(Received for publication, September 7, 1995; and in revised form, October 16, 1995)
From the
Model building studies have intimated a role for aspartic acid 301 in the substrate binding of cytochrome P450 2D6 (CYP2D6). We have tested this hypothesis by generating a range of CYP2D6 mutants substituting a variety of amino acids at this site. The mutant proteins, which included substitution with a negatively charged glutamic acid residue or neutral asparagine, alanine, or glycine residues, were expressed in Saccharomyces cerevisiae. In addition, a mutant where aspartic acid 301 was deleted was also tested. All the mutants expressed approximately equivalent amounts of recombinant apoprotein and, apart from the alanine 301 and the aspartic acid 301 deletion mutants, gave carbon monoxide difference spectra of similar magnitude to the wild type. In the cases of the alanine and deletion mutants, the amount of holoprotein was significantly reduced or absent relative to the amount of apoprotein, indicating restricted heme incorporation. The glutamic acid mutant was shown to have similar catalytic properties to the wild type enzyme toward the substrates debrisoquine and metoprolol; however, some differences in regioselectivity and ligand binding were observed. The mutants containing neutral amino acids at position 301 exhibited marked reductions in catalytic activity. At low substrate concentrations little, if any, activity toward debrisoquine and metoprolol was measured. However, at a higher substrate concentration (2 mM) some activity was observed (about 10-20% of wild type levels). Consistent with the above findings, the debrisoquine-induced spin changes in the mutant proteins were markedly reduced. These data collectively demonstrate that aspartic acid 301 plays an important role in determining the substrate specificity and activity of CYP2D6 and provide experimental evidence supporting the role of this amino acid in forming an electrostatic interaction between the basic nitrogen atom in CYP2D6 substrates and the carboxylate group of aspartic acid 301.
Cytochrome P450 2D6 (CYP2D6) ()mediates the
metabolism of over 30 drugs of wide therapeutic use including many
antiarrhythmics, antidepressants,
-adrenergic antagonists,
neuroleptics, and analgesics(1) . Although structurally
diverse, all known ligands (substrates and inhibitors) of CYP2D6
possess a basic nitrogen, usually either an amine or a guanidino group,
which is presumed to be protonated when the ligand is bound in the
active site of the enzyme(2) . Furthermore, substrate-template
models (3, 4, 5) have revealed that this
basic nitrogen is normally located 5-7 Å from the site of
oxidation in the substrate molecule. A pharmacophore based on
competitive inhibitors of CYP2D6 (6) also complies with this
model; the positively charged nitrogen of such inhibitors is distanced
up to 7.5 Å from a flat hydrophobic region of the inhibitor.
Based on this conformity, it has been proposed that the basic nitrogen
of CYP2D6 substrates and inhibitors interacts with a negatively charged
residue, such as aspartate or glutamate, in the active site of enzyme
and that this electrostatic interaction facilitates binding and
orientation of the ligand in the active
site(3, 4, 5, 6, 7) .
Recent computer-derived homology models of the active site of CYP2D6 (4, 8, 9) , ()based on alignment
with the crystal structures of the bacterial P450s CYP101
(P450
) or CYP102 (P450
) have identified
aspartic acid 301 (Asp
) as a candidate residue for the
proposed electrostatic interaction with the ligand. Such homology
models and a recent structure-based alignment (10) locate
Asp
in the central region of the I-helix of CYP2D6. This
region maps to one of the substrate-recognition sites (SRS4) identified
by Gotoh (11) as being important in substrate binding in the
CYP2 family of P450s; these substrate-recognition site regions may also
be predictive across other P450 families(10) .
In three bacterial P450s for which crystal structures are known (12, 13, 14) , the central region of the I-helix is one of the most spatially conserved areas of the P450 core(10) . In these P450s, it is located close to the heme moiety and runs across the distal face of the heme, completely or partially covering pyrrole ring B(10) . Several residues in the central region of the I-helix have also been shown by mutation to play a role in substrate specificity and/or reaction kinetics(15, 16, 17) . Recent work also indicates that residues in this region may play a role in the supply of catalytic protons via helix-associated solvent molecules(10) .
In this report we provide experimental evidence, through the use of
site-directed mutagenesis, that Asp is of critical
importance in the efficient oxidation of substrates by CYP2D6. The data
are consistent with the proposal that Asp
forms an ion
pair with the basic nitrogen of CYP2D6 ligands, facilitating binding
and orientation in the active site.
Yeast cells transformed with wild type and Asp mutant forms of CYP2D6, with the exception of D301
,
expressed similar amounts of immunodetectable microsomal bound CYP2D6
apoprotein (Fig. 1). The CYP2D6 holoprotein (heme-containing)
content of microsomes, as determined by carbon monoxide-difference
spectroscopy, varied depending on the mutant (representative spectra
are shown in Fig. 2). No holoprotein was detectable in
microsomes from the D301
mutant, and a marked decrease (90%) was
found in microsomes prepared from D301A (8 ± 4 pmol/mg protein)
relative to wild type (49 ± 15 pmol/mg protein). The holoprotein
content of microsomes prepared from the other mutants (D301E, 45
± 22 pmol/mg protein; D301N, 29 ± 9 pmol/mg protein;
D301G, 40 ± 12 pmol/mg protein) was comparable with that of wild
type (Fig. 2). (The preceding mean ± S.D. P450 values
were determined from at least 10 microsomal preparations in each case.)
A Soret absorption maximum of 448 nm was observed with the carbon
monoxide complex of wild type and D301E microsomes. This was shifted
slightly to 450 nm with microsomes prepared from the D301N and D301G
mutants and to 454 nm with the D301A mutant. Variable amounts of P420
were detectable in microsomes prepared from the D301A mutant only (Fig. 2).
Figure 1:
Immunoblots of microsomes derived from S. cerevisiae expressing wild type and Asp
mutant forms of CYP2D6. 20 µg of microsomal protein was applied to
each lane. The control lane refers to microsomes derived from
yeast cells transformed with plasmid lacking a CYP2D6 gene.
Positions of molecular mass markers are indicated
(kDa).
Figure 2:
Reduced carbon monoxide difference spectra
of microsomes derived from yeast cells expressing wild type and
Asp mutant forms of CYP2D6. Wild type (-),
D301E(- - -), D301N (
), D301A
(
-
), D301G (- - -), and D301
(
) spectra were conducted with an equivalent amount of
microsomal protein (5 mg) except D301A (15
mg).
Debrisoquine-induced difference spectra (type I)
were obtainable only with microsomes prepared from yeast cells
expressing wild type and D301E forms of CYP2D6 (K,
13 ± 5 and 35 ± 21 µM, respectively, and
A
, 55 ± 2 µmol and 41 ±
19 µmol P450
, respectively; each is a mean
± S.D. of three microsome preparations). Debrisoquine
concentrations in excess of 500 µM failed to induce
discernible spectral changes with microsomes from the D301N or D301A
mutants. In contrast, quinidine, a potent specific inhibitor of CYP2D6,
induced a type I difference spectrum with microsomes from the D301N (K
, 99 ± 80 µM;
A
, 35 ± 12 µmol
P450
) and D301A (K
,
163 ± 113 µM;
A
, 208
± 103 µmol P450
) mutants, in addition to
wild type (K
, 0.08 ± 0.06 µM;
A
, 97 ± 8 µmol
P450
) and D301GE mutant (K
, 0.72 ± 0.31 µM;
A
, 51 ± 10 µmol
P450
; all values represent mean ± S.D. of
three microsome preparations). Microsomes from D301G and D301
mutants were not tested in these binding studies.
Catalytic
activity, as assessed by the oxidation of 250 µM debrisoquine and 40 µM (±)-metoprolol, was
virtually absent (1-2% of wild type) in microsomes prepared from
yeast cells expressing the mutant forms of CYP2D6, with the exception
of D301E, which retained rates of activity comparable with that of the
wild type (see Table 2). However, the regioselective oxidation of
(±)-metoprolol, as assessed by the ratio of formation of ODM and
-OH metabolites, was significantly different with microsomes
prepared from the D301E mutant compared with the wild type (8.5:1 and
3.8:1, respectively; p < 0.005) (Table 1). An
alteration in the regioselective oxidation of metoprolol was also
apparent with the R-(+)- and S-(-)-enantiomers (Table 2). In contrast,
enantioselective oxidation was not altered by the substitution of
Asp
with Glu (Table 3). Thus, although O-demethylation was significantly R-enantioselective
and
-hydroxylation showed a preference for S-(-)-metoprolol, the pattern and extent of this
enantioselectivity were similar with microsomes prepared from wild type
and D301E mutant (Table 2).
When the substrate concentration
of (±)-metoprolol was increased 50-fold from 40 µM to 2 mM, significant formation of ODM and -OH
metabolites was observed in microsomes derived from all of the
Asp
mutants, with the exception of the D301
(Table 3). In contrast to the insignificant level of oxidation of
metoprolol at 40 µM, rates of formation of ODM and
-OH equivalent to 20-22% and 10-20% of wild type
rates, respectively, were observed with microsomes derived from the
D301N, D301A, and D301G mutants. Although highly variable, the
regioselective oxidation of the substrate was also altered in the D301N
and D301A mutants, while D301G retained the same regioselectivity as
wild type (Table 3). The regioselectivity of metoprolol oxidation
by D301E at a concentration of 2 mM was the same as that
determined at 40 µM ( Table 1and Table 3).
The results demonstrate that Asp is a critical
residue in the catalytic function of CYP2D6. Substitution of this
carboxylate residue with a similar functional moiety (Glu) did not
influence the catalytic competence of the enzyme significantly,
although a subtle change in the regioselective oxidation of metoprolol
and a 10-fold reduction in quinidine binding was observed. In contrast,
substitution of Asp
with neutral amino acids (Asn, Ala,
Gly), differing in size and polarity, resulted in marked reductions in
catalytic activity. While it appears that a negative charge at amino
acid residue 301 is important for efficient CYP2D6 catalysis, the
precise role of the negative charge cannot be ascertained from these
results alone. Nevertheless, the data do support the proposal that the
carboxylate anion of Asp
forms a charge pair with the
positively charged substrate nitrogen and, by doing so, facilitates the
binding and orientation of the ligand in the active site.
The ligand
binding role of Asp is substantiated by the observation
that replacement of the negatively charged residue with a neutral side
chain results in substantial decreases in the binding capacity of
debrisoquine (loss of type I spectrum) and quinidine (1000-fold greater K
value). The requirement of a negative
charge at position 301 for substrate binding can also be inferred from
the catalytic data, which show that substantially higher concentrations
of metoprolol were required to achieve significant catalytic activity
in the mutants in which Asp
was replaced with a neutral
residue. Although full kinetic analyses have not been conducted, these
results are indicative of a decrease in the affinity of the enzyme for
the substrate. In addition, a reduction in the V
value of the mutated enzyme cannot be precluded.
The altered
regioselective oxidation of metoprolol in the D301E mutant compared
with wild type suggests a slightly different orientation of the
substrate in the active site of the enzyme. As no gross change in the
integrity of the active site was apparent in this mutant (as evidenced
by a normal Soret absorption maximum of 448 nm, good heme
incorporation, and retention of catalytic activity), the altered
regioselectivity could be due to a subtle difference in the location of
the substrate oxidation sites relative to the (Fe-O) entity, as a consequence of the extension of the carboxylate
residue by a methylene group. Implicit in such a rationale is an
interaction between a carboxylate residue in the active site of the
enzyme and a positive charge of the substrate molecule. Thus the
observed alteration in the regioselective oxidation of metoprolol by
the D301E mutant adds weight to the proposal that Asp
serves as a negatively charged substrate-contact residue in the
active site of CYP2D6. The lack of effect of the D301E substitution on
the enantioselective oxidation of metoprolol indicates that a
residue(s) other than Asp
is a determinant of CYP2D6
chiral selectivity. A candidate amino acid residue for such a role is
Ser-304.
When Asp was replaced with a
neutral residue (Asn, Ala, or Gly), the structural integrity of the
active site was also perturbed to varying degrees (as seen by the
slight shift in the Soret absorption maximum of the carbon monoxide
complex and in the different extent of heme incorporation). Thus an
alternative explanation for the requirement of an anionic residue at
position 301 for substrate binding is that this amino acid helps to
maintain the integrity of the active site and that in its absence the
topography of the site is altered. The different effects of the Ala and
Gly substitutions on heme incorporation are difficult to explain.
However, while both residues can be classed as neutral, Gly with only a
hydrogen atom as a side chain can adopt a much wider range of main
chain conformations than other residues and hence may accommodate and
minimize potentially deleterious structural changes in the active site.
The greater conformational flexibility of glycine is due to the lack of
steric hindrance between its side chain atoms and the polypeptide main
chain, thus allowing greater rotation around the C
-C` and
the N-C
bonds of a glycine residue(27) . In this
context, it may be significant that the central region of the I-helix
of CYP2D6, in which Asp
is located, comprises a sequence
of non-polar residues (Leu
-Val
)
interspersed at regular intervals with polar residues
(Arg
, Asp
, Ser
). Modeling
indicates that these polar residues all point in the direction of the
proposed active site and potentially form one of its boundaries. (
)Thus, the introduction of a non-polar residue (Ala) into
this polar zone may drastically perturb the local environment adjacent
to the active site and directly or indirectly influence heme
incorporation. This may not arise when Gly is the substituent due to
the adaptable nature of this residue resulting in minimal
conformational change of the active site.
Koymans et al. (8) have proposed Asp as an alternative
carboxylate residue for the interaction with the basic nitrogen of
CYP2D6 ligands. However, a recent homology model of the active
site
was unable to rationalize the involvement of this
residue because of its peripheral location in the site. Furthermore,
site-directed mutagenesis studies of Asp
have confirmed
that the substitution of this residue with neutral amino acids (Asn or
Ala) does not adversely influence the catalytic competence of the
enzyme. (
)
In the absence of a substrate-bound crystal
structure of CYP2D6, the precise role(s) of Asp in CYP2D6
catalysis will remain unproven. Nevertheless, the present data strongly
support the proposal that Asp
is a ligand-binding residue
in the active site of CYP2D6, interacting via a charge pair with the
positively charged nitrogen of CYP2D6 ligands.