Mechanism of biochemical action of substituted 4-methylbenzopyran-2- ones. Part 6: Hydrolysis of 7,8-diacetoxy-4-methylcoumarin by a novel deacetylase in rat liver microsomes - A simple method for assay and characterisation

Article abstract of DOI:10.1016/S0968-0896(99)00272-2

The existence of a novel microsomal deacetylase in rat liver catalysing deacetylation of diacetoxy 4-methylcoumarins has been reported. A simple method is outlined for the enzyme assay based upon the quantification of the dihydroxy derivative by measuring the UV absorption of its complex with ADP and Fe³⁺ at 600 nm. The enzyme can be routinely assayed using 7,8- diacetoxy-4-methylcoumarin (DAMC) as the substrate and demonstrated hyperbolic kinetics and yielded K(m) and V(max) values of 1250 μM and 500 units, respectively. The pH optima was found to be 7.5 for the enzyme. No DAMC deacetylase activity was found in hepatic cytosol and the enzyme activity was not discernible in extrahepatic tissues. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00272-2

Bioorganic & Medicinal Chemistry 8 (2000) 233±237
Mechanism of Biochemical Action of Substituted
4-Methylbenzopyran-2-ones. Part 6: Hydrolysis of
7,8-Diacetoxy-4-methylcoumarin by a Novel Deacetylase in
Rat Liver Microsomes Ð a Simple Method for Assay and
Characterisation
a
b,
b
a
Hanumantha Rao G. Raj, Virinder S. Parmar, * Subhash C. Jain, Sanjay Goel,
a
b
c
d
Yogesh K. Tyagi, Sunil K. Sharma, Carl E. Olsen and Jesper Wengel
aDepartment of Biochemistry, V.P. Chest Institute, University of Delhi, Delhi-110 007, India
bDepartment of Chemistry, University of Delhi, Delhi-110 007, India
cChemistry Department, Royal Veterinary & Agricultural University, 40 Thorvaldsensvej, Frederiksberg C, DK-1871 Copenhagen,
Denmark
d
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK 2100 Copenhagen, Denmark
Received 2 August 1999; accepted 21 September 1999
AbstractÐThe existence of a novel microsomal deacetylase in rat liver catalysing deacetylation of diacetoxy 4-methylcoumarins has
been reported. A simple method is outlined for the enzyme assay based upon the quanti®cation of the dihydroxy derivative by
measuring the UV absorption of its complex with ADP and Fe3 at 600 nm. The enzyme can be routinely assayed using 7,8-dia-
cetoxy-4-methylcoumarin (DAMC) as the substrate and demonstrated hyperbolic kinetics and yielded Km and ꢀmax values of 1250
mM and 500 units, respectively. The pH optima was found to be 7.5 for the enzyme. No DAMC deacetylase activity was found in
hepatic cytosol and the enzyme activity was not discernible in extrahepatic tissues. # 2000 Elsevier Science Ltd. All rights reserved.
+
3
Introduction
Cardona and King demonstrated that O-glucuronide
of N-hydroxy-N-2-¯uorenylacetamide was deacetylated
by liver microsomes of guinea pigs. Deacetylation of O-
glycoside of N-hydroxyacetanilide was found to be
localized in liver microsomes of guinea pigs.4
As a part of our ongoing research programme towards
screening of various lipases/esterases for carrying out
selective protection/deprotection of polyphenolics, we
explored the possibility of using tissue esterases for
selective protection/deprotection of coumarins. A very
large number of esterases are known to occur in human
We herein report the deacetylation of diacetoxy 4-
methylcoumarins using rat liver microsomes (Fig. 1(A)).
The assay and characterisation of the enzyme is carried
out principally using 7,8-diacetoxy-4-methylcoumarin
(DAMC) (1) as the substrate and the method is exten-
ded to assay the deacetylase with other diacetoxy-
coumarins as substrates.
1
tissues. However, the knowledge on tissue microsomal
esterases is rather incomplete and complex, as analysis
and characterisation of tissue esterases has not only
been complicated in terms of puri®cation but also due
to their existence in multiple molecular forms. Mostly
the pattern of substrate speci®city and sensitivity to
various inhibitors have been the basis for characterisa-
tion of the esterases. Hammer and Ladu2 employed
extraction of human liver microsomes by glycerol, fol-
lowed by gel ®lteration for the isolation of esterases.
Materials and Methods
ADP was purchased from Sigma Chemical Co., St.
Louis (MO, USA). p-Hydroxymercuribenzoate (PHMB)
was procured from E-Merck, Germany. FeCl , Tris and
3
DMSO were purchased from local suppliers and were of
high purity. DAMC (1), 7,8-dihydroxy-4-methylcoumarin
*
7
Corresponding author. Tel.: +91-11-725-6555/7206; fax: +91-11-
25-7206; e-mail: vparmar.duchem@axcess.net.in
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00272-2

2
34
H. R. G. Raj et al. / Bioorg. Med. Chem. 8 (2000) 233±237
Kinetics of rat liver microsomal DAMC deacetylase
Initial velocity as a function of substrate concentration.
Several concentrations of DAMC (25 to 250 mM) were
separately incubated with microsomes (1 mg protein),
Tris±HCl (0.025 M, pH 7.5) and water in a total volume
ꢀ
of 1.6 mL for 10 min at 37 C. The progress of the
reaction was assayed by the quanti®cation of DHMC as
described above. The reciprocal of initial rate (1/v) was
plotted against that of substrate concentration (l/[S]) in
order to calculate K and ꢀ
values.
m
max
E€ect of pH. The reaction mixture consisting of DAMC
125 mM), liver microsomes (1 mg protein), di€erent
bu€ers (0.025 M) of varying pH (4.0 to 11.0) and water
(
Figure 1. (A) Enzymatic deactylation of diacetoxy 4-methylcoumarin;
B) formation of mixed ligand complex.
in a total volume of 1.6 mL was incubated for 10 min at
ꢀ
(
3
7 C. The enzyme was inactivated by heat exposure as
described above and pH adjusted to 7.5 before the
development of colour by the addition of ADP and
Fe as described above.
(DHMC), 6,7-diacetoxy-4-methylcoumarin (2) and 7,8-
diacetoxycoumarin (3) were synthesized in our laboratory.
3+
Quantitation of DHMC
Substrate speci®city
Standard solution (2 mM) of DHMC was prepared in
DMSO. Aliquots of the standard solution (10 to 300
mL) were pipetted out into di€erent tubes taking care
that each concentration was taken in triplicate, 0.2 mL
of 0.25 M Tris±HCl (pH 7.5) was added to all the tubes
followed by the addition of 0.2 mL of 30 mM ADP and
Several structural analogues of DAMC were separately
incubated at several concentrations (25±250 mM) with
microsomes (1 mg protein), Tris±HCl (0.025 M, pH 7.5)
and water in a total volume of 1.6 mL. The activity of
the enzyme was quanti®ed as described earlier.
0.2 mL of 1.5 mM FeCl . The total volume of the reac-
3
tion mixture was ensured to be 2.0 mL by the addition
of appropriate amount of water. The contents of the
tubes were mixed thoroughly on a vortex mixer and
intensity of the green colour developed was measured
at 600 nm using Beckman spectrophotometer (Model
DU 64).
Results and Discussion
The existence of microsomal deacetylase that catalyses
the deacetylation of diacetoxy 4-methylcoumarins has
been reported here for the ®rst time. A simple method
for the assay of this deacetylase is described which
enables preliminary characterisation of the enzyme.
Preparation of rat liver microsomes
The enzyme assay is based upon the quanti®cation of
the deacetylated product of DAMC (or any other ortho
diacetoxycoumarin) resulting in the formation of
DHMC (or corresponding ortho dihydroxy derivatives)
Male albino rats of Wistar strain weighing 190±200 g
fed on rat chow supplied by Hindustan Lever Ltd.,
Mumbai (India) were sacri®ced, liver excised and
5
microsomes prepared as described earlier. Protein was
6
estimated by the method of Lowry et al.
Assay of rat liver microsomal DAMC deacetylase
activity
The assay mixture consisting of Tris±HCl (0.025 M, pH
7.5), microsomes (1 mg protein), DAMC (50 to 600 mM)
and water to make total volume of 1.6 mL was incu-
bated for 10 min at 37 C. The tubes were covered later
ꢀ
with glass marbles and placed in a boiling water bath
for 10 min, cooled, ADP (3 mM) and FeCl (0.15 mM)
3
added, the ®nal volume of reaction mixture was adjus-
ted to 2.0 mL and the concentration of DHMC was
calculated by measuring the absorption at 600 nm. One
unit of deacetylase catalysed the hydrolysis of 1 mmol
ortho diacetoxycoumarin derivatives under the condi-
tions of the assay. The conditions for the assay of liver
microsomal DAMC deacetylase were chosen to ensure
that the initial rate was linear with respect to time of
incubation and enzyme concentration.
Figure 2. Quanti®cation of DHMC.

H. R. G. Raj et al. / Bioorg. Med. Chem. 8 (2000) 233±237
235
(Fig. 1(A)). We earlier demonstrated that DHMC forms
a stable mixed ligand complex of ADP-Fe ±DHMC
procedure. It is worth mentioning that 6,7-dihydroxy-4-
methylcoumarin exhibited the same extinction as
DHMC. Hence DHMC can be employed as a standard
when di€erent ortho diacetoxy derivatives are used as
the substrates for the deacetylase described here.
DAMC deacetylase was assayed by incubating DAMC
with rat liver microsomes under the speci®ed conditions
and the progress of the reaction was followed by quan-
tifying the product DHMC as described earlier. The
linearity observed with respect to time of incubation
and enzyme concentration (Figs 3 and 4) con®rm the
enzymatic hydrolysis of DAMC by rat liver micro-
somes. DAMC deacetylase exhibited hyperbolic kinetics
and application of double reciprocal plot to rat liver
3+
3+
(
Fig. 1(B)) with ADP and Fe which had intense UV
5
absorption at 600 nm. The data depicted in Fig. 2
reveals linearity of absorption at 600 nm with respect
to the concentration of ADP-Fe3 ±DHMC complex
which served to quantify the concentration of DHMC.
+
The method a€orded an accuracy of estimation up to a
minimum of 25 nmols of DHMC (4.8 mg). The molar
extinction coecient of DHMC worked out to be
3
� 1
� 1
cm under the conditions of the assay
2
Â10
M
Figure 3. E€ect of time of incubation on DAMC deacetylase activity.
Rat liver microsomes (1 mg protein) was incubated with DAMC
(125 mM). DAMC deacetylase was assayed as described under Mate-
rials and Methods.
Figure 4. E€ect of enzyme concentration on DAMC deacetylase
activity. Rat liver microsomes (0.5±3.0 mg protein) was separately
incubated with DAMC (250 mM) for 10 min and DAMC deacetylase
was assayed as described under Materials and Methods.
Figure 5. E€ect of DAMC concentration on DAMC deacetylase activity.

2
36
H. R. G. Raj et al. / Bioorg. Med. Chem. 8 (2000) 233±237
Figure 6. E€ect of pH on DAMC deacetylase activity.
Table 1. Substrate speci®city for rat liver microsomal diacetoxy 4-
methylcoumarin deacetylase
Table 2. Tissue speci®city of microsomal diacetoxy 4-methylcou-
marin deacetylase of rat tissues
Compound
Kinetic constants
Tissue
Diacetoxy 4-methylcoumarin deacetylase
K
m
(mM)
250
ꢀ
max (units)
Liver
Lung
Kidney
Intestine
++++
0
0
0
1
500
deacetylase action can impart green colour with ADP
and Fe3 . 7,8-Diacetoxycoumarin (3) was found to be a
better substrate as compared to DAMC for the deace-
tylase (Table 1). An e€ort was made to examine the
e€ect of the thiol binding agent PHMB on deacetylase
by including the mercurial compound at a concentration
+
1
437
450
�
2
of 10
M in the enzyme assay mixture. The result
demonstrated that PHMB had no e€ect on the enzyme
activity (data not shown) indicating that no thiol group
is involved at the active centre of DAMC deacetylase.
The DAMC deacetylase reported here was found to be
very speci®cally present in the rat liver, other extra-
hepatic tissues such as lung, kidney and intestine
revealed no measurable deacetylase activity (Table 2). It
is worth noting that no deacetylase activity towards
DAMC was detected in rat liver cytosol although the
cytosol has been shown to catalyse a variety of deace-
8
25
670
1
tylation reactions. Hepatic microsomes have been
reported to hydrolyse carboxy ester groups,7 DAMC
deacetylase can possibly belong to the family of aryl
carboxyl esterases. Puri®cation of DAMC deacetylase is
being undertaken to con®rm such possibilities and to
help further characterisation.
±10
microsomal DAMC deacetylase (Fig. 5) yielded K and
m
ꢀmax values of 1250 mM and 500 units, respectively.
3
+
Since the chromophoric complex ADP-Fe ±DHMC is
decolorised at the acidic pH, care was taken to readjust
the pH of di€erent bu€ers used to 7.5 during the study
of the e€ect of pH on the DAMC deacetylase activity
pro®le which revealed pH optima of 7.5 (Fig. 6). An
e€ort was made to assess the substrate speci®city for the
rat liver microsome-catalysed deacetylation of diace-
toxycoumarins (Table 1). In this investigation, ortho
diacetoxy derivatives were taken as the substrates with a
view that the ortho dihydroxy moiety generated upon
Acknowledgements
The ®nancial assistance of the Danish International
Development Agency (DANIDA) is gratefully acknowl-
edged. The authors acknowledge the help and coopera-
tion of Dr V. K. Vijayan, Director, VP Chest Institute.

H. R. G. Raj et al. / Bioorg. Med. Chem. 8 (2000) 233±237
237
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