250 × 4.6 mm)
column with a flow rate of 1 ml/min. Step gradient separation using
acetonitrile (Buffer A), water (Buffer B), and methanol (Buffer C) was
used. The gradient profile was: 0 min = 2.5% A:45% B:52.5% C; 8 min = 2.5% A:25% B:72.5% C; 11.5 min = 2.5% A:45%
B:52.5% C (all v/v/v). The run was terminated after 15 min. Products
were detected at 240 nm and quantified with respect to an authentic
6
-hydroxytestosterone standard.
P450s cam, terp, eryF, BM3, and 2C5.
The substrates were then docked into the active site of CYP2D6 using
GOLD (29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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[in a new window]
Fig. 2.
Structures of probe substrates used in this
study. Sites of metabolism investigated are indicated with an
asterisk.
Kinetic parameters for bufuralol 1'-hydroxylation and dextromethorphan
O-demethylation
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Fig. 3.
Relative amounts of dextrorphan and
3-methoxymorphinan produced from dextromethorphan by the action of wild
type and mutant CYP2D6. Assays were performed using 100 µM dextromethorphan as described under "Materials and
Methods." The error bars represent the average deviation
from duplicate incubations.
-hydroxytestosterone and 4'-hydroxy diclofenac, the products formed
from these substrates by CYP3A4 and CYP2C9, respectively, are
shown in Table II.
Specific activities of E. coli membranes expressing wild type and
mutant CYP2D6 for testosterone and diclofenac
100-fold less than the rates
observed with bufuralol or dextromethorphan. Most of the mutants showed
only modest increases (up to 4-fold) in the rate of formation of
6
-hydroxy-testosterone compared with wild type; the largest effect
was seen for the E216F mutant, which showed a 10-fold increase. These
relatively modest effects most probably reflect the steric constraints
on the binding of the bulky testosterone molecule as discussed earlier
(23).
1, around 1000-fold higher than wild
type; interestingly, its testosterone 6
-hydroxylase activity was
increased only 2-fold over wild type. The rate of formation of
4'-hydroxy diclofenac was not significantly greater with the E216K
mutant than with E216A, suggesting that the carboxylate group of the
substrate is not positioned near this residue.
1, indicating that the mutations
have substantially altered the specificity of the enzyme. Although the
E216Q/D301Q mutant showed a 2-fold increase in testosterone
6
-hydroxylase activity compared with the wild type enzyme (Table
II), this is still 1000-fold lower than that observed for than CYP3A4
expressed in an equivalent system (30). By contrast, the
N-oxidation of nifedipine by the E216Q/D301Q mutant is
described by kinetic parameters much closer to values previously
reported for CYP3A4 (Km 28 versus 9 µM, kcat 2 versus 6.5 min
1; Ref. 27). For the two CYP2C9 substrates, the
kcat values measured with the CYP2D6 E216Q/D301Q
mutant are only a factor of 2-5 lower than those reported for CYP2C9
itself (31, 32), and the Km value for tolbutamide is
comparable to that reported for CYP2C9 purified from human liver (32).
However, it should be noted that the Km value for
diclofenac is significantly higher (36-fold) than the average reported
values (33) and that the catalytic efficiency
(kcat/Km) much lower (around
1%) than that of CYP2C9.
Kinetic parameters of CYP2D6 E216Q/D301Q acting on atypical
CYP2D6 substrates
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
10 Å from the site of
oxidation (34-37). The proposed role for Glu-216 is consistent with
our recent modeling study of CYP2D6 (21). This model also suggests a
structural role for Asp-301, through the formation of a hydrogen bond
with a residue in the flexible B'-C loop. This latter hypothesis is
consistent with the fact that we and others (20) find that mutations to Asp-301 generally result in a less stable protein. It should be noted,
however, that these mutations do not lead to a marked decrease in the
kcat of the enzyme toward substrates having a
basic nitrogen or to the specific activity toward atypical substrates
(Tables I and II); this suggests that any structural changes produced by mutation of this residue are unlikely to be gross.
an interaction that is removed when Glu-216 is replaced by anything other than Asp, hence the ~100-fold increase in Km.
amide is repelled by the backbone amide of Phe-120, and therefore in the D301N mutant the B-C loop, also known as substrate recognition site 1 (SRS 1), adopts a different conformation to that in
the wild type enzyme. The D301Q mutant has an extra degree of freedom
with respect to D301N, and modeling suggest that this substitution can
be accommodated without the same need for structural rearrangement of
the B-C loop as with D301N. The relative rigidity of dextromethorphan,
and thus its decreased ability to adapt to changes in the active site,
could explain why mutation of Asp-301 has a greater effect on the
binding of dextromethorphan than on that of bufuralol. It is notable
that the kcat values are relatively unchanged
while the Km values are markedly increased. This
suggests that, although these substrates bind much more weakly, they
bind in a position and orientation appropriate for catalysis.
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ACKNOWLEDGEMENTS |
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This work was funded by the Drug Metabolism Consortium (AstraZeneca, Aventis, Boehringer-Ingelheim, Celltech, GlaxoSmithKline, Hoffmann-La Roche, Johnston and Johnston Pharmaceuticals, Merck Sharp and Dohme, Novartis, Novo Nordisk, Pfizer, Pharmacia, and Wyeth) and United Kingdom Medical Research Council Grant G9203175.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 44-0-1382-632-621; Fax: 44-0-1382-668278; E-mail: margaret.rooney@cancer.org.uk.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M209519200
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ABBREVIATIONS |
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The abbreviations used are: CYP2D6, cytochrome P450 2D6; HPLC, high pressure liquid chromatography; 3MM, 3-methoxymorphinan.
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