Comparative inhibition of tetrameric carbonyl reductase activity in pig heart cytosol by alkyl 4-pyridyl ketones

Article abstract of DOI:10.3109/14756366.2013.790021

Context and objective: The present study is to elucidate the comparative inhibition of tetrameric carbonyl reductase (TCBR) activity by alkyl 4-pyridyl ketones, and to characterize its substrate-binding domain. Materials and methods: The inhibitory effect

Full text of DOI:10.3109/14756366.2013.790021

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J Enzyme Inhib Med Chem, Early Online: 1–4
2013 Informa UK Ltd. DOI: 10.3109/14756366.2013.790021
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RESEARCH ARTICLE
Comparative inhibition of tetrameric carbonyl reductase activity in pig
heart cytosol by alkyl 4-pyridyl ketones
Hideaki Shimada1, Takahiro Tanigawa1, Kazunori Matayoshi2, Kazufumi Katakura2, Ken Babazono2,
Hiroyuki Takayama2, Tsuyoshi Murahashi2, Hiroyuki Akita2, Toshiyuki Higuchi2, Masashi Eto3, and
Yorishige Imamura2
2
1Faculty of Education, Kumamoto University, Chuo-ku, Kumamoto, Japan, Nihon Pharmaceutical University, Komuro, Saitama, Japan, and
3Faculty of Pharmaceutical Sciences, Sojo University, Nishi-ku, Kumamoto, Japan
Abstract
Keywords
Context and objective: The present study is to elucidate the comparative inhibition of tetrameric
carbonyl reductase (TCBR) activity by alkyl 4-pyridyl ketones, and to characterize its substrate-
binding domain.
4-Benzoylpyridine, 4-hexanoylpyridine,
competitive inhibition, cyclohexyl pentyl
ketone, stereoselective reduction
Materials and methods: The inhibitory effects of alkyl 4-pyridyl ketones on the stereoselective
reduction of 4-benzoylpyridine (4-BP) catalyzed by TCBR were examined in the cytosolic
fraction of pig heart.
Results: Of alkyl 4-pyridyl ketones, 4-hexanoylpyridine, which has a straight-chain alkyl group of
five carbon atoms, inhibited most potently TCBR activity and was a competitive inhibitor.
Furthermore, cyclohexyl pentyl ketone, which is substituted by cyclohexyl group instead of
phenyl group of hexanophenone, had much lower ability to be reduced than hexanophenone.
Discussion and conclusion: These results suggest that in addition to a hydrophobic cleft
corresponding to a straight-chain alkyl group of five carbon atoms, a hydrophobic pocket with
affinity for an aromatic group is located in the substrate-binding domain of TCBR.
History
Received 12 February 2013
Revised 11 March 2013
Accepted 22 March 2013
Published online 8 May 2013
Introduction
cytosolic fraction of pig heart, 4-BP was stereoselectively reduced
to S(ꢀ)-a-phenyl-4-pyridylmethanol (S(ꢀ)-PPOL), as shown in
Figure 116. These findings suggest that in the cytosolic fraction
of pig heart, 4-BP is mainly reduced to S(ꢀ)-PPOL by TCBR17,
and that the subcellular fraction is useful as the reaction system
of TCBR when 4-BP is used as the substrate.
Our previous article18 has examined the substrate inhibition
of TCBR activity by a variety of alkyl phenyl ketones, by using
the cytosolic fraction of pig heart. Of alkyl phenyl ketones,
hexanophenone having a straight-chain alkyl group of five
carbon atoms inhibited TCBR activity most potently and was a
competitive inhibitor. These results suggest that a hydrophobic
cleft corresponding to straight-chain alkyl group of five carbon
atoms in length is located in the substrate-binding domain of
TCBR18. As shown in Figure 1, 4-BP used as the substrate has
4-pyridyl group and phenyl group across a ketone group in the
chemical structure. The purpose of the present study is to
elucidate the comparative inhibition of TCBR activity by alkyl
4-pyridyl ketones (Figure 2), and to characterize its substrate-
binding domain.
Carbonyl reductase (EC 1.1.1.184) is an enzyme that catalyzes
NADPH-dependent reduction of endogenous and xenobiotic
carbonyl compounds1–4. The cDNA for carbonyl reductase was
first cloned by Wermuth et al.5, and the enzyme belongs to the
short-chain dehydrogenase/reductase (SDR) superfamily. So far,
four kinds of mammalian carbonyl reductases (CBR1, CBR2,
CBR3 and CBR4) are characterized5–8. Carbonyl reductases
have the ability to catalyze stereoselective reduction of ketone-
containing drugs, such as acetohexamide and loxoprofen, to their
alcohol metabolites9–12
.
We have recently purified a new tetrameric carbonyl reductase
(TCBR) from the cytosolic fraction of pig heart, using
4-benzoylpyridine (4-BP) as a substrate13. The purified TCBR
is now designated dehydrogenase/reductase (SDR family)
member 4 (DHRS4) and the catalytic properties of mammalian
DHRS4 including human enzyme have been reported2,3,13,14
The purified TCBR (pig DHRS4), like human DHRS4, has the
ability to reduce ketones, quinones and retinals efficiently13,14
Interestingly, when the cytosolic fraction of pig heart was applied
to a DEAE-Sephacel column, the activity of 4-BP-reducing
enzyme appeared as a single peak, similar to the result obtained
from the cytosolic fraction of rabbit heart15. Furthermore, in the
.
.
Materials and methods
Materials
4-BP was purchased from Wako Pure Chemicals (Osaka, Japan).
S(ꢀ)-PPOL and R(þ)-PPOL were synthesized from 4-BP16.
Hexanophenone and 4-acetylpyridine were obtained from Aldrich
(Milwaukee, WI). All other chemicals were of reagent grade.
Address for correspondence: Hideaki Shimada, Faculty of Education,
Kumamoto University, 2-40-1 Kurokami, Chuo-ku, Kumamoto 860-8555,
Japan. E-mail: hshimada@gpo.kumamoto-u.ac.jp

2
H. Shimada et al.
J Enzyme Inhib Med Chem, Early Online: 1–4
O
quenched with aqueous saturated NH4Cl solution (100 ml). The
resulting mixture was extracted with CHCl3 (3 ꢁ 50 ml) and the
CHCl3 layer was washed with brine. The extract was dried over
anhydrous MgSO4. After evaporation of the solvent, the residue
was chromatographed on silica gel to give alkyl 4-pyridyl
alcohols (n-hexane-ethyl acetate, 5:1 (v/v)). To the mixture of
the alkyl 4-pyridyl alcohols (15 mmol) in acetone (15 ml) and
MgSO4 (3.9 g, 32 mmol) in water (70 ml) was added to KMnO4
(4.7 g, 30 mmol). The mixture was stirred at 60 ꢂC for 1 h and then
quenched with methanol (about 10 ml). The resulting mixture was
filtered with celite and the filtrate was extracted with CHCl3
(3 ꢁ 50 ml). The organic layer was washed with brine, dried over
MgSO4 and concentrated under reduced pressure. The residue was
purified by chromatography on silica gel to give alkyl 4-pyridyl
ketones (n-hexane-ethyl acetate, 4:1 (v/v)). The structures of alkyl
4-pyridyl ketones were confirmed by IR, 1H-NMR and mass
spectrometry.
N
4-BP
NADPH
TCBR
NADP+
OH
N
Synthesis of cyclohexyl pentyl ketone
S(-)-PPOL
A solution of cyclohexanecarboxaldehyde (1.0 g, 8.9 mmol) in
anhydrous tetrahydrofuran (5 ml) was added to a solution of 2.0 M
pentylmagnesium bromide in diethyl ether (4.5 ml, 9.0 mmol) at
ꢀ15 ꢂC under argon atmosphere. The reaction mixture was stirred
at the same temperature for 1 h and then diluted with aqueous
NH4Cl solution (50 ml). The resulting mixture was extracted with
ethyl acetate (50 ml). The organic layer was washed with brine
and dried over MgSO4. The organic layer was evaporated to give a
residue, which was chromatographed on silica gel (n-hexane-ethyl
acetate, 75:1) to afford alcohol (786.6 mg, 48%). The structure of
Figure 1. Stereoselective reduction of 4-BP to S(ꢀ)-PPOL.
O
O
N
N
4-Acetylpyridine
4-Propionylpyridine
1
alcohol was confirmed by IR, H-NMR and 13C-NMR spectros-
copy. A mixture of alcohol (100 mg, 0.54 mmol) and Dess–Martin
reagent (460 mg, 1.08 mmol) in CH2Cl2 (2 ml) was stirred for
30 min at room temperature. The reaction mixture was diluted
with diethyl ether (30 ml). The organic layer was washed with 7%
aqueous NaHCO3 (30 ml) and dried over MgSO4. Evaporation of
organic solvent gave a crude residue, which was chromatographed
on silica gel (n-hexane-ethyl acetate, 50:1) to afford cyclohexyl
pentyl ketone (86.3 mg, 87%). The structure of cyclohexyl pentyl
ketone was confirmed by IR, 1H-NMR and 13C-NMR
spectroscopy.
O
O
N
N
4-Butyrylpyridine
O
4-Valerylpyridine
O
Preparation of the cytosolic fraction from pig heart
N
N
Pig hearts were supplied from a slaughterhouse and stored at
ꢀ80 ꢂC. The tissues were homogenized in three volumes of
10 mM sodium potassium phosphate buffer containing 1.15% KCl
(pH 6.0). The homogenates were centrifuged at 105 000 ꢁ g for
60 min to obtain the cytosolic fraction which was used for the
determination of TCBR activity, as described below.
4-Hexanoylpyridine
4-Heptanoylpyridine
O
N
Assay of TCBR activity
4-Octanoylpyridine
TCBR activity was assayed by determining S(ꢀ)-PPOL
formed from 4-BP in the cytosolic fraction of pig heart.
The reaction mixture consisted of substrate (500 mM 4-BP),
NADPH-generation system (50 mM NADPþ, 1.25 mM glucose-
6-phosphate, 50 mU glucose-6-phosphate dehydrogenase and
1.25 mM MgCl2), pig heart cytosol and 100 mM sodium potas-
sium phosphate buffer (pH 6.0) in a final volume of 0.5 ml. The
reaction mixture was incubated at 37 ꢂC for 10 min and boiled
for 2 min to stop the reaction. After centrifugation at 5000 rpm,
the supernatant (20 ml) was subjected to high-performance liquid
chromatography (HPLC) for determination of the reduction
products (S(ꢀ)-PPOL and R(þ)-PPOL) of 4-BP.
Figure 2. Chemical structures of alkyl 4-pyridyl ketones. Alkyl 4-pyridyl
ketones, except 4-acetylpyridine, were synthesized by us.
Synthesis of alkyl 4-pyridyl ketones
The synthesis of alkyl 4-pyridyl ketones, except 4-acetylpyridine,
was carried out according to a slight modification of the method
reported by us19. A solution of pyridine-4-aldehyde (5.4 g,
50 mmol) in anhydrous tetrahydrofuran (30 ml) was added to an
ice-cooled solution of the corresponding Grignard reagent
(freshly prepared from Mg and alkyl bromide in anhydrous
tetrahydrofuran (65 mmol)) under a nitrogen atmosphere. The
mixture was stirred at room temperature for 12 h and then
HPLC was carried out using a Tosoh DP-8020 HPLC
apparatus (Tosoh, Tokyo, Japan) equipped with a Daicel

DOI: 10.3109/14756366.2013.790021
Inhibition of tetrameric carbonyl reductase activity in pig heart
3
60
Chiralpak AD-RH column (Daicel, Tokyo, Japan) and a Tosoh
UV-8020 monitor (254 nm). A mixture of 20 mM borate buffer
(pH 9.0)–acetonitrile (6:4, v/v) was used as a mobile phase at a
flow rate of 0.5 ml/min. In the case of inhibition experiments,
alkyl 4-pyridyl ketones were dissolved in methanol and added
to the reaction mixture at a concentration of 500 mM. The final
concentration of methanol did not exceed 2% (v/v), and this
concentration did not affect the enzyme reaction.
Furthermore, the inhibition for the stereoselective reduction
of 4-BP to S(ꢀ)-PPOL by 4-hexanoylpyridine was kinetically
analyzed using Lineweaver-Burk plots. Protein concentration was
determined by the method of Lowry et al.20 with bovine serum
albumin as the standard.
O
50
R
N
40
30
20
10
0
0
1
2
3
4
5
6
7
Number of carbon atoms in R
Figure 3. Relationship between the number of carbon atoms in alkyl
4-pyridyl ketones and their inhibitory potencies for TCBR activity in the
cytosolic fraction of pig heart. 4-BP at a concentration of 500 mM was
used as the substrate. The concentration of these ketones added as
inhibitors was 500 mM. Each bar represents the mean ꢃ SD of three or
four experiments.
Determination of kinetic parameters for
4-hexanoylpyridine and hexanophenone
The reduction of 4-hexanoylpyridine and hexanophenone was
measured spectrophotometrically by monitoring the decrease
in the absorbance of NADPH at 340 nm (" ¼ 6220 Mꢀ1 cmꢀ1).
The reaction mixture consisted of substrate, 0.3 mM NADPH, pig
heart cytosol and 100 mM sodium potassium phosphate buffer
(pH 6.0) in a final volume of 0.5 ml. The enzyme reaction was
initiated by the addition of 4-hexanoylpyridine or hexanophenone
at various concentrations to the reaction mixture. The parameters
(Km, Vmax and Vmax/Km) in the reduction of 4-hexanoylpyridine
and hexanophenone were kinetically determined. One unit of
enzyme activity was defined as the amount catalyzing the
oxidation of 1 mmol of NADPH/min at 37 ꢂC.
3.0
2.5
2.0
1.5
1.0
0.5
Reduction of cyclohexyl pentyl ketone and
hexanophenone
The reduction of cyclohexyl pentyl ketone and hexanophenone
was measured spectrophotometrically, as described above. The
reaction mixture consisted of substrate, 0.3 mM NADPH, pig
heart cytosol and 100 mM sodium potassium phosphate buffer
(pH 6.0) in a final volume of 0.5 ml. The enzyme reaction
was initiated by the addition of cyclohexyl pentyl ketone or
hexanophenone at a concentration of 100 mM to the reaction
mixture.
-4
-2
0
2
4
6
8
10
-1
1/[4-BP] (mM)
Figure 4. Lineweaver-Burk plots for the reduction of 4-BP to S(ꢀ)-PPOL
in the absence and in the presence of 4-hexanoylpyridine. Open circle
*
(
), in the absence of 4-hexanoylpyridine; closed circle (ꢄ), in the
presence of 4-hexanoylpyridine (500 mM). Each bar represents the
mean ꢃ SD of four experiments.
Results
Reduction of cyclohexyl pentyl ketone and
hexanophenone
Inhibition of TCBR activity by alkyl 4-pyridyl ketones
The reduction of cyclohexyl pentyl ketone, which is substituted
by cyclohexyl group (ring of six carbon atoms) instead of phenyl
group (ring of six carbon atoms) of hexanophenone, was
examined spectrophotometrically by monitoring the decrease in
the absorbance of NADPH at 340 nm. As shown in Figure 5,
cyclohexyl pentyl ketone had much lower ability to be reduced in
pig heart cytosol than hexanophenone.
We synthesized alkyl 4-pyridyl ketones having a straight-chain
alkyl groups (Figure 2) and examined the inhibitory potencies of
these ketones for TCBR activity. TCBR activity was estimated as
the ability to reduce 4-BP to S(ꢀ)-PPOL in the cytosolic fraction
of pig heart, since the subcellular fraction is useful as the reaction
system of TCBR when 4-BP is used as the substrate. As shown in
Figure 3, the inhibitory potencies of alkyl 4-pyridyl ketones for
TCBR activity increased with an increase in the number up to five
carbon atoms in the alkyl group, and 4-hexanoylpyridine was the
most potent inhibitor of the enzyme activity. The inhibitory
potencies of 4-heptanoylpyridine and 4-octanoylpyridine, which
have respective six and seven carbon atoms in the alkyl groups,
were lower than that of 4-hexanoylpyridine.
Discussion
Our recent study18 has demonstrated that alkyl phenyl ketones
having a straight-chain alkyl group can inhibit TCBR activity (the
stereoselective reduction of 4-BP to S(ꢀ)-PPOL) in the cytosolic
fraction of pig heart. Interestingly, the inhibitory potencies of
alkyl phenyl ketones for TCBR activity were found to increase
with an increase in the number up to five carbon atoms in the
alkyl group. Of alkyl phenyl ketones examined, hexanophenone
was the most potent inhibitor for the enzyme activity and a
Kinetic mechanism for inhibition of TCBR activity by
4-hexanoylpyridine
The kinetic mechanism for inhibition of TCBR activity by competitive inhibitor. On the basis of these results, we have
4-hexanoylpyridine was analyzed using Lineweaver-Burk plots. proposed that an alkyl hydrophobic cleft, which fits most
As expected, 4-hexanoylpyridine was confirmed to inhibit the efficiently to a straight-chain alkyl group of five carbon atoms,
enzyme activity competitively (Figure 4).
is located in the substrate-binding domain of TCBR18.

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H. Shimada et al.
J Enzyme Inhib Med Chem, Early Online: 1–4
Declaration of interest
The authors report no declarations of interest.
0.90
A
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B
0.80
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Time (min)
Figure 5. The time courses of NADPH oxidation during the reduction of
cyclohexyl pentyl ketone and hexanophenone. A, cyclohexyl pentyl
ketone; B, hexanophenone. The concentration of cyclohexyl pentyl ketone
and hexanophenone was 100 mM.
In the present study, the inhibitory potencies of alkyl 4-pyridyl
ketones having a straight-chain alkyl group for TCBR activity
were further examined in the cytosolic fraction of pig heart.
Of alkyl 4-pyridyl ketones, 4-hexanoylpyridine having a straight-
chain alkyl group of five carbon atoms inhibited the enzyme
activity most potently and was a competitive inhibitor. These
results provide further evidence that a hydrophobic cleft corres-
ponding to a straight-chain alkyl group of five carbon atoms in
length is located in the substrate-binding domain of TCBR18.
In addition to the alkyl hydrophobic cleft, it is likely that an aryl
hydrophobic pocket is located in substrate-binding domain of
TCBR. The pocket probably has affinity for an aromatic group.
This is supported from the facts that cyclohexyl pentyl ketone,
which is substituted by cyclohexyl group instead of phenyl group
of hexanophenone, is little reduced in the cytosolic fraction of
pig heart.
The inhibitory potencies of alkyl 4-pyridyl ketones for TCBR
activity were confirmed to be lower than those of alkyl phenyl
ketones having a straight-chain alkyl group of the correspond-
ing carbon atoms, by comparing with the experimental data
reported previously18. Furthermore, the Vmax/Km value (186 ꢃ 30
mU/mg/min, n ¼ 4) for the reduction of 4-hexanoylpyridine
was significantly smaller than that (410 ꢃ 13 mU/mg/min, n ¼ 4)
of hexanophenone. These may be because 4-pyridyl group
exhibits lower hydrophobicity than phenyl group. 4-BP used as
the substrate in this study has 4-pyridyl group and phenyl group
across a ketone group in the chemical structure (Figure 1).
Judging from the hydrophobicity, it is reasonable to assume
that phenyl group rather than 4-pyridyl group in 4-BP binds
predominantly to the aryl hydrophobic pocket located in the
substrate-binding domain of TCBR. As a result, 4-BP may be
reduced stereoselectively to S(ꢀ)-PPOL by TCBR.
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In conclusion, the present study demonstrates that a hydro-
phobic cleft corresponding to a straight-chain alkyl group of five
carbon atoms in length and a hydrophobic pocket with affinity for
an aromatic group are located in the substrate-binding domain of
TCBR. Further studies are in progress to elucidate the character-
istics in substrate-binding domain of TCBR.