Significant increase in phenacetin oxidation on L382V substitution in human cytochrome P450 1A2

Article abstract of DOI:10.1124/dmd.109.030767

Human CYP1A2 is an important drug-metabolizing enzyme, similar in sequence to CYP1A1 but with distinct substrate specificity. We have previously shown that residue 382 affected CYP1A1 and CYP1A2 specificities with alkoxyresorufins. To determine whether this residue is also important for the metabolism of other substrates, we have investigated phenacetin oxidation by single (T124S, T223N, V227G, N312L, and L382V) and multiple (L382V/T223N, L382V/N312L, L382V/T223N/N312L, and L382V/T124S/N312L) mutants of CYP1A2. The enzymes were expressed in Escherichia coli and purified. All the CYP1A2 mutants that contained the L382V substitution displayed much higher activities than the wildtype enzyme, with k_cat values 3-fold higher, in contrast to other mutants, for which k_cat decreased. Likewise, a significant increase in specificity, expressed as the k_cat/K_m ratio, was observed for the mutants containing the L382V substitution. The efficiency of coupling of reducing equivalents to acetaminophen formation was decreased for all the single mutants except L382V, for which the coupling increased. This effect was also observed with multiple CYP1A2 mutants containing the L382V substitution. Low activities of the four other single mutants were likely caused by dramatically increased uncoupling to water. In contrast, the increase in activity of the L382V-containing mutants resulted from decreased water formation. This finding is consistent with molecular dynamics results, which showed decreased phenacetin mobility leading to increased product formation. The results of these studies confirm the importance of residue 382 in CYP1A2-catalyzed oxidations and show that a single residue substitution can dramatically affect enzymatic activity. Copyright

Full text of DOI:10.1124/dmd.109.030767

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090-9556/10/3807-1039–1045$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:1039–1045, 2010
Vol. 38, No. 7
30767/3593046
Printed in U.S.A.
Significant Increase in Phenacetin Oxidation on L382V Substitution
in Human Cytochrome P450 1A2
Qingbiao Huang and Grazyna D. Szklarz
Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia
Received October 13, 2009; accepted March 24, 2010
ABSTRACT:
Human CYP1A2 is an important drug-metabolizing enzyme, similar mutants containing the L382V substitution. The efficiency of cou-
in sequence to CYP1A1 but with distinct substrate specificity. We pling of reducing equivalents to acetaminophen formation was
have previously shown that residue 382 affected CYP1A1 and decreased for all the single mutants except L382V, for which the
CYP1A2 specificities with alkoxyresorufins. To determine whether coupling increased. This effect was also observed with multiple
this residue is also important for the metabolism of other sub-
strates, we have investigated phenacetin oxidation by single of the four other single mutants were likely caused by dramatically
T124S, T223N, V227G, N312L, and L382V) and multiple (L382V/ increased uncoupling to water. In contrast, the increase in activity
CYP1A2 mutants containing the L382V substitution. Low activities
(
T223N, L382V/N312L, L382V/T223N/N312L, and L382V/T124S/ of the L382V-containing mutants resulted from decreased water
N312L) mutants of CYP1A2. The enzymes were expressed in Esch- formation. This finding is consistent with molecular dynamics re-
erichia coli and purified. All the CYP1A2 mutants that contained the sults, which showed decreased phenacetin mobility leading to
L382V substitution displayed much higher activities than the wild- increased product formation. The results of these studies confirm
type enzyme, with kcat values 3-fold higher, in contrast to other the importance of residue 382 in CYP1A2-catalyzed oxidations and
mutants, for which kcat decreased. Likewise, a significant increase show that a single residue substitution can dramatically affect
in specificity, expressed as the kcat/Km ratio, was observed for the enzymatic activity.
Cytochromes P450 (P450s) are heme-containing monooxygenase al., 1993; Burke et al., 1994). The structural basis for such functional
enzymes, which are involved in the metabolism of numerous exoge- differences between highly related enzymes can be investigated using
nous and endogenous compounds. P450s are ubiquitous in living a variety of techniques, including molecular modeling and experimen-
organisms, with at least 50 families and 82 subfamilies found in tal methods, such as site-directed mutagenesis and NMR. More re-
different species. Human CYP1A subfamily has two major isoforms: cently, the crystal structure of CYP1A2 was solved by X-ray crystal-
CYP1A1 and CYP1A2. CYP1A2, one of the major P450s in the lography (Sansen et al., 2007); thus, it provides a practical model for
human liver, was first characterized as a phenacetin O-deethylase structure-function studies.
(
Distlerath et al., 1985). Currently, it is estimated that this enzyme
Our previous studies on structure-function relationships of CYP1A1
indicated that Val382 played an important role in binding of
alkoxyresorufin substrates (Liu et al., 2003). The sequence alignment
between CYP1A1 and CYP1A2 indicated that five active site residues
that are different between these enzymes (Ser122, Asn221, Gly225,
Leu312, and Val382 in CYP1A1 and the corresponding residues in
CYP1A2: Thr124, Thr223, Val227, Asn312, and Leu382) might be
involved in determining substrate specificity (Liu et al., 2004). This
result was confirmed by the finding that five reciprocal mutations in
CYP1A1 and CYP1A2 altered enzymatic activity with alkoxyresoru-
fins as substrates. Moreover, mutations at position 382 in both
CYP1A1 and CYP1A2 shifted substrate specificity from one enzyme
to another (Liu et al., 2004). Further computational and experimental
studies with multiple CYP1A2 mutants confirmed the importance of
this residue for alkoxyresorufin oxidation (Tu et al., 2008). Therefore,
it would be of interest to examine the effect of these mutations on
oxidation of other types of substrates.
metabolizes approximately 11% of all drugs in humans (Shimada et
al., 1994). Despite the fact that CYP1A2 participates in the deactiva-
tion and detoxification of xenobiotics, the main interest in this enzyme
is because of the metabolic activation of a large number of chemical
carcinogens (Guengerich and Shimada, 1991; Lewis et al., 1994).
In humans, CYP1A2 shares 72% amino acid sequence identity with
CYP1A1, but the substrate specificities and inhibitor susceptibilities
of these enzymes are different. For example, substrates such as
phenacetin and 7-methoxyresorufin are primarily metabolized by
CYP1A2 with high catalytic efficiency, whereas CYP1A1 displays
weak capability to oxidize those substrates. On the other hand,
7
-ethoxyresorufin is preferentially oxidized by CYP1A1 (Nerurkar et
This work was supported in part by the National Institutes of Health National
Center for Research Resources [Grant RR16440]; and the National Institutes of
Health National Institute of General Medical Sciences [Grant GM079724].
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
In the current study, we chose phenacetin as a substrate. This
compound has been used as the most common marker for CYP1A2
doi:10.1124/dmd.109.030767.
ABBREVIATIONS: P450, cytochrome P450; MD, molecular dynamics; DLPC, dilauroyl-L-3-phosphatidyl-choline; HPLC, high-performance liquid
chromatography; WT, wild type; RMSD, root mean square deviation.
1039

1
040
HUANG AND SZKLARZ
activity in the in vitro studies of 45% of new drugs by investigators in obtained using difference visible spectroscopy (Modi et al., 1995). Solutions
(
800 ␮l) contained 0.5 ␮M CYP1A2 WT or the mutants in 100 mM phosphate
the pharmaceutical industry (Yuan et al., 2002). The objective of the
present study was to investigate whether any reciprocal mutations in
CYP1A2 may alter phenacetin oxidation and to examine the potential
mechanism(s) that might be involved. Five single mutants and four
multiple mutants containing the L382V substitution were evaluated
using a combination of molecular modeling and experimental meth-
ods. These included enzyme kinetics and stoichiometry studies, as
buffer, containing 20% glycerol and 0.1 mM EDTA, pH 7.4. Two microliters
of different concentrations of solutions of phenacetin in menthol was added to
the sample cuvette, and the same volume of menthol was added to the
reference, and UV spectra were then recorded. The data were analyzed by
nonlinear regression analysis using Microsoft (Redmond, WA) Excel software.
NADPH Oxidation. The rate of NADPH oxidation was determined spec-
trophotometrically at 340 nm in a cuvette thermostated at 37°C. The reaction
well as molecular dynamics (MD) simulations of phenacetin in the mixture was similar to that used for the phenacetin assay and contained 0.5 ␮M
active site of CYP1A2 mutants to facilitate the interpretation of P450 enzyme, 1 ␮M P450 reductase, 45 ␮M DLPC, and 1 mM phenacetin in
experimental results. This study should provide an increased under- a 100 mM potassium phosphate buffer, pH 7.5, in a volume of 980 ␮l. The
reaction was initiated by the addition of 20 ␮l of 50 mM NADPH. The
standing of the biochemical aspects of substrate specificity in the
NADPH oxidation rates were recorded for approximately 3 min at 340 nm
P450 family of enzymes.
from the beginning of the reaction. The molar extinction coefficient of 6.22 per
mM/cm for NADPH at 340 nm was used to obtain oxidation rates in nanomole
per minute per nanomole of P450. Three 50-␮l aliquots of the reaction mixture
Materials and Methods
Materials. Phenacetin, acetaminophen, 2-hydroxy acetanilide, sodium were removed after 1, 2, and 3 min and quenched with 50 ␮l of 10%
dithionite, NADPH, ampicillin, isopropyl-␤-D-thiogalactopyranoside, ␦-
aminolevulinic acid, CHAPS, dilauroyl-L-3-phosphatidyl choline (DLPC), hydrogen peroxide (H O ).
CF COOH. The triplicate-quenched reaction mixture was then used to measure
3
2
2
and phenylmethanesulfonyl fluoride were from Sigma-Aldrich (St. Louis,
H O Production. The reaction mixtures from the NADPH oxidation assay
2 2
MO). Nickel-nitrilotriacetic acid agarose and a gel extraction kit were
were used to measure the production of H O using the xylenol orange iron
2
2
purchased from QIAGEN (Valencia, CA). Potassium phosphate, EDTA, (III) assay (Jiang et al., 1990; Fang et al., 1997) with slight modifications. The
acetic acid, and high-performance liquid chromatography-grade methanol
coloring agent was prepared by mixing 100 volumes of 125 ␮M xylenol
were purchased from Thermo Fisher Scientific (Waltham, MA). All the orange in 100 mM sorbitol and 1 volume of 25 mM fresh ferrous (Fe2ϩ
other chemicals used were of analytical grade and were obtained from ammonium sulfate in 2.5 M H SO . The calibration curve was prepared using
)
2
4
standard commercial sources.
the quenched reaction mixture, which was supplemented with H O at con-
2 2
Protein Expression and Purification. The clones of CYP1A1 wild type centrations ranging from 0 to 10 ␮M. The H O standard solutions were
2
2
(
WT), CYP1A2 WT, and CYP1A2 single mutants, T124S, T223N, V227G, prepared fresh on the day of the assay by dilution of a 30% H O stock
2 2
N312L, and L382V, all of them containing a His-tag for easy purification, were
constructed earlier (Liu et al., 2003, 2004). CYP1A2 His-tag multiple mutants,
solution. The reaction mixture was incubated at room temperature for 1 h.
Absorbance was recorded using a Beckman Coulter, Inc. (Fullerton, CA)
L382V/T223N, L382V/N312L, L382V/T223N/N312L, and L382V/T124S/ spectrophotometer set at 560 nm to obtain the concentration of H O produced
2
2
N312L, were also constructed previously (Tu et al., 2008). The P450 enzymes
were expressed in Escherichia coli DH5␣ cells and purified essentially as
described previously (Liu et al., 2004; Tu et al., 2008). During the purification,
the addition of 5 mM caffeine in the purification buffers helped to stabilize
CYP1A2 proteins. The substrate caffeine was removed completely from the
enzyme preparation during the ultrafiltration stage, as verified by HPLC. Rat
in nanomole per minute per nanomole of P450.
Oxygen Consumption. The reaction was conducted using a Mitocell
(Strathkelvin Instruments Ltd., Glasgow, UK), which was connected to a water
bath thermostated at 37°C. The reaction mixture was prepared in a similar way
to that for the NADPH oxidation assay. Nine hundred eighty microliters of the
sample was placed in the chamber of the Mitocell, and once a steady baseline
P450 reductase was expressed in E. coli and purified according to an estab- was established, the reaction was initiated by the addition of 20 ␮l of NADPH.
lished procedure (Liu et al., 2003). The final purity of the enzymes was
The oxygen consumption was recorded over 5 min as micromolar per hour,
assessed by SDS-polyacrylamide gel electrophoresis. Western blots were per- which was then converted to nanomole per minute per nanomole of P450.
formed using anti-human CYP1A1/1A2 (Oxford Biomedical Research, Ox-
General Molecular Modeling Methods. Molecular modeling simulations
ford, MI), and P450 proteins were visualized as described previously (Kedzie were conducted using a Silicon Graphics Octane workstation with Insight II
et al., 1991). P450 content was determined by reduced CO/reduced difference software (Accelrys, San Diego, CA). The crystal structure of CYP1A2 (Protein
spectra (Omura and Sato, 1964), and protein was measured using Folin phenol Data Bank code 2hi4) was obtained courtesy of Dr. Eric F. Johnson (The
reagent (Lowry et al., 1951).
Scripps Research Institute, La Jolla, CA) (Sansen et al., 2007). The heme
P450 Activity Assay. Phenacetin O-dealkylase activities of CYP1A2 WT cofactor was removed and replaced with the oxoheme cofactor. Substrate
and mutants were determined by HPLC measurements as described previously
von Moltke et al., 1996) with some modifications. The reaction mixtures
phenacetin was constructed with Insight II/Builder module and optimized.
The models of CYP1A2 single and multiple mutants were constructed from the
(
contained 0.5 ␮M CYP1A2, 1 ␮M P450 reductase, and 45 ␮M DLPC in 100 crystal structure of CYP1A2 WT by the replacement of selected amino acid(s)
mM potassium phosphate buffer, pH 7.5. The enzymes and DLPC were
preincubated for 2 min at 37°C before the dilution. For kinetic assays, phen-
and further refinement of the structures according to the previously established
procedure (Liu et al., 2003, 2004; Tu et al., 2008). MD simulations and energy
acetin was added at concentrations ranging from 0 to 1000 ␮M, and the minimization were carried out using the Insight II/Discover module with the
mixture was incubated for another 3 min at 37°C. The reaction was initiated by
adding NADPH to a final concentration of 1 mM in a total volume of 1 ml and
consistent valence force field supplemented with parameters for heme and
ferryl oxygen, as described earlier (Paulsen and Ornstein, 1991, 1992). The
conducted for 30 min. The reaction was terminated by the addition of 5 ␮l of nonbond cutoff was 16 Å, and all the other parameters were set at their default
0% HClO , and the reaction mixture was put on ice for 10 min. Ten
values. Structural refinement of CYP1A2 WT and mutants involved 1000 steps
6
4
microliters of 2-hydroxy acetanilide (100 ␮M) was then added as an internal of minimization using steepest descent gradient followed by 10-ps MD and
standard for HPLC determination. The reaction mixture was centrifuged at then another 1000 steps of steepest descent minimization. The optimized
1
000g for 5 min, and 100 ␮l of the supernatant was removed and used directly
structures were used for the subsequent docking studies.
for HPLC analysis. The product acetaminophen was eluted from a C18 column
Docking of Phenacetin into the Active Site of CYP1A2 WT and Mu-
(
Alltech Associates, Deerfield, IL) with a mobile phase of methanol/0.1% tants. Initially, phenacetin was manually placed into the active site of CYP1A2
acetic acid (30:70, v/v; flow rate, 1.5 ml/min) and monitored at 254 nm. The
product was quantified using acetaminophen standards. The kinetic parameters
WT and the mutants on the distal side of the oxoheme. Docking of phenacetin
was performed with Insight II (Accelrys)/Affinity module using default pa-
(
Vmax and kcat) were calculated using nonlinear regression with GraphPad rameters, as described previously (Liu et al., 2004; Ericksen and Szklarz, 2005;
Software Inc. (San Diego, CA) Prism software.
Tu et al., 2008). Residues within 10 Å of the initial phenacetin position
Binding Constant Determination. Spectral binding constants for phenac- comprised the flexible region of the receptor (CYP1A2 WT and mutants)
etin bound in the active site of CYP1A1 WT and CYP1A2 enzymes were during all the docking runs. The Affinity docking method uses both the Monte

INCREASED PHENACETIN OXIDATION IN CYP1A2 MUTANTS
1041
Carlo search technique and simulated annealing approach, followed by the The overall yield of the procedure was approximately 20 to 40%,
minimization protocol to generate low-energy substrate binding orientations. A
similar to that reported previously (Liu et al., 2004; Tu et al., 2008).
distance-dependent dielectric constant was applied to simulate charge screen-
The purity of CYP1A2 WT and mutants verified by SDS-polyacryl-
ing by water molecules. The 10 lowest-energy phenacetin binding orientations
obtained from Affinity docking were selected for further analysis.
amide gel electrophoresis and Western blots indicated that they were
at least 95% pure. The spectrum of the Fe -CO complex exhibited a
characteristic peak at 450 nm, with little or no P420 formation. The
holoenzyme content of the enzymes was usually in the range of 40 to
II
MD Simulations of Enzyme-Substrate Complexes. MD simulations were
performed to investigate phenacetin mobility in the active site and the effect of
mutations on substrate orientation. The starting configuration for MD simula-
tions chosen from Affinity docking represented the productive binding orien-
tation of phenacetin leading to its O-dealkylation and had the lowest potential
6
0%, as previously observed in our laboratory.
Kinetic parameters, kcat, K , and substrate specificity (kcat/K ),
m
m
energy rank. The MD simulations of each phenacetin-enzyme complex were were determined for purified CYP1A2 WT and mutants using a range
performed at 310 K in vacuo essentially as described earlier (Liu et al., 2004; of substrate phenacetin concentrations. Phenacetin undergoes O-
Tu et al., 2008). The substrate, heme, and protein residues within 10 Å from
deethylation to form acetaminophen as the main product of the reac-
the initial substrate position were flexible without any restraints, whereas the
tion. Kinetic parameters for CYP1A2 WT and mutants are shown in
remainder of the protein was fixed. A distance-dependent dielectric constant
Table 1. Compared with CYP1A2 WT, the mutations affected both
was used to simulate aqueous environment, and the nonbond cutoff distance
was 16 Å. After 5-ps MD equilibration phase, the MD simulations were
continued for 100 ps, and 400 frames obtained every 250 fs were extracted to
record the snapshots of each enzyme-substrate complex.
kcat and K . The CYP1A2 L382V mutant and multiple mutants
containing the L382V mutation displayed 2- or 3-fold higher kcat than
the WT enzyme. Four other single mutants, namely, T124S, T223N,
m
V227G, and N312L, exhibited much lower kcat than the WT.
As shown in Table 1, the K values for CYP1A2 WT and mutants
In addition, to evaluate the effect of explicit solvent on phenacetin dynamics in
the active site, we performed 100-ps MD simulations on solvated enzyme-
m
substrate complexes using a similar protocol. The enzymes chosen were CYP1A2 varied greatly. In general, the Km values for all the mutants were more
WT and the L382V mutant. The enzyme-substrate complexes were solvated using than 50% lower than for the WT (ϳ60 ␮M). The lowest values of Km
crystallographic water molecules from CYP1A2 crystal structure and optimized
before MD with 1000 steps of steepest descent minimization using a nonbond
cutoff of 16 Å and dielectric constant of 1. Similar to previous MD simulations,
only protein residues, heme, substrate, and solvent within a 10-Å radius of the
significantly increased the binding affinity of the enzyme for phen-
initial substrate position were permitted to move, whereas the remainder of
the protein and other water molecules were fixed. This ensures that none of the
moving solvent molecules escape from the vicinity of the active site. In contrast to
previous MD simulations, the flexible region also included 21 water molecules
were observed for several mutants containing the L382V substitution,
including L382V (ϳ6 ␮M), L382V/T223N (ϳ8 ␮M), and L382V/
T124S/N312L (ϳ10 ␮M), which suggests that the L382V mutation
acetin. The substrate specificities of the L382V mutant and multiple
mutants containing the L382V mutation, expressed as kcat/K , were at
m
least 5-fold higher than that of the WT (Table 1). In particular, the
surrounding phenacetin. For the aqueous simulations, the dielectric constant was relative substrate specificities of L382V and L382V/T223N mutants
set to 1. All the other parameters were the same as for the previous enzyme- were more than 20-fold higher, which is extremely high. Incidentally,
substrate simulations without explicit solvent present.
phenacetin is less efficiently metabolized by CYP1A1 WT (kcat 0.5/
min; K , 66 ␮M), with k close to one third of the value observed
with CYP1A2 WT (see Table 1) and the kcat/Km ratio lower than 0.01.
Phenacetin binding constants were also determined for CYP1A2
WT and some mutants. CYP1A2 WT and the N312L mutant showed
similar phenacetin binding, with binding constants of 17.1 and 10.2
All the MD trajectories were examined using Insight II (Accelrys)/Analysis
module. To score the likelihood of hydroxylation, each sampled frame of a given
enzyme-substrate complex was evaluated using the following geometric criterion:
r Յ 3.5 Å and ␪ Ն 120°, where r represents the distance between the ferryl oxygen
and the hydrogen of the substrate to be abstracted; ␪ represents the angle between
ferryl oxygen, the hydrogen atom to be abstracted, and the carbon at the oxidation
site, as described previously (Ericksen and Szklarz, 2005; Tu et al., 2008).
Trajectory data from 100-ps MD simulations were extracted and graphed using
M
cat
␮M, respectively. In contrast, the L382V and the L282V/N312L
mutants displayed much lower values for binding constants, namely,
Microsoft Excel. The MD frames where the geometric criterion, r Յ 3.5 Å and ␪ Ն 0.7 and 3.5 ␮M, indicating tighter substrate binding. CYP1A1 WT
120°, was satisfied were counted as hits. The number of hits is a useful indicator exhibited much weaker binding, with a binding constant of 57 ␮M.
of whether a CYP1A2-mediated phenacetin O-deethylation occurred.
Stoichiometry of Phenacetin O-Deethylation. To assess whether
the L382V mutation affected CYP1A2 coupling efficiency of reduc-
ing equivalents to acetaminophen formation, stoichiometry experi-
Results
Kinetics of Phenacetin O-Deethylation by CYP1A2 WT and ments were conducted. The rates of NADPH oxidation, hydrogen
Mutants. CYP1A2 enzymes were expressed in E. coli and purified. consumption, product formation, H O , and excess water production
2
2
TABLE 1
Kinetic parameters for phenacetin O-deethylation by purified CYP1A2 WT and mutants
Phenacetin O-Deethylation
CYP1A2
a
cat
a
b
k
K
m
k
cat/K
m
͓(kcat/K
m
m
)mutant/(kcat/K )WT͔
min
␮M
␮M/min
WT
1.28 Ϯ 0.08
0.29 Ϯ 0.03
0.30 Ϯ 0.01
0.38 Ϯ 0.02
0.31 Ϯ 0.01
3.03 Ϯ 0.06
3.91 Ϯ 0.10
3.05 Ϯ 0.15
3.67 Ϯ 0.10
2.81 Ϯ 0.03
59.70 Ϯ 1.82
28.90 Ϯ 1.10
26.99 Ϯ 0.01
32.36 Ϯ 2.93
25.26 Ϯ 0.01
5.75 Ϯ 0.57
7.77 Ϯ 0.80
16.14 Ϯ 2.05
32.42 Ϯ 11.31
10.13 Ϯ 2.40
0.02 Ϯ 0.01
0.01 Ϯ 0.01
0.01 Ϯ 0.01
0.01 Ϯ 0.01
0.01 Ϯ 0.01
0.53 Ϯ 0.04
0.51 Ϯ 0.06
0.19 Ϯ 0.02
0.12 Ϯ 0.04
0.28 Ϯ 0.07
1.00
0.47
0.52
0.55
0.57
24.77
23.65
8.87
5.61
13.34
T124S
T223N
V227G
N312L
L382V
L382V/T223N
L382V/N312L
L382V/T223N/N312L
L382V/T124S/N312L
a
b
Data are means of triplicate determinations.
k
m
cat/K represents substrate specificity.

1
042
HUANG AND SZKLARZ
TABLE 2
a
Rates [nmol/min/(nmol of P450)] determined for phenacetin metabolism by CYP1A2 WT and mutants
CYP1A2
NADPH Oxidized
O
2
Consumed
Product Formed
H
2
O
2
Produced
Excess H
2
Ob
WT
43 Ϯ 4
27 Ϯ 2
41 Ϯ 3
40 Ϯ 3
32 Ϯ 2
84 Ϯ 6
98 Ϯ 7
70 Ϯ 4
72 Ϯ 5
42 Ϯ 3
22.7 Ϯ 0.9
14.8 Ϯ 0.4
17.3 Ϯ 0.7
16.9 Ϯ 0.5
18.3 Ϯ 0.3
47.0 Ϯ 1.3
57.9 Ϯ 2.5
41.0 Ϯ 2.0
41.7 Ϯ 1.1
34.2 Ϯ 0.9
1.28 Ϯ 0.08
0.29 Ϯ 0.03
0.30 Ϯ 0.00
0.38 Ϯ 0.02
0.31 Ϯ 0.00
3.03 Ϯ 0.06
3.91 Ϯ 0.10
3.05 Ϯ 0.15
2.18 Ϯ 0.03
3.67 Ϯ 0.10
14 Ϯ 2
10 Ϯ 0
10 Ϯ 1
9 Ϯ 1
13 Ϯ 2
29 Ϯ 3
38 Ϯ 3
29 Ϯ 3
28 Ϯ 3
26 Ϯ 2
15 Ϯ 2
9 Ϯ 1
T124S
T223N
V227G
N312L
L382V
L382V/T223N
L382V/N312L
L382V/T223N/N312L
L382V/T124S/N312L
14 Ϯ 2
15 Ϯ 2
10 Ϯ 1
30 Ϯ 4
32 Ϯ 5
18 Ϯ 3
23 Ϯ 3
9 Ϯ 2
a
b
Data are means of triplicate determinations.
Excess water (H O) was calculated from the equation: H O ϭ NADPH Ϫ H O Ϫ product.
2 2 2 2
for phenacetin oxidation by CYP1A2 WT and mutants are shown in zyme (20–30% of WT). All of the enzymes displayed similar ratios of
Table 2. The excess water formation was calculated from the differ- H O production to O consumption, which suggests that the muta-
2
2
2
ence between the rates of NADPH oxidation and rates of H O and tions had no effect on uncoupling at the first and second branching
2
2
product formation (excess H O ϭ NADPH Ϫ H O Ϫ product). The points of the P450 cycle. On the other hand, significant differences
2
2 2
amount of water was also obtained from the difference between the between the enzymes were observed with respect to the H O/product
2
rate of oxygen consumption and rates of H O plus product formation ratios used to measure uncoupling at the third branching point. Thus,
2
2
[
H O ϭ 2(O Ϫ H O Ϫ product)], as reported by others (Fang et al., for the L382V mutant and multiple mutants containing the L382V
2 2 2 2
1
997), giving values that were very similar (within 5%) to those substitution, these ratios were generally lower than that for the WT,
derived from the previous equation (data not shown). The coincuba- whereas for the other single mutants, these ratios were 3-fold higher.
tion of phenacetin with CYP1A2 L382V and three multiple mutants, Therefore, low activities of the four single mutants were likely the
L382V/T223N, L382V/N312L, and L382V/T223N/N312L, resulted result of dramatically increased uncoupling to water, whereas the
in consumption of both NADPH and oxygen by the mutants at rates increase in activity in the L382V-containing mutants resulted from
ϳ2-fold greater than those with the WT enzyme. Likewise, the decreased water formation.
formation of product acetaminophen, as well as byproducts such as
Molecular Modeling Analyses. Using the crystal structure of
H O and water, was 2 to 3 times higher in the case of these mutants. CYP1A2, molecular modeling studies have been conducted to under-
2
2
The exception was the L382V/T124S/N312L mutant, which seemed stand the effects of single and multiple mutations on enzyme-substrate
to use NADPH at a rate similar to the WT enzyme but displayed interactions and substrate mobility, as well as to explain the alterations
increased consumption of oxygen, along with increased product and of catalytic efficiency. Figure 1 depicts binding orientations of phen-
H O formation. On the other hand, four single mutants, T124S, acetin within the active sites of CYP1A2 WT and the L382V mutant.
2
2
T223N, V227G, and N312L, are similar or less efficient than the In general, the binding orientation of phenacetin in both enzymes was
WT with respect to NADPH oxidation, oxygen consumption, very similar. However, the replacement of Leu382 by a smaller Val
H O , and water production but exhibit a substantial decrease in increased the volume of the active site and allowed the hydrogens at
2
2
product formation.
the oxidation site of phenacetin to approach closer to the ferryl oxygen
Table 3 presents the effects of mutations on the coupling efficiency of the heme, which facilities hydrogen abstraction. The average dis-
of CYP1A2, both overall and at specific P450 uncoupling branching tance between the hydrogens at the oxidation site of phenacetin and
points. The ratios of product formation to NADPH oxidation, account- the ferryl oxygen of the L382V mutant was 3.1 Å, compared with 3.7
ing for the overall efficiency of CYP1A2 coupling of reducing equiv- Å for the WT enzyme. Similar results were also obtained for the
alents to product, were higher for L382V and multiple mutants con- L382V/T223N and L382V/T223N/N312L mutants. In contrast, both
taining the L382V substitution than for the WT. In contrast, other hydrogens at the oxidation site of phenacetin were much farther away
single mutants, T124S, T223N, V227G, and N312L, exhibited signif- from the ferryl oxygen in the active sites of T124S, T223N, V227G,
icantly decreased coupling efficiencies compared with the WT en-
TABLE 3
Effect of mutations on coupling efficiency of CYP1A2 at different branching
a
points of P450 cycle
Product/NADPHb
H
O
/O
c
H
2
O/Productd
CYP1A2
2
2
2
WT
0.030
0.011
0.007
0.010
0.010
0.036
0.040
0.044
0.030
0.087
0.62
0.68
0.58
0.53
0.71
0.62
0.66
0.71
0.67
0.76
11.72
31.03
46.67
39.47
32.26
9.9
8.18
5.9
10.55
2.45
T124S
T223N
V227G
N312L
L382V
L382V/T223N
L382V/N312L
L382V/T223N/N312L
L382V/T124S/N312L
FIG. 1. Binding orientation of phenacetin within the active site of CYP1A2 WT (A)
and the L382V mutant (B). The protein backbone is depicted as a blue ribbon; the
side chain of residue 382 is green, with van der Waals surface displayed; heme is
red; and phenacetin is yellow, with hydrogens at the oxidation site shown in red. The
distance between the hydrogen to be abstracted and the ferryl oxygen (marked with
a black line) is 3.6 Å in the WT and 2.9 Å in the L382V mutant.
a
Ratios were calculated from parameters in Table 2.
Efficiency of coupling reducing equivalents to product.
Effect on uncoupling at first or second branch point.
Effect on uncoupling at third branch point.
b
c
d

INCREASED PHENACETIN OXIDATION IN CYP1A2 MUTANTS
1043
FIG. 2. Ensembles of substrate orientations ob-
tained from 100-ps MD simulations of phenac-
etin-enzyme complexes described by geometric
parameters, distance r and angle ␪. H1 in
-
OCH - group of phenacetin is blue, and H is
2
2
red. Gray regions, where the criterion (r Յ 3.5
Å and ␪ Ն 120°) is satisfied, represent produc-
tive binding orientations of phenacetin within
the active site. CYP1A2 enzymes were WT (A),
N312L mutant (B), L382V mutant (C), and
L382V/T223N mutant (D).
and N312L mutants (data not shown). These findings correlate well 4-, and 5-fold higher, respectively, than that for the WT (Table 4). In
with the results of kinetic and stoichiometric studies described previ- contrast, zero or few hits were obtained for T124S, T223N, V227G,
ously and suggest that the L382V substitution not only increases and N312L mutants during 100-ps dynamics. It is possible that some
catalytic efficiency (kcat) of CYP1A2 but also decreases Km and hits might be recorded during a longer simulation time. Overall, the
enzyme uncoupling.
results of MD simulations are, like those from docking experiments,
consistent with the kinetic and stoichiometric analyses of CYP1A2
WT and mutants.
To examine the tendency of phenacetin to remain in the productive
binding orientation in the active site of the enzyme, 100 ps of MD
simulations were performed as described under Materials and Meth-
ods. After the 5-ps MD equilibration phase, the energy of the system
remained constant throughout the simulations. The mobility of the
substrate varied with the mutant: we observed fairly low mobility for
the WT enzyme, L382V, T124S, and L382V/T223N/N312L mutants,
with root mean square deviation (RMSD) from the initial substrate
orientation of 1 to 2 Å, moderate mobility for V227L and N312L
mutants with RMSD of 2 to 4 Å, and a high phenacetin mobility in the
case of T223N and L382V/T223N mutants (RMSD, Ͼ4 Å).
The productive binding orientations of phenacetin were determined
using geometric parameters, distance r and angle ␪, as described under
Materials and Methods. These parameters were then plotted based on
A possible drawback of the simulations described previously might
have been the use of the distance-dependent dielectric constant instead
of explicit solvent. Therefore, we have also conducted MD simula-
tions of phenacetin docked in the active site of CYP1A2 WT and the
L382V mutant with the solvent molecules present and the dielectric
constant of 1. In the presence of water, 70 hits were recorded for the
WT enzyme and 144 hits for the L382V mutant. Thus, in both cases,
the percentage of hits increased less than 10% and to a very similar
extent (9.4% for the WT and 7.5% for the mutant) compared with the
results from MD simulations without water (see Table 4). This result
indicates that, although some changes may be observed with solvent
present, simulations with distance-dependent dielectric provide a rea-
4
00 recorded snapshots for each enzyme-substrate complex. The
representative plots, those for CYP1A2 WT, N312L, L382V, and sonable approach to explain the observed experimental findings.
L382V/T223N mutants, showing the ensembles of substrate orienta-
tions, are presented in Fig. 2. The region where the geometric criterion
(
r Յ 3.5 Å and ␪ Ն 120°) is satisfied is gray, and the points (or
TABLE 4
frames) located within represent all the snapshots of each enzyme-
substrate complex where phenacetin was bound in the productive
binding orientation. The distribution of the MD frames for each
enzyme-substrate complex displayed varied. Overall, phenacetin
showed higher occupancy within a productive binding region
Geometric analysis of MD results for CYP1A2 WT and mutants
Hits represent the MD frames where phenacetin was in the productive binding orientation,
as evaluated by the geometric criterion: r Յ 3.5 Å and ␪ Ն 120°.
CYP1A2
Number of Hits
(
counted as hits) in the L382V and L382V/T223N mutants than in the
WT
64
0
0
4
0
134
252
308
WT. Similar results were also seen in the case of other multiple
mutants containing L382V (data not shown), whereas few hits, if any,
were observed for the remaining four single mutants, as shown for the
N312L mutant (Fig. 2B). A quantitative analysis of the hits for each
enzyme-substrate complex revealed that the number of hits for
L382V, L382V/T223N, and L382V/T223N/N312L mutants were 2-,
T124S
T223N
V227G
N312L
L382V
L382V/T223N
L382V/T223N/N312L

1
044
HUANG AND SZKLARZ
observed in all the mutants, those that contained the L382V substitu-
Discussion
Our previous studies indicated that residue 382 plays an important
tion showed less uncoupling to water (decreased H O/product ratio;
2
Table 3). Thus, it seems reasonable to suggest that the L382V sub-
role in controlling the specificity of alkoxyresorufin O-dealkylation
by CYP1A1 and CYP1A2 (Liu et al., 2003, 2004; Tu et al., 2008).
Because phenacetin is another important probe substrate for CYP1A2,
the studies to address the effects of reciprocal mutations, particularly
L382V, on phenacetin oxidation may broaden our understanding of
the role of this residue in substrate specificity. In the present study,
nine single and multiple mutants were investigated using a battery of
complementary approaches, such as kinetic assays, stoichiometry
measurements, and molecular modeling methods. The results showed
that the L382V substitution resulted in a significant increase in cata-
lytic activity and substrate specificity of CYP1A2. All of the multiple
mutants that contained this substitution displayed very similar kinet-
ics, stoichiometry, and dynamic mobility as the single L382V mutant.
Thus, the presence of this single residue was critical for dramatically
improving the efficiency of phenacetin oxidation by CYP1A2.
stitution in CYP1A2 yields the mutants that use NADPH and O more
2
efficiently to oxidize phenacetin to products and display less uncou-
pling to water, so that the overall coupling efficiency of the enzyme
increases, which is consistent with enzyme kinetics results (Table 1).
Molecular modeling studies provide another possible explanation
of the effects of mutations on phenacetin specificity, as indicated by
changes in kinetic parameters. For these simulations, we used the
X-ray structure of CYP1A2, and the structures of the mutants were
derived from the crystal. As reported by Sansen et al. (2007), this
CYP1A2 structure has a closed compact active site, without clear
solvent or substrate access channels, with a relatively small volume of
3
the cavity, estimated at 375 Å . The active site of CYP1A2 is
3
approximately 44% larger than that of CYP2A6 (260 Å ) and signif-
3
icantly smaller than that of CYP3A4 (1385 Å ) (Sansen et al., 2007).
To date, many residues of CYP1A2 have been identified that may Consequently, only planar compounds, such as ␣-naphthoflavone, and
play a role in enzyme-ligand interactions by site-directed mutagenesis typical CYP1A2 substrates such as phenacetin, 7-ethoxyresorfin, caf-
and/or molecular modeling studies (Yun et al., 2000; Liu et al., 2004; feine, tacrine, or theophylline can be fitted well with the narrow and
Zhou et al., 2009). In the case of single mutants, most of the muta- flat active site cavity of enzyme. Leu382 of CYP1A2 is a critical
tions, including T124S, T223N, V227G, and N312L in the present residue located close to the heme iron, and its replacement with a
study, resulted in decreased catalytic activity and substrate specificity smaller Val increases the volume of the active site near heme. This
compared with the WT enzyme (Parikh et al., 1999; Liu et al., 2004). activity allows phenacetin to move closer to heme, so that one of the
A number of CYP1A2 mutants obtained from random mutagenesis hydrogens at the oxidation site is within a hydrogen-bonding distance
showed increased catalytic activities and substrate specificities toward from the ferryl oxygen (Fig. 1), which promotes hydrogen abstraction.
phenacetin (Parikh et al., 1999), but none of them was as highly active This is consistent with the MD results (Table 4; Fig. 2). Consequently,
as the L382V mutant reported in this study (Table 1). It is worth the movement of phenacetin closer to the ferryl oxygen in the L382V
mentioning that the kinetic parameters for phenacetin O-dealkylation mutants helps to explain not only the substantial increase in kcat and
by CYP1A2 WT determined in the present investigation were very a decrease in Km for the production of acetaminophen (Table 1) but
similar to those reported by Parikh et al. (1999). Although the L382V also more efficient coupling of the P450 reaction cycle with less water
mutation dramatically increased oxidation of phenacetin, it led to a formation (Table 3).
significant decrease in 7-methoxyresorufin O-dealkylation (Liu et al.,
MD simulations similar to those described in this work have been
004). Thus, the functional effect of residue substitution appears to be successfully used in our previous studies for fairly rapid predictions or
2
dependent on the substrate (Zhou et al., 2009).
interpretation of experimental results (Ericksen and Szklarz, 2005; Tu
Moreover, the binding constants determined for CYP1A2 WT and et al., 2008). The present studies suggest that the use of the distance-
mutants showed that the L382V substitution leads to tighter phenac-
etin binding in the active site of the L382V-containing mutants,
consistent with enzyme kinetics results. This effect may increase
phenacetin residence time, resulting in higher activity.
To better explain the effects of mutations on catalytic activity and
substrate specificity of CYP1A2, stoichiometry studies were per-
formed. The coupling efficiencies of different mutants, expressed in
dependent dielectric constant can be a reasonable substitute for the
presence of explicit water molecules. We have observed only a small
increase in the percentage of hits (less than 10%) from MD simula-
tions with water. These conclusions may be further verified by more
extensive MD simulations of the complete protein-substrate com-
plexes on nanosecond timescales. More recently, longer 2-ns MD
simulations have been successfully used to predict the effect of
mutations on the catalytic efficiency of CYP2B6 (Nguyen et al.,
terms of product/NADPH, H O /O , and H O/product ratios, were
2
2
2
2
compared with those of WT. A similar approach has been used by
Fang et al. (1997) and Kobayashi et al. (1998) to evaluate coupling
efficiencies of CYP2B1 mutants. Frequently, the mutation decreases
the coupling efficiency of the P450, as observed with CYP2B1 (Fang
et al., 1997; Kobayashi et al., 1998) and P450cam (French et al.,
2
008).
In summary, our results show that the L382V substitution in
CYP1A2 can alter the binding orientation of phenacetin within the
active site of the enzyme, so that the site of metabolism moves closer
to the heme iron. This provides a good mechanistic explanation for the
increased catalytic efficiency and coupling efficiency of phenacetin
O-dealkylation in CYP1A2 mutants containing the L382V substitu-
tion. The current studies also show that a combination of several
experimental approaches with molecular modeling methods can im-
prove our understanding of P450 catalysis. These different method-
ologies complement each other well to offer a mechanistic interpre-
tation of P450 function on a molecular level.
2
002), but the effect depends on the substrate. Previously studied
CYP1A2 mutants showed only a small increase in coupling efficiency
with phenacetin as a substrate (Yun et al., 2000), in contrast to our
results with the L382V-containing mutants. In the present studies, the
L382V mutant and multiple mutants containing the L382V substitu-
tion were similar or more efficient at coupling reducing equivalents to
acetaminophen formation than the WT (Table 3). In general, the
increased ratios of product/NADPH for the L382V mutant and mul-
tiple mutants and the decreased ratios for T124S, T223N, V227G, and
Acknowledgments. We thank Dr. Rahul Deshmukh for help and
N312L mutants were in agreement with the kinetic data regarding advice concerning expression and purification of P450 enzymes.
phenacetin turnover rates. Although no significant changes of uncou- Molecular modeling studies were performed at the Computational
pling to H O at the first and the second branching points were Chemistry and Molecular Modeling Laboratory, Department of Basic
2
2

INCREASED PHENACETIN OXIDATION IN CYP1A2 MUTANTS
1045
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Address correspondence to: Grazyna D. Szklarz, Department of Basic Phar-
maceutical Sciences, School of Pharmacy, West Virginia University, P.O. Box
9530, Morgantown, WV 26506-9530. E-mail: gszklarz@hsc.wvu.edu