Sialic acid 9-O-acetylesterase catalyzes the hydrolyzing reaction from alacepril to deacetylalacepril

Article abstract of DOI:10.1023/A:1025073720126

Purpose. In this work, the alacepril thiolesterase, which catalyzes the hydrolyzing reaction of the thiolester linkage in alacepril and the conversion from alacepril to deacetylalacepril, was purified from rat liver cytosol and characterized. Methods. A purification procedure for the thiolesterase consisted of ammonium sulfate fractionation and chromatographies with phenyl Sepharose CL-4B, Q Sepharose FF, ceramic hydroxylapatite, and phenyl Sepharose HP. The thiolesterase activity was assayed for alacepril as a substrate and the reaction product, deacetylalacepril, was measured using high-performance liquid chromatography. Results. The purified thiolesterase is heterodimeric with a molecular mass of 29 and 36 kDa subunits as estimated by sodium dodecyl sulfate -polyacrylamide gel electrophoresis. N-terminal amino acid sequence of these subunits reveals that the thiolesterase is identical to sialic acid 9-O-acetylesterase. The thiolesterase hydrolyzes not only the thiolester bond in alacepril, spironolactone, and acetyl coenzyme A but also the carboxylester bond in α-naphtyl acetate. The alacepril thiolestrase activity is competitively inhibited by α-naphtyl acetate. Conclusion. The thiolesterase, i.e., sialic acid 9-O-acetylesterase, seems to be involved in the metabolism of certain drugs such as alacepril and spironolactone. However, drugs having ester-type and amide-type linkages, for example dilazep, aniracetam, and benazepril, are not substrates for the thiolestrase.

Full text of DOI:10.1023/A:1025073720126

Pharmaceutical Research, Vol. 20, No. 8, August 2003 (© 2003)
Research Paper
inhibitor (4). Therefore, the conversion from alacepril to
deacetylalacepril to hydrolyze the thiolester linkage in alace-
pril seems to be an important reaction step for alacepril to
exhibit the antihypertension effects as an ACE inhibitor.
Esterases are well known to catalyze the hydrolyzing re-
action of carboxylester, thiolester, and amide linkages (5).
These enzymes are widely distributed in various tissues of
animals and hydrolyze a variety of endogenous and exog-
enous compounds (6–8). Several esterases have been purified
from many sources of animals and their properties have been
investigated (9–12). In drug metabolism, ester-linkages in
many prodrugs are hydrolyzed by esterases, converting pro-
drugs to active forms (13,14). Other drugs with the linkage are
degraded to inactivate forms by the enzymes (15). Many find-
Sialic Acid 9-O-Acetylesterase
Catalyzes the Hydrolyzing Reaction
from Alacepril to Deacetylalacepril
1
1
Shigeyuki Usui, Masafumi Kubota,
1
1
2
Kazuhiro Iguchi, Tadashi Kiho, Tadashi Sugiyama,
Yoshihiro Katagiri, and Kazuyuki Hirano
2
1, 3
Received March 25, 2003; accepted April 30, 2003
Purpose. In this work, the alacepril thiolesterase, which catalyzes the ings on inhibitors (16) and inducers (17,18) for esterases and
hydrolyzing reaction of the thiolester linkage in alacepril and the polymorphism of these enzymes (19,20) have been accumu-
conversion from alacepril to deacetylalacepril, was purified from rat lated and are for analyzing pharmacokinetics of drugs with
liver cytosol and characterized.
Methods. A purification procedure for the thiolesterase consisted of
the ester linkage in human. However, the substrate specificity
of each esterase for ester-type drugs are not well known be-
ammonium sulfate fractionation and chromatographies with phenyl
cause esterases generally appear to show broad substrate
Sepharose CL-4B, Q Sepharose FF, ceramic hydroxylapatite, and
specificity.
phenyl Sepharose HP. The thiolesterase activity was assayed for ala-
Species differences of esterases have not been elucidated
cepril as a substrate and the reaction product, deacetylalacepril, was
sufficiently. However, it is important to speculate the metabo-
measured using high-performance liquid chromatography.
lizing pathway of drugs with ester linkage in humans to apply
drug metabolism utilizing animal experiments in pre-clinical
study of drug development. We previously reported that
pranlukast, an antagonist for the leukotriene receptor, was
Results. The purified thiolesterase is heterodimeric with a molecular
mass of 29 and 36 kDa subunits as estimated by sodium dodecyl
sulfate -polyacrylamide gel electrophoresis. N-terminal amino acid
sequence of these subunits reveals that the thiolesterase is identical to
sialic acid 9-O-acetylesterase. The thiolesterase hydrolyzes not only hydrolyzed by rat esterases RL1 and RH1, but not by human
the thiolester bond in alacepril, spironolactone, and acetyl coenzyme esterase HU1 and hCE-2 (21). Indomethacin was hydrolyzed
A but also the carboxylester bond in ␣-naphtyl acetate. The alacepril by a pig esterase but not by rat and human esterases (22).
thiolestrase activity is competitively inhibited by ␣-naphtyl acetate.
Conclusion. The thiolesterase, i.e., sialic acid 9-O-acetylesterase,
seems to be involved in the metabolism of certain drugs such as
alacepril and spironolactone. However, drugs having ester-type and
amide-type linkages, for example dilazep, aniracetam, and benaz-
epril, are not substrates for the thiolestrase.
Therefore, it is useful to clarify the species differences of
esterases for the study of drug metabolism as substrate speci-
ficity of esterases for some drugs with the ester-linkage seems
to be different between species.
In this study, we identified and characterize an alacepril
thiolesterase that catalyzes the hydrolyzing reaction of thi-
olester linkage in alacepril. The substrate specificity of the
thiolesterase for drugs with ester linkage is investigated.
KEY WORDS: alacepril; thiolesterase; carboxylesterase; hydrolase;
sialic acid 9-O-acetylesterase.
MATERIALS AND METHODS
Materials
INTRODUCTION
Alacepril, which is used clinically as an anti-hypertension
drug, is a prodrug of captopril, an angiotensin I-converting
enzyme (ACE) inhibitor (1). Alacepril is known to be me-
tabolized mainly in the liver and its metabolizing pathway has
been reported (2,3). Alacepril first receives the deacetylation
of thiolester and is converted to deacetylalacepril. Second,
amide linkage in deacetylalacepril is hydrolyzed and phenyl-
alanine is released to produce captopril. Because the sulfhy-
dryl group in captopril chelates zinc that is located in the
active center of ACE, a metalloenzyme containing zinc, this
group is believed to be essential for the drug effect as an ACE
Alacepril was a generous gift from Dainippon Pharma-
ceutical Co. Ltd. (Osaka Japan), and authentic materials of
clinically used preparations were kindly supplied by their re-
spective manufactures. Q-Sepharose FF, Phenyl Sepharose
CL-4B, and Phenyl Sepharose HP were obtained from Amer-
Sham Pharmacia Biotech (Buckinghamshire, UK). Ceramic
Hydroxylapatite was purchased from Bio-Rad Laboratories
(Hercules, CA, USA). Cosmosil 5-C18 MS, a reversed phase
high-performance liquid chromatography (HPLC) column,
was from Nacalai Tesque Inc. (Kyoto, Japan). All other
chemicals and reagents were of analytical grade or liquid
chromatographic grade.
1
Department of Pharmaceutics, Gifu Pharmaceutical University,
5
-6-1 Mitahora-higashi, Gifu 502-8585, Japan.
Purification of Alacepril Thiolesterase
2
3
Department of Pharmacy, Gifu University Hospital, 40 Tsukasa-
machi, Gifu 502-8705, Japan.
Livers from Sprague–Dawley male rats were obtained
To whom correspondence should be addressed. (E-mail: hirano@ according to a protocol approved by the Review Board in
gifu-pu.ac.jp) Gifu Pharmaceutical University and stored at −80°C until use.
1309
0724-8741/03/0800-1309/0 © 2003 Plenum Publishing Corporation

1
310
Usui et al.
Table I. Subcellular Distribution of Alacepril Thiolesterase in Rat Liver
Total protein
mg)
Total activity
(units)
Specific activity
(units/mg)
(
Cytosol fraction from frozen livers
From fresh livers
Microsomal fraction from frozen livers
From fresh livers
5,810
5,234
13,626
13,455
21,202
17,849
5,062
3.649
3.410
0.371
0.582
7,826
All purification procedures were performed at 0–4°C. Frozen with an equal volume of the same buffer, the thiolesterase was
rat livers were homogenized with 6 volumes of 10 mM potas- eluted with a linear gradient formed from 1 to 0 M ammo-
sium phosphate, pH 8.0, containing 0.32 M sucrose. The ho- nium sulfate in 5 mM potassium phosphate, pH 7.0. The frac-
mogenate was centrifuged at 9000 × g for 10 min. and the tions containing the enzyme were collected, concentrated by
supernatant was further centrifuged at 105,000 × g for 1 h. Centricon YM-10 (Amicon-Millipore), and dialyzed over-
The supernatant obtained was designated as the cytosol frac- night against 5 mM potassium phosphate, pH 7.0.
tion. The precipitate was suspended with 10 mM potassium
Assay for Enzyme Activities
phosphate, pH 8.0, containing 0.5% Triton X-100 and then
centrifuged at 105,000 × g for 1 h. The supernatant prepared
was designated as the solubilized microsomal fraction.
The cytosol fraction was subjected to ammonium sulfate
fractionation. The precipitate formed between 45 and 60%
ammonium sulfate saturation was collected by centrifugation
at 12,000 × g for 20 min and redissolved in 5 mM potassium
phosphate, pH 7.0, containing 0.8 M ammonium sulfate and
then dialyzed overnight against the same buffer. The dialyzed
solution was applied to a Phenyl Sepharose CL-4B column
Forty microliters of the reaction mixture consisted of 50
mM potassium phosphate, pH 7.5, containing 1 mM alacepril,
1
3
2
.25% DMSO, and the enzyme solution was incubated at
7°C for 60 min. The enzyme reaction was stopped by adding
00 ␮L of acetonitril. To the reaction mixture, 200 ␮L of 20
␮
g/mL p-hydroxy benzoic acid n-propyl as an internal stan-
dard was added and the supernatant was separated by cen-
trifugation. Thirty microliters of the supernatant was subse-
quently applied to Cosmosil 5-C18 MS (4.6 i.d. × 250 mm), a
reversed-phase HPLC column, and then alacepril, deacet-
ylalacepril, which is a product of the enzyme reaction, and
p-hydroxy benzoic acid n-propyl were detected by monitoring
at a wavelength of 220 nm. The analytical condition of these
compounds by HPLC was as follows: the mobile phase con-
sisted of three parts of 10 mM potassium phosphate, pH 2.5,
and two parts of acetonitril was used and separation of com-
pounds was performed at 50°C and the flow-rate of 1.5 mL/
min. One unit of enzyme activity was defined as the amount
to hydrolyze the thiolester bond in alacepril at a rate of 1
nmol per min. The activities of calboxylesterase and acetyl
coenzyme A thiolesterase were measured by the methods of
Sugiura et al. (23) and Prass et al. (24), respectively.
(2.5 × 20 cm) equilibrated with 5 mM potassium phosphate,
pH 7.0, containing 0.8 M ammonium sulfate. After washing
the column with the same buffer, the alacepril thiolesterase
was eluted with 5 mM potassium phosphate, pH 7.0. Fractions
with thiolesterase activity were collected and dialyzed over-
night against 10 mM Tris-HCl, pH 8.0. The dialysate was
loaded to a Q Sepharose FF column (2.5 × 40 cm) equili-
brated with 10 mM Tris-HCl, pH 8.0, and the column was
washed with 2 volumes of the same buffer. The thiolesterase
was eluted with a linear gradient formed from 0 to 0.15 M
NaCl in 10 mM Tris-HCl, pH 8.0. The fractions exhibiting the
enzyme activity were pooled and dialyzed overnight against 5
mM potassium phosphate, pH 7.0. The dialysate was applied
to a Hydroxylapatite column (1.5 × 0.8 cm) equilibrated with
5
mM potassium phosphate, pH 7.0, and the column was
Protein Determination
washed with 3 volumes of the same buffer. The thiolesterase
was eluted with a linear gradient formed from 5 to 100 mM
potassium phosphate, pH 7.0, and the fractions with the ac-
tivity were combined. Ammonium sulfate was added to the
fractions to bring the concentration to 1 M and the solution
was loaded on a phenyl Sepharose HP column (0.7 × 15 cm)
equilibrated with 5 mM potassium phosphate, pH 7.0, con-
Protein concentration was measured with bovine serum
albumin as a standard according to the method of Lowry
et al. (25).
Protein Blotting and Amino Acid Sequence Analysis
The purified enzyme preparation was resolved by elec-
taining 1 M ammonium sulfate. After the column was washed trophoresis with 12.5% polyacrylamide gel containing 1% so-
Table II. Purification of Alacepril Thiolesterase from Rat Liver
Total protein
(mg)
Total activity
(units)
Specific activity
(units/mg)
Yield
(%)
Purification
(fold)
Step
Crude extract
5,810
1,508
468
25.9
1.47
21,202
9,122
8,174
1,348
513
3.65
6.05
17.5
52.1
349
100
1.00
1.66
4.79
14.3
95.5
190
Ammonium sulfate fractionation
Phenyl Sepharose CL-4B
Q Sepharose FF
Hydroxylapatite
Phenyl Sepharose HP
43.0
38.6
6.36
2.42
0.62
0.190
132
695

Alacepril Thiolesterase
1311
Fig. 1. Hydroxylapatite column chromatography of alacepril thiolesterase from rat
liver. The fraction containing thiolesterase, which was obtained from Q Sepharose
column chromatography, was applied to a Hydroxylapatite column equilibrated with 5
mM potassium phosphate buffer, pH 7.0. After the column was washed with the buffer,
the enzyme was eluted with a linear gradient formed between 5 and 100 mM potassium
phosphate. Solid and broken lines indicate the absorbance at 280 nm and the concen-
tration of potassium phosphate, respectively. Open and closed circles indicate the
carboxylesterase activity for ␣-naphtylacetate as a substrate and the alacepril thioles-
terase activity, respectively.
dium dodecyl sulfate (SDS) under a reducing condition by reversed phase HPLC column by the linear gradient of ace-
-mercuptoethanol according to the method described by tonitril and 1% triethylamine, pH 2.5. The atmospheric pres-
2
Laemmli (26). The enzyme in the gel was electroblotted onto sure chemical ionization method was used as an ionization
a polyvinylidene difluoride membrane according to the method of the product in the mass measurement and the
method of Towbin et al. (27). The protein blotted on the
membrane was visualized with coomassie brilliant blue R 250.
The protein band was cut out, briefly wash with methanol,
and applied to a protein sequencer (model 473 protein se-
quencer, Applied Biosystems).
Molecular Mass Determination
The molecular mass of the native thiolesterase was esti-
mated by gel filtration using a Superdex 200HR column (1 ×
2
5 cm) equilibrated with 10 mM potassium phosphate, pH 7.0,
containing 0.15 M NaCl. Ovalbumin (M 43,000), bovine se-
r
rum albumin (68,000), and ␤-galactosidase (130,000) were
used as molecular mass standards. The molecular mass of the
denatured thiolesterase was determined by SDS-(12.5%)
polyacrylamide gel electrophoresis by the method of Laemmli
(26). The sample and standards were treated with 2% SDS
and 5% 2-mercuptoethanol at 100°C for 5 min before they
were subjected to electrophoresis. The protein standards used
were lysozyme (14,000), carbonic anhydrase (31,000), ovalbu-
min, bovine serum albumin, and phospholylase B (94,000).
Liquid Chromatography-Mass Spectroscopy
(LC-MS) Analysis
Fig. 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of
the purified alacepril thiolesterase. Lanes 1 and 2 show molecular
mass standards and the purified thiolesterase, respectively. About 1
The product of the thiolesterase reaction using alacepril
as a substrate was identified by LC-MS (Hewlett Packard
model HP 1100 series LC/MSD). The enzyme reaction was
performed in a standard reaction mixture. The separation of
␮
g of the purified enzyme was resolved with 12.5% sodium dodecyl
sulfate polyacrylamide gel under a reducing condition. Proteins on
the reaction product using HPLC was conducted by the the gel were visualized by staining with coomassie brilliant blue
method described above, but the product was eluted form the R-250.

1
312
Usui et al.
analysis condition was optimized by the flow injection analy- thiolesterase activity coeluted with the carboxylesterase ac-
sis function equipped in the LC-MS system.
RESULTS
tivity for ␣-naphtyl acetate as a substrate in the hydroxylap-
atite column chromatography, the thiolesterase was separated
from the carboxylesterase, shown as a major fraction of the
activity for ␣-naphtyl acetate (Fig. 1). The chromatographies
with PBA-30, concanavaline A (Con A) Sepharose, and
wheat germ lectin (WGA) agarose were performed to deter-
mine whether or not the thiolesterase contained sugar chains.
The solution of the purified thiolesterase was dialyzed against
Subcellular Distribution of Thiolesterase in Rat Liver
The cytosol and solubilized microsomal fractions were
prepared from fresh and frozen rat livers and the alacepril
thiolesterase activity in these fractions was assayed for alace-
pril as a substrate (Table I). The specific activity of the thi-
olesterase in the cytosol fraction was found to increase by
freezing rat livers, whereas it decreased in the microsomal
fraction. This implies that a part of the thiolesterase is re-
leased from the microsomal fraction to the cytosol fraction by
freezing and thawing. Because the total and specific activities
of the thiolesterase in the cytosol fraction from frozen livers
were observed to be about four and ten times higher, respec-
tively, than those in the solubilized microsomal fraction, the
thiolesterase was purified using the cytosol fraction from fro-
zen livers.
2
5 mM HEPES-NaOH, pH 7.5 containing 1 mM MgCl and
2
then applied to a PBA-30 column equilibrated with the same
buffer. The thiolesterase was retained on this column and
eluted with 25 mM HEPES-NaOH, pH 7.5, containing 100
mM sorbitol. However, the thiolesterase was not absorbed by
Con A Sepharose or WGA agarose, although the enzyme
solution was dialyzed against 10 mM potassium phosphate,
pH 7.4, containing 0.15 M NaCl and then applied to these
columns.
The native thiolesterase eluted as a single peak by gel
filtration with Superdex 200HR and the molecular mass was
estimated as about 60 kDa. Two stained protein bands cor-
responding to the molecular mass of 29 and 36 kDa were
observed by SDS-polyacrylamide gel electrophoresis under a
reducing condition (Fig. 2). When the thiolesterase was incu-
bated at room temperature for 8 h with 10 mM 2-mercapto-
thanol or dithiothreitol, the enzyme activity completely dis-
appeared.
Purification of Thiolesterase from Rat Liver
The alacepril thiolesterase was purified by sequential ap-
plication of ammonium sulfate precipitation and four serial
chromatographies. About 190 ␮g of the aracepril thiolester-
ase was obtained from 67 g of frozen rat livers. The overall
yield was approximately 0.62%. Using aracepril as a sub-
strate, a 190-fold purification was obtained (Table II). The
yield of the enzyme activity was shown to be relatively low in
Identification of Enzymatic Reaction Product
The enzymatic reaction product with alacepril as a sub-
the step of Q-Sepharose column chromatography. This was strate for thiolesterase was identified by LC-MS analysis. Ala-
caused by the enzyme eluating broadly from the column and cepril, deacetylalacepril (product), and internal standard were
the part of the fractions exhibiting the activity were collected completely separated by HPLC with a reversed-phase col-
by raising the purification efficiency. Although the alacepril umn. Alacepril and deacetylalacepril were confirmed by the
Fig. 3. (A) High-performance liquid chromatography profile and (B) mass spectra of
alacepril and deacetylalacepril. The standard reaction mixture containing the purified
alacepril thiolesterase was incubated at 37°C for 60 min. Acetonitril and n-propyl
p-hydroxybenzoate as an internal standard were added to the mixture. After centrifu-
gation, 30 ␮L of the supernatant was analyzed by high-performance liquid chromatog-
raphy and liquid chromatography-mass spectroscopy under the conditions described in
Materials and Methods section.

Alacepril Thiolesterase
1313
mass of the molecular ion peak, which were estimated as Table IV. List of Drugs Not Hydrolyzed by Aracepril Thiolesterase
407.5 and 365.5 (m/z), respectively (Fig. 3).
Linkage
Drugs
Catalytic Properties of Thiolesterase
Ester type
Dilazep, Flavoxate, Propiverine, Oxybutynin
Amide type Aniracetam, Flutamide, Indomethacin, Posatireline,
Pranlukast, Prazosin, Tiaramide
Substrate specificity of the alacepril thiolesterase are
shown in Table III. The thiolesterase catalyzed the hydrolyz-
ing reaction of not only the thiolester bond in alacepril, spi-
ronolactone, and acetyl coenzyme A but also the carboxyl-
ester bond in ␣-naphthyl acetate. ␣-Naphthyl acetate was
found to be a competitive inhibitor for this enzyme when the
activity was assayed for alacepril as a substrate. The inhibition
constant of ␣-naphthyl acetate was calculated to be about 10
mM. The thiolester bond in palmitoyl coenzyme A was not
hydrolyzed by this enzyme. Whether the thiolesterase cata-
lyzes the hydrolysis of thiolester-, carboxylester-, and amide-
type bonds in various drugs clinically used was investigated,
as alacepril is a drug administered frequently in Japan. As
shown in Table IV, this enzyme was not observed to hydro-
lyze the bonds in all the drugs examined.
Mixed type
Camostat mesilate, Benazepril, Ditiazem, Irinotecan,
Quinapril, Temocapril
masses of the native and the denatured thiolesterase were
calculated as 60 and, 29 and 36 kDa, respectively, indicating
that this enzyme is heterodimeric. Both subunits of the thi-
olesterase appear to be necessary for the enzyme activity be-
cause the thiolesterase activity disappeared by the reduction
of the enzyme with the reducing reagents for the disulfide
bond, 2-mercaptoethanol and dithiothreitol. Thiolesterase
seems to be different from calboxylesterases RL1, RH1, and
RL2 (28–31) from the result of the hydroxylapatite column
chromatography; the thiolesterase activity for alacepril and
the major calboxylesterase activity for ␣-naphtyl acetate
eluted separately from this column. N-terminal amino acid
sequences of 29- and 36-kDa subunits of the purified thioles-
terase were found to be identical to those of the sialic acid
Optimal pH of the enzymatic reaction was determined to
be between 7.5 and 8.0 in potassium phosphate and Tris-HCl
buffers when alacepril was used as a substrate, and similar
optimal pH was observed with ␣-naphtyl acetate as a sub-
strate.
9
-O-acetylesterase reported as a membrane associated ester-
ase, LSE (32,33).
Sialic acid 9-O-acetylesterase was reported to be distrib-
Effects of Metal Ions and Inhibitors
Table V lists metal ions and compounds which affected uted widely to variety of rat tissues including the liver, kidney,
the thiolesterase activity. The enzyme activity was inhibited heart, lung, spleen, intestine, testis, and brain (34). Acetyles-
by diisopropyl fluorophosphate and phenylmethylsulfonyl terase activity was observed in influenza C virus and mam-
fluoride, which are well known as serine residue modifiers, mals such as human and mouse (35). Two acetylesterases
2+
and the enzyme was also inactivated strongly by Hg and were reported to show a similar substrate specificity but dif-
2
+
Cu
.
ferent subcellular distribution (34). One was a membrane as-
sociated intralumenal sialic acid 9-O-acetylesterase localized
mainly in lysosome (LSE) and the other was a cytosolic sialic
acid 9-O-acetylesterase (CSE). The alacepril thiolesterase
was purified from the cytosol fraction prepared from frozen
N-Terminal Amino Acid Sequence of Thiolesterase
After two subunits, corresponding to the molecular mass
of 29 and 36 kDa, of the thiolesterase were electroblotted on
a polyvinylidene difluoride membrane, the N-terminal amino
acid sequences of these subunits were analyzed separately. As
shown in Fig. 4, sequences of 29 and 36 kDa subunits partly
agreed with those of the small and large subunits of sialic acid
Table V. Effects of Metal Ions and Compounds on Alacepril
Thiolesterase Activity
Concentration
(mM)
Remaining
activity (%)
9
-O-acetylesterase from rat, although several unidentified
amino acids were observed in these sequences.
Metal ion and compound
2
+
Hg
1
0
3.9
85.9
0.6
34.9
73.3
81.3
89.8
92.7
97.0
101.4
115.3
0
DISCUSSION
0
0
1
0
1
1
1
1
1
1
1
1
.1
.01
Alacepril thiolesterase, which catalyzes the hydrolyzing
reaction of the thiolester bond in aracepril and the conversion
to deacetylalacepril, was purified to apparent homogeneity
2+
Cu
.1
2+
Ni
2
+
from the cytosol fraction of frozen rat livers. Molecular Co
2
+
Fe
2
+
Mg
Table III. Substrate Specificity of Alacepril Thiolesterase
2
+
Zn
2
+
Mn
Km
Vmax
2
+
Cd
Substrate
Alacepril
(mM)
(␮mol/min/mg)
Diisopropyl fluorophosphate
22.0
0.652
1.71
1.28
28.6
0.0156
143
0.1
0.01
0.001
10
1
1
5.4
Spironolactone
-Naphtyl acetate
28.8
69.1
46.4
77.9
93.4
␣
Acetyl coenzyme A
Palmitoyl coenzyme A
0.998
Phenylmethylsulfonyl fluoride
Ethylendiaminetetraacetic acid
a
a
ND
ND
a
ND, not detected.

1
314
Usui et al.
Fig. 4. N-terminal amino acid sequences of two subunits of the purified alacepril thi-
olesterase from rat liver. Two subunits of the purified thiolesterase was electroblotted
on a polyvinylidene difluoride membrane after electrophoresis with sodium dodecyl
sulfate polyacrylamide gel. The amino acid sequence of the blotted protein was ana-
lyzed by automated Edman degradation using a gas-phase protein sequencer. LSE,
lysosomal sialic acid 9-O-acetylesterase (33); X, unidentified amino acid
rat livers despite the consistency of N-terminal amino acid seem to resemble partly those of LSE reported, the effects of
sequence between this enzyme and the membranous LSE. the fluoride and the metal ions on the thiolesterase were dif-
This contradiction may be solved by the following explana- ferent from those on LSE (34). The reason why this difference
tions. The molecular mass of CSE was reported as about 82 occurs is not clear at present. However, the result that the
kDa with a single polypeptide. The N-terminal amino acid thiolesterase was inhibited at a concentration range from 1 to
sequence of CSE reported, MAQQTLGTMNFTLRV-, was 10 ␮M diisopropyl fluorophosphate is consistent with the re-
apparently different from that of LSE, but LSE and CSE are port about the sialic acid 9-O-acetylesterase (38). The sialic
suggested to be encoded by one gene via differential usage of acid 9-O-acetylesterase including LSE was also reported to
a single peptide-encoding exon at the N terminus (36). The not contain the active site sequence Gly-X-Ser-X-Gly com-
sialic acid 9-O-acetylesterase from mammalian sources ap- monly found in serine hydrolases and its putative sequence
peared to be a soluble form even though two enzymes of LSE Gly-Asp-Ser-Arg-Thr in the acetylesterase was proposed.
and CSE, which were localized in different subcelluar frac-
It seems certain that thiolesterase hydrolyzes both thi-
tions, were found (35). Because frozen rat livers were used as olester and carboxylester bonds since alacepril, coenzyme A,
the starting material for purifying the thiolesterase, this en- and ␣-naphtyl acetate were apparent substrates for the thi-
zyme could efficiently traverse the damaged membrane from olesterase and the thiolesterase activity with alacepril as a
lysosome to cytosol. Therefore, the alacepril thiolesterase is substrate was inhibited competitively by ␣-naphtyl acetate.
believed to be identical to the sialic acid 9-O-acetylesterase of However, the thiolesterase did not catalyze the hydrolyzing
LSE, from the result of the N-terminal amino acid sequencing reaction of the thiolester bond in palmitoyl CoA and the
of the thiolesterase.
carboxylester bonds in dilazep, flavoxate, proplverine, and
LSE was observed to bind to Con A Sepahrose because oxybutynin. These results suggest that the ester bonds con-
of the N-linked sugar chains in the enzyme, but CSE did not, sisted of an acetyl group, a small molecule, which are only
as it lacks the sugar chains (34). Therefore, LSE seemed to be hydrolyzed by this enzyme. Schauer et al. (35,39) described
a glycoprotein with high mannose-type and complex-type that the substrate specificity of sialic acid 9-O-acetylesterase
sugar chains. It was suggested that these esterases were in- seemed to be relatively specific for acetic acid esters, such as
volved in regulation of membrane sialic acid O-acetylation N-acetyl-9-O-acetylneuraminic acid and 4-methylumbelliferyl
and acted as a scavenger of recycled free O-acetylsialic acid acetate. It was demonstrated especially that this acetylester-
(37). The alacepril thiolesterase was not retained on Con A ase from rat liver had strict substrate specificity for O-
Sepharose and WGA agarose in our conditions, although the acetylsialic acids and broad specificity for small synthetic es-
thiolesterase was identified to LSE. Nonetheless, the thioles- ters, such as p-nitrophenyl acetate, ␣-naphthyl acetate, and
terase bound to PBA-30 which couples agarose with m- 4-methylumbelliferyl acetate (34). The alacepril thiolesterase
aminophenyl boronic acid. Because a boronate group in seems to show similar substrate specificity for synthetic ace-
PBA-30 was reported to form a complex with hydroxyl tylesters to LSE, although 9-O-acetyl-N-acetyl-neuraminic
groups in a sugar, this suggests the possibility that the thioles- acid was not examined at present because of its commercial
terease contains sugar chains. It is unfortunately unclear unavailability.
whether N-linked sugar chains proposed in LSE are involved
in the thiolesterase.
The alacepril thiolesterase was demonstrated to hydro-
lyze alacepril and spironolactone, but carboxylester-type and
The alacepril thiolesterase activity was shown to be sen- amide-type linkage in the drugs tested were not substrates for
sitive for serine modifiers, diisopropyl fluorophosphate and this enzyme in this report. The apparent Km value for the
2
+
phenylmethylsulfonyl fluoride, and heavy metal ions, Hg
endogenous substrate, 9-O-acetyl-N-acetyl-neuraminic acid,
2
+
and Cu , suggesting that this enzyme is one of the serine in LSE was estimated as 8.8 mM with a Vmax of 48 nmol/min/
hydrolases. The optimal pH of the thiolesterase activity was mg (34) and the value for alacepril in the thiolesterase was 22
observed to be around pH 7.8. Although these properties mM with 28.6 ␮mol/min/mg. It is likely for this enzyme to

Alacepril Thiolesterase
1315
metabolize alacepril at a similar rate to the endogenous sub-
strate in liver, although it may not be able to compare these
values directly because of the different assay system. Thus,
the alacepril thiolesterase, i.e., LSE, is suggested to take part
in the metabolism of certain drugs with acetylester and
acetylthiolester bonds. It is very beneficial to clarify what
enzymes are involved in metabolism of drugs and prodrugs
with thiolester, carboxylester, and amide linkages for studying
the pharmacokinetics of these drugs. LSE was distributed in a
variety of rat organs and its specific activity was observed to
be high in the brain, testis, kidney, and colon mucosa (34).
Therefore, thiolesterase is expected to be applied to the de-
cancer agent, by carboxylesterase. Biol. Pharm. Bull. 17:662–664
(
1994).
1
1
1
1
5. R. Mentlein and E. Heymann. Hydrolysis of ester- and amide-
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