(Received for publication, August 18, 1994; and in revised form, October 18, 1994)
From the
The stereoselectivity of cytochrome P450 hydroxylation has been investigated with the enantiomerically
pure substrate analog norcamphor. (1R)- and
(1S)-norcamphor (>92 enantiomeric excess) were
characterized in the hydroxylation reaction with cytochrome
P450
with respect to the product profile, steady state
kinetics, coupling efficiency, and free energy of substrate
dissociation. The experimental results demonstrate regiospecificity
that is enantiomer-specific and confirm our previously reported
prediction that (1R)-norcamphor is hydroxylated preferentially
at the 5-carbon and (1S)-norcamphor at the 6-carbon (Bass, M.
B., and Ornstein, R. L.(1993) J. Comput. Chem. 14,
541-548); these simulation results are now compared with
simulations involving a ferryl oxygen intermediate. Hydroxylation of
(1R)-norcamphor was found at the 5-, 6-, and 3-carbons in a
ratio of 65:30:5 (respectively), whereas (1S)-norcamphor was
oxidized to produce a 28:62:10 ratio of the same products. With the
exception of the regiospecificity, all of the reaction and physical
parameters are similar for each enantiomer of norcamphor. These results
show that the position of the carbonyl group on the hydrocarbon
skeleton of norcamphor plays a role in determining the average
orientation of this substrate in the active site and suggests that
hydrogen bonding interactions can aid in directing the regiospecificity
and stereospecificity of the hydroxylation reaction catalyzed by
cytochrome P450
.
The superfamily of monooxygenase enzymes referred to as cytochrome P450 plays a critical role in a variety of biological processes, including clearance of xenobiotics, steroid synthesis, and lipid peroxygenation(1) . Many of the exogenous small molecules metabolized by this class of enzymes are of great clinical and commercial importance in both animals and plants(2) . While different enzymes, or ``forms,'' of the cytochrome P450 system are responsible for oxidizing various substrates, the chemical substituent (heme-thiolate ligation) and mechanism of oxidation appear to be largely invariant(3, 4) . Thus, the device by which the protein matrix determines the substrate specificity of a given enzyme within the expansive superfamily of cytochrome P450s has been an area of active investigation(5, 6, 7, 8, 9, 10, 11) .
The mammalian enzymes are membrane-bound and as such have been resistant to crystallization and detailed structural characterization. In the absence of atomic coordinates, topology mapping of the active site by means of suicide substrates and amino acid scanning via site-directed mutagenesis are being used to determine the general characteristics of cytochrome P450 active sites(9, 10, 11, 12, 13, 14, 15) . Still, the analysis of specific electrostatic, hydrophobic, and steric contributions to the reaction efficiency and oxidative specificity cannot be accomplished without detailed structural information.
The
x-ray structure of the bacterial cytochrome, P450, was
the first to become available nearly a decade ago(16) . This
enzyme catalyzes the 5-exo hydroxylation of d-camphor
as the first in a series of oxidative steps which allow its host, Pseudomonas putida, to live with camphor as its sole source of
carbon. As it has been available in large quantities for many years,
P450
has served as the prototypical cytochrome P450 in
providing detailed chemical, physical, and structural information for
the superfamily(7) . The x-ray structure of cytochrome
P450
reveals that the active site is a buried cleft,
essentially isolated from bulk solvent(17) . There is a single
hydrogen bond formed between Tyr-96 and the ketone of camphor that
enhances substrate affinity and influences substrate positioning in the
active site(30, 32) . Extensive hydrophobic contacts
are also made with camphor, and the shape of the active site is highly
complementary to the three-dimensional structure of this molecule.
Recently, cytochrome P450
has been the subject of several
theoretical studies of protein-small molecule
recognition(18, 19, 20, 21, 22, 23, 24, 25) .
Our efforts to characterize structure-function relationships in the
enzyme-substrate complex of P450
have focused on
utilizing camphor analogs to probe specific features of the active
site(26, 27, 28, 29, 30, 31, 32, 33, 34) .
In previous reports, racemic norcamphor was used to characterize the
contribution of hydrophobic contacts to the regiospecificity of the
hydroxylation reaction(33) . However, the interpretation of
these experiments is complicated by the fact that racemic substrate was
used; therefore, the observed changes in the reaction specificity
cannot be unequivocally attributed to specific structural features of
the alternate substrate(35, 36) . Herein we describe
investigations which utilize the pure stereoisomers of norcamphor to
unambiguously evaluate the role of steric contacts and hydrogen bonding
interactions in the P450
-substrate complex. The
experimental results verify predictions made previously by Bass and
Ornstein (37) that (1R)-norcamphor is hydroxylated
preferentially at the 5-carbon and (1S)-norcamphor at the
6-carbon.
The preparation of (1R)- and
(1S)-norcamphor has been reported previously. ()Other chemicals were purchased from Aldrich and Sigma.
Cytochrome P450
, putidaredoxin, and putidaredoxin
reductase were overexpressed in E. coli and purified according
to previously established procedures(38, 39) .
The
cytochrome P450 reaction system was reconstituted under the following
conditions: 1 µM P450, 5 µM putidaredoxin, 1
µM putidaredoxin reductase, 50 mM Tris-HCl, pH
7.0, 200 mM KCl, 500 µM substrate, 250 µM NADH in a final volume of 1 ml. Steady state reaction kinetics
were monitored spectrophotometrically by the oxidation of NADH
( = 6.23 mM
cm
). Each reaction mixture was extracted three
times with an equal volume of methylene chloride, and the combined
organic portion was dried over anhydrous magnesium sulfate and
concentrated under a slow stream of dry nitrogen. The product profile
was determined by GC analysis of the reaction extract (J & W DB-5,
inner diameter: 0.32 mm, 30 m; 130 °C for 5 min, 15
°C/min
). Retention times are 5.2 min,
norcamphor; 6.3 min, 3-hydroxynorcamphor; 7.6 min, 5-hydroxynorcamphor;
8.1 min, 6-hydroxynorcamphor. The total hydroxylated product was
quantitated by adding a known amount of internal standard (norborneol,
retention time: 5.6 min) to the reaction mixture immediately after the
complete oxidation of NADH. The ferric spin equilibrium and equilibrium
dissociation constants (K
) were
determined as reported previously(34) .
Simulations were
performed on substrate-bound P450 using the cvff force
field (40, 41) and version 2.9 of the Discover
simulation package. The x-ray structure of
(1R)-norcamphor-bound P450
(42) ,
obtained from the Protein Data Bank(43) , served as the
starting point for the simulations. An all hydrogen representation for
the protein was used, and all acidic and basic groups were modeled in
their ionized forms. A 3-Å layer of solvent was also included in
the simulations, and a constant dielectric of 1.0 (40, 41) was used. Group-based twin cutoffs of 12.0
and 15.0 Å were employed with no switching function. Prior to the
dynamics trajectories, minimization was performed first on the added
hydrogens, then on the solvent, and finally on the entire protein. For
the trajectories, velocities were initialized at 50 K and the system
slowly warmed to 300 K. For each enantiomer, four separate trajectories
of 200 ps each were calculated. In the simulations of the
(1S)-enantiomer, the substrate was superimposed on
(1R)-norcamphor in the crystal structure of substrate-bound
cytochrome P450
. The current simulations differ from our
earlier simulations (37) in that a ferryl oxygen intermediate
has now been included and a more widely used Coulombic -hydration model
has now been used. Previously the iron was unligated, a
distance-dependent dielectric constant was used, physiologically
charged residues were made net neutral, and only waters of
crystallization were explicitly treated.
The steady state rate of NADH oxidation (nmol
min NADH/nmol/min P450), stoichiometry of NADH
consumption to hydroxynorcamphor production (expressed as percent
coupling), and product profile are summarized in Table 1.
Cytochrome P450
hydroxylates (1R)-norcamphor and
(1S)-norcamphor to form three regioisomers,
5-hydroxynorcamphor, 6-hydroxynorcamphor, and 3-hydroxynorcamphor. The
(1R)-enantiomer is structurally analogous to the physiologic
substrate d-camphor and is preferentially hydroxylated at the
5-exo position: 65:30:5 for the 5-, 6-, and 3-carbons. This is
in contrast to the racemic mixture which results in a ratio of
47:45:8(31) . As expected, the product profile of the
(1S)-enantiomer compensates for the difference with a ratio of
28:62:10 for 5-, 6-, and 3-hydroxylation. The steady state NADH
oxidation rate, 90 nmol min
NADH/nmol/min
P450
, and the overall efficiency of NADH consumption,
15%, is nearly the same for each enantiomer, and all values are similar
to those reported previously for the racemic
mixture(33, 44) . Under saturating conditions, both
(1R)- and (1S)-norcamphor result in an equilibrium
spin conversion of about 45%. The free energy of binding is also
similar with
G
of approximately
-5.6 kcal mol
and -5.3 kcal
mol
, respectively. With the exception of the
regiospecificity, all of the reaction and physical parameters are very
similar for each enantiomer of norcamphor.
For comparison with the
experimental results, product profiles for (1R)- and
(1S)-norcamphor were predicted from an analysis of molecular
dynamics simulations. Structures saved at 0.5-ps intervals from the
final 125 ps of 200-ps molecular dynamics simulations of (1R)-
and (1S)-norcamphor bound P450 were analyzed to
determine the frequency of reactive substrate conformations. The
geometric criteria used to define a reactive geometry were the same as
had been used previously (23) and included the substrate
hydrogen-ferryl oxygen distance and the corresponding
carbon-hydrogen-ferryl oxygen angle. A substrate orientation was
considered reactive if the oxygen distance was less than 3.5 Å
and the C-H-O bond angle was within 45 °C of linear.
For both (1R)- and (1S)-norcamphor, in a large
majority of structures examined, neither the 3-, 5-, nor 6-carbon was
in a reactive orientation relative to the ferryl oxygen. Thus, a
predicted product profile was based on a very small number of
structures. To improve the regiospecificity statistics, four separate
trajectories were calculated for each substrate. The number of reactive
conformations found for the 3-, 5-, and 6-carbon of norcamphor in each
of the trajectories is shown in Table 2along with the total
number of reactive conformations. The predicted product profiles
calculated from these trajectories are reported in Table 1. The
(1R)-enantiomer is predicted to be preferentially hydroxylated
at the 5-carbon, whereas the (1S)-enantiomer is predicted to
form excess 6-hydroxynorcamphor. The values reported in Table 1were obtained by averaging the profiles from each set of
trajectories. Also shown in Table 1is the fraction of
conformations classified as reactive (shown as ``Percent
Coupling''). Previous calculations suggested that this quantity is
correlated with the experimentally observed uncoupling (23) .
In the case of the physiological substrate, d-camphor, the high resolution x-ray structure and
experimental measures of the reaction efficiency provide convincing
evidence that the three-dimensional features of the P450 active site are complementary to those of this small
molecule(17, 32, 33, 42, 44, 45) .
The active site is completely buried, and multiple contacts are made
between each d-camphor carbon and the heme and amino acid
residues of the protein. (1R)-d-camphor essentially
fills the negative image of the active site without atomic overlap and
without leaving open space which might be occupied by solvent.
(1R)-norcamphor is an exceptionally good camphor analog for
probing the steric contacts in the binary complex because it retains
the (2.2.1) bicyclic skeleton and the ketone of camphor, yet lacks the
8, 9, and 10 methyl groups which protrude from the center of the
molecule. These methyl groups account for approximately 36% of the
total surface area of camphor. Even without an x-ray structure of
P450
with norcamphor bound(42) , one could
imagine this substrate oriented in the active site with a hydrogen bond
formed between Tyr-96 and the substrate ketone and the carbons 1
through 7 in approximately the same positions as the corresponding
atoms of d-camphor. In this orientation the 5-carbon of
norcamphor is adjacent to the iron as is the case with d-camphor, and the active site contacts between Val-295,
Ile-395, Val-396, Thr-185, and Val-247 of P450
and the
8-, 9-, and 10-carbon of norcamphor are absent(42) .
Previous investigations using the racemic mixture of norcamphor have
shown that the loss of the specific contacts results in a less
efficient catalytic system(31, 33) . Not only is the
steady state rate of NADH oxidation slowed by half, but the overall
conversion of reducing equivalents to hydroxylated product is decreased
by a factor of 10(31) . The spectrally determined K is 100-fold larger, and the spin state
equilibrium is reduced by 50%(33) . These experimentally
measured parameters are known to be sensitive markers for detailed
features of the substrate bound complex, such as active site hydration,
substrate positioning and orientation in the active site,
substrate-active site complementarity, and relative free energy of
noncovalent interactions(30, 34, 46) . Thus,
the loss of steric contacts, or hydrophobic interactions, apparently
results in a binary complex in which norcamphor is not bound in a
``hand-in-glove'' fashion. Additional experimental evidence
in this regard is the change in the product profile from 100%
5-hydroxycamphor with d-camphor to multiple hydroxylation
products with norcamphor: 5-hydroxynorcamphor, 6-hydroxynorcamphor, and
3-hydroxynorcamphor in a ratio of 47:45:8, respectively(31) .
Theoretical calculations of radical stability show that the norcamphor
3-carbon is the most reactive position and suggest that the 3-carbon
hydroxylation is favored in solution phase(18) . The flattening
of the product profile and corresponding increase in
3-hydroxynorcamphor is due to a loss of stereochemical constraints on
the substrate by the surrounding amino acid residues in the
P450
active site. Norcamphor is either mobile in the
active site or is bound in multiple orientations such that not only the
5-carbon, but also the 6- and 3-carbon can approach the putative
oxidizing intermediate ([FeO]
) and be
positioned for hydroxylation. While the ratio of products is not taken
as an absolute measure of the relative residence time of each substrate
carbon near the [FeO]
(due to complex
geometric and chemical constraints), the values do reflect a loss of
substrate recognition by P450
and serve as a rough
measure of the structural complementarity between substrate and active
site. The fact that 1-methylnorcamphor exhibits an intermediate
decrease in 5-hydroxy product provides additional support for this line
of reasoning(33) . The results are consistent with a conceptual
model of enzyme-small molecule recognition in which substrate mobility
and/or accessible binding orientations increase as the structure of the
substrate diverges from that of d-camphor and
protein-substrate contacts are lost.
Although preliminary
investigations utilizing the racemic mixture of norcamphor have
provided many important insights into the mechanism of substrate
recognition by P450, extending this line of investigation
requires the purified enantiomers of norcamphor. For example, in
previous reports, active site mutations were shown to perturb the
regiospecificity of the reaction in a predictable manner (33) .
The prediction was based on molecular modeling of
(1R)-norcamphor, the structural analog of d-camphor.
Any rationalization of the results cannot be accurate without
acknowledging the possibility that the experimentally observed change
in product profile was a result of the (1S)-enantiomer. The
percent 5-hydroxy product formed from the (1R)-enantiomer may
be altered in a counter intuitive fashion and this fact masked by
corresponding changes in the product profile of
(1S)-norcamphor. Therefore, the pure stereoisomers are needed
to accomplish the detailed mapping of specific interactions in the
binary complex.
This problem was first addressed theoretically by
Bass and Ornstein (37) who foresaw the stereoselective
oxidation of (1R)- and (1S)-norcamphor based on
molecular dynamics simulations. These calculations predicted
(1R)-norcamphor to be hydroxylated preferentially at the
5-carbon and (1S)-norcamphor at the 6-carbon. Additional
calculations using a more sophisticated molecular model are reported
here and are in agreement with the results of the initial simulations (Table 1). The predicted regiospecificity of (1R)- and
(1S)-norcamphor hydroxylation is confirmed by the experimental
determination of the product profile as summarized in Table 1.
This is one of the few instances that a theoretical prediction of
stereoselective and regiospecific oxidation by P450 has
actually preceded the experimental account. The initial reports were
published almost 2 years before the preparation of the pure enantiomers
of norcamphor
; therefore, the validity of the original
predictions is undisputed(37) . The results suggest that the
dynamics of a substrate-active site complex can be accurately
represented over a limited time scale by a molecular mechanics level
calculation. These interpretations are consistent with a series of
similar investigations which, collectively, establish computational
analysis as a valid approach to the characterization and prediction of
small molecule binding and metabolism with
P450
(18, 19, 20, 21, 22, 23, 24, 25, 48, 49, 50) .
Theoretically determined ratios for the 5-, 6-, and 3-hydroxy
products presented herein are consistent with experiment in a
qualitative sense and are also in good agreement quantitatively. The
calculations reported differ most significantly from those described
previously by the inclusion of a single oxygen atom as a distal ligand
to the iron, intended to explicitly model the putative
[FeO] intermediate. In addition, the
previous study used a distance-dependent dielectric and included only
crystallographic waters, whereas the present set of trajectories was
calculated with a constant dielectric and a layer of added solvent.
These differences in the model do result in some quantitative
differences between the two sets of predicted product profiles, but do
not affect the qualitative trends. To obtain an estimate of the
precision of our predicted product profiles, multiple trajectories were
analyzed for each enantiomer. For both of the enantiomers, one of the
four trajectories predicted a product profile in qualitative
disagreement with the other three trajectories (see Table 2). For
(1R)-norcamphor this resulted in a particularly large estimate
of the uncertainty in the predicted product profile. Although
norcamphor is significantly more mobile than d-camphor, the
simulation time for a single trajectory may be insufficient to properly
sample substrate orientations. For highly mobile substrates such as
norcamphor(21) , the vast majority of a trajectory is spent
sampling unreactive conformations, making a precise prediction for the
product profile difficult. A substantially large investment of computer
resources is required to attain the same number of reactive
conformations with norcamphor as a highly coupled substrate such as d-camphor.
(1R)- and (1S)-norcamphor
might be expected to behave similarly in P450-catalyzed
hydroxylation due to the high degree of symmetry between the pair of
stereoisomers. In this light it is not surprising that the efficiency
of the P450
-catalyzed hydroxylation of (1R)- and
(1S)-norcamphor is nearly identical as measured by: 1) the
steady state rate of NADH oxidation, 2) the coupling of NADH to
hydroxylated product, 3) the equilibrium spin conversion, and 4) the
spectrally determined dissociation constant. The unique difference is
in the product profile. The ratio of 5-hydroxynorcamphor to
6-hydroxynorcamphor produced is inverted for the pair of stereoisomers
with (1R)-norcamphor favoring the 5-carbon and
(1S)-norcamphor the 6-carbon (Table 1). The reversal in
the regiospecificity is especially interesting in light of the
observation that l-camphor, as well as d-camphor, is
oxidized to form only 5-hydroxycamphor(51, 52) . This
may be due to the smaller size of norcamphor, a property which could
allow it to undergo more extensive rearrangements relative to the
iron(53) . Inasmuch as hydroxylation regiospecificity reports
on individual orientations of the substrate in the active site, the
ratio of 5-carbon to 6-carbon hydroxylation can be expressed as a
relative free energy. The value of 0.8 kcal mol
reflects a quantitative measure of the difference in the
recognition of (1R)- versus (1S)-norcamphor
by P450
and is comparable with recent reports of the
stereospecific hydroxylation of ethylbenzene and epoxidation of styrene
and stereoselective hydroxylation of (1R)- and
(1S)-nicotine(19, 20, 47) . Given
the fact that 3-hydroxynorcamphor is the favored product free in
solution, the predominance of 5-carbon hydroxylation suggests that
extensive constraints are placed on (1R)-norcamphor despite
the loss of several protein-substrate contacts. The increased 3-carbon
oxidation observed with the (1S)-enantiomer and the larger K
may be an indication of increased
mobility in the active site. Nevertheless, the 34% excess of
6-hydroxynorcamphor production is evidence that
(1S)-norcamphor makes a significant number of specific
interactions with P450
and is highly oriented in the
active site, regardless of its stereochemistry.
When the hydrocarbon
portions of (1R)- and (1S)-norcamphor are
superimposed, the structural difference between the stereoisomers is
the position of the ketone on the hydrocarbon ring. In consideration of
the experimentally determined reversal in the product profile, this
observation indicates that the position of the carbonyl on the
hydrocarbon skeleton of norcamphor contributes the most to the average
orientation of these substrates in the active site. Molecular docking
of (1S)-norcamphor in the P450 active site shows
that when the ketone oxygen is positioned on the same atom of d-camphor the 6-carbon ends up adjacent to the iron (Fig. 1). Moving the carbonyl by one carbon appears to rotate
the hydrocarbon skeleton of (1S)-norcamphor in the active
site. These observations suggest that hydrogen bonding interactions can
be used to direct the regiospecificity of the hydroxylation reaction.
To test this hypothesis, genetically engineered variants of cytochrome
P450
which contain novel hydrogen bonding groups are
being constructed and characterized as an approach to the rational
redesign of the enzyme active site.
Figure 1:
The cytochrome P450 active
site with (1R)- and (1S)-norcamphor bound (A and B, respectively). A, side-on view of the
cytochrome P450
active site with (1R)-norcamphor
bound(24) . The hydrogen bond between the substrate ketone and
Tyr-96 is intact and the 5-carbon is adjacent to the iron. B,
same view of the active site with (1S)-norcamphor modeled in.
The substrate is positioned to form the hydrogen bond with Tyr-96. Note
that the 6-carbon of the substrate is oriented near the
iron.
In summary, the reaction of
(1R)- and (1S)-norcamphor with cytochrome
P450 is stereoselective with respect to the
regiospecificity of hydroxylation. The experimental characterization
confirms the previously reported prediction that the product profile
resulting from the (1R)-enantiomer would reflect a preference
for 5-hydroxynorcamphor and the major product with the
(1S)-enantiomer would be 6-hydroxynorcamphor. The results
provide evidence that hydrogen bonding interactions in the
P450
-substrate complex make a significant contribution to
the specificity of the oxygenation reaction and play an important role
in the mechanism of substrate recognition.