Exfoliated nanosheets of a CuII coordination polymer modulate enzyme activity of α-chymotrypsin

Article abstract of DOI:10.1002/chem.201802376

A 2D coordination polymer derived from 5-azi-doisophthalic acid (AIA) and Cu(NO₃)₂ was designed with the aim of modulating the activity of a digestive enzyme α-chymotrypsin (ChT). The coordination polymer namely {[Cu_0.5(m-AIA)_0.5(H₂ O)]·2 H₂O}_α (CP1) was successfully synthesized and fully characterized by single-crystal X-ray diffraction (SXRD). An exfoliated nanosheet (ENS) of CP1 was readily produced by overnight stirring of hand-ground CP1 crystals dispersed in DMSO. ENS(CP1) was demonstrated to be acting as an inhibitor of ChT; as much as ≡ 97 % inhibition of ChT was achieved with 100 mm of ENS(CP1) using N-succinyl-l-phenylalanine-p-nitroanilide (SPNA) as substrate. Enzyme kinetics data revealed that the inhibition of ChT followed a competitive pathway. An enzyme assay under varying ionic strength and varying concentration of free histidine revealed that the active site His-57 participated in coordination with the Cu^II metal center of ENS(CP1) thereby preventing the substrate (SPNA) from binding with the enzyme resulting in efficient inhibition.

Full text of DOI:10.1002/chem.201802376

A Journal of
Accepted Article
Title: A Cu(II) coordination polymer based exfoliated nano-sheet
inhibits ￿ -chymotrypsin
Authors: Parthasarathi Dastidar and Koushik Sarkar
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To be cited as: Chem. Eur. J. 10.1002/chem.201802376
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COMMUNICATION
A Cu(II) Coordination Polymer Based Exfoliated Nano-sheet
Inhibits -chymotrypsin
Koushik Sarkar and Parthasarathi Dastidar*
[
a]
Abstract:
azidoisophthalic acid (AIA) and Cu(NO
of modulating the activity of a digestive enzyme -chymotrypsin
A
2D coordination polymer derived from 5-
3 2
)
was designed with the aim
(ChT).
The
coordination
polymer
namely
{[Cu0.5(µ-
AIA)0.5(H
2
O)].2H
2
O} (CP1) was successfully synthesized and fully
α
characterized by single crystal X-ray diffraction (SXRD). Exfoliated
nano-sheet (ENS) of CP1 was readily produced by overnight stirring
of hand-ground CP1 crystals dispersed in DMSO. ENS(CP1) was
demonstrated to be acting as an inhibitor of ChT; as much as ~97%
inhibition of ChT was achieved with 100 M of ENS(CP1) using N-
succinyl-L-phenylalanine-p-nitroanilide
(SPNA)
as
substrate.
Scheme 1: a) catalytic triad and b) its mode of action in ChT.
Enzyme kinetics data revealed that the inhibition of ChT followed a
competitive pathway. Enzyme assay under varying ionic strength
and varying concentration of free histidine revealed that the active
site His-57 participated in coordination with Cu(II) metal center of
ENS(CP1) thereby preventing the substrate (SPNA) from binding
with the enzyme resulting in efficient inhibition.
[
12]
[13]
[14]
dendrimers, small organic molecules, macro-assemblies,
nanotubes,
[
15]
[16]
metal-organic complexes
have been used to
modulate enzyme activity. The structures of CPs, on the other
hand, are highly tuneable; it can offer high surface area and
various contact sites such as unsaturated coordinating metal
centers, periodic array of counter ions, hydrophobic or
hydrophilic surface etc. to promote enzyme-CP binding.
However, CPs have hitherto been exploited scarcely to
[
1]
Coordination polymers (CPs) have been exploited in many
[
2]
diverse fields, like gas adsorption and separation, anion
[
3]
[4]
[5]
separation,
catalysis,
magnetism,
post
synthetic
[
6]
8]
[7]
[17]
modification , bio-mineralization, drug delivery and healthcare
modulate enzyme activity.
[
applications. CPs are usually synthesized via building block
approach wherein organic linkers (ligands) are reacted with
suitable metal centers; the resulting crystalline CPs are
characterized by single crystal X-ray diffraction (SXRD)
providing details of the structures at atomic resolution; virtually
infinite combinations of ligands and metals offer unmatched
compositional and structural diversities allowing tuneable
physicochemical properties that consequently help in developing
In this communication, we have demonstrated that a 2D
exfoliated nano-sheet (ENS) derived from a Cu(II) CP can
indeed be accessed by design to inhibit the activity of -
chymotrypsin (ChT) as much as ~97 %.
Modulating the activity of ChT has been a popular research
target as immoderate activity of ChT is linked with various
[18]
[19]
disorders such as cystic fibrosis, inflammatory arthritis etc.
Various materials such as dendrimers,
[20]
[21]
2
nano MoS ,
[
2-8]
[22]
[23]
[24]
materials for such diverse applications.
Modulating enzyme activity is important as it can provide means
nanoparticles, graphene oxides, carbon nanotube, metal
[
25]
[26]
complexes, calix-peptide conjugates have been reported as
ChT inhibitors.
[
9]
to control various important biological processes; it can be
achieved by designing artificial receptors that specifically bind to
the enzyme surface or its active site thereby preventing enzyme-
ChT is a digestive enzyme available in the pancreatic juice. It is
a serine protease that catalyses the breakdown of proteins and
polypeptides. Its 3D structure is well characterized by SXRD.
[
10]
[27]
enzyme or enzyme-substrate binding.
Complex topology of
enzyme surface induced by hydrophobic, electrostatic and
hydrophilic residues, large interfacial area required for
preventing enzyme-enzyme interactions and relatively small (in
the order of a few nm) opening to the active site make the
The active site is surrounded by cationic residues such as
arginine (Arg) and lysine (Lys). The so called ‘catalytic triad’ of
the enzyme contains three residues namely Ser-195, His-57 and
…
Asp-102. While Asp-102 holds His-57 in position via N-H O
[
11]
design particularly difficult. Nevertheless, nanoparticles,
hydrogen bonding, the lone pair of electron of the imidazole
moiety of His-57 abstracts proton from the hydroxyl group of
Ser-195 making it a powerful nucleophile which attacks the
carbonyl of amide moiety of the substrate thereby catalysing the
proteolysis process (Scheme 1). Since the active side is located
within a hydrophobic pocket, it preferably catalyses the cleavage
of peptide bonds having hydrophobic side chains such as phenyl
alanine, tyrosine and tryptophan. It is interesting to note that the
perpendicular distance from the protein surface to the imidazole
N of His-57 is ~3 Å thereby making it accessible to coordination
to metal center in a CP (the calculation was based on the
coordinates of ChT retrieved from protein data bank). Thus,
[
a]
K. Sarkar and Prof. P. Dastidar
Department of Organic Chemistry
Indian Association for the Cultivation of Science (IACS)
2A and 2B, Raja S. C. Mullick Road
Jadavpur, Kolkata, 700032, West Bengal (India)
Fax: (+91) 33-2473-2805
E-mail: ocpd@iacs.res.in
Supporting information for this article is given via a link at the end of the
document.
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bonded water cluster having four water molecules around the 4-
fold symmetry axis (Figure 1a).
Scheme 2: Schematic representation of inhibition of ChT by activated
exfoliated nano-sheet of the coordination polymer CP1.
periodic array of coordinatively unsaturated metal centers which
can easily be envisaged in a 2D CP can effectively bind
imidazole N of His-57 thereby preventing proton abstraction from
Ser-195 consequently modulating the activity of ChT towards
effective inhibition. It is also understandable that the efficiency of
such metal-N coordination leading to ChT inhibition would be
maximum in nanoscale due to large surface area. With this
background, we set out our effort to design a counter ion free 2D
Cu(II) CP, the ENS of which would expectedly inhibit ChT by
accessing the coordinatively unsaturated Cu(II) metal center.
For this purpose, we chose to work with an azido functionalized
aromatic dicarboxylic acid ligand namely 5-azidoisophthalic acid
Figure 1: a) Crystal structure illustration of CP1 displaying 2D polymeric
network; b) TEM image of the nano-sheet; c) Tyndall scattering of the
dispersed nano-sheets in DMSO/PBS; d) DLS trace of the suspended nano-
sheets in DMSO/PBS.
Therefore, the 2D corrugated sheets were primarily held
…
together via O-H O hydrogen bond which, under suitable
conditions, may be exfoliated to produce the targeted 2D
exfolidated nano-sheets i.e. ENS(CP1). A small amount of
crystals of CP1 was hand-ground in a mortar and pestle and
stirred overnight in DMSO. The resulting suspension of CP1 in
DMSO was drop casted on a carbon coated Cu grid (300 mesh)
and the corresponding TEM images were recorded. 2D nano-
sheets (~ 450 ˣ 850 nm) were observed in the TEM images
indicating the formation of exfoliated nano-sheets of CP1. AFM
images of the exfoliated nano-sheets revealed that the average
thickness was ~1.7 nm which was in close agreement with the
value obtained from SXRD (Figure S13). Tyndall scattering of
suspended nano-sheets of CP1 both in DMSO and
DMSO/Phosphate Buffered Saline (PBS) for a period of 24 h
indicated good dispersion and stability of the 2D exfoliated nano-
sheets (Figure S4, Supporting information). Particles having
hydrodynamic diameter of ~850 nm were evident from DLS data
that supported the size observed in TEM images (Figure 1b-d).
Having successfully made the exfoliated nano-sheets of CP1,
we then undertook ChT activity assay to study the extent of
inhibition of ChT activity by the nano-sheet. For this purpose, we
have taken N-succinyl-L-phenylalanine-p-nitroanilide (SPNA) as
a model substrate. ChT catalyzes the hydrolysis of the amide
(
AIA) and Cu(NO
salt is expected to produce counter ion free 2D CP via
Cu (COO) paddle-wheel secondary building unit (SBU).
3 2
) . The reaction between AIA and the Cu(II)
2
4
Covalently linked azido moiety being hydrophobic and non-
coordinative, AIA moiety should provide the required
hydrophobicity for effective binding with ChT; the coordinatively
unsaturated Cu(II) center of the paddle-wheel SBU obtained
after removing axially coordinated solvent molecule by mild
heating may facilitate coordination with His-57 of the active site
thereby effectively inhibiting the activity of ChT (Scheme 2).
Slow evaporation of a EtOH-water (4:1) solution containing
2 3 2
disodium AIA (Na AIA) and Cu(NO ) in 1:1 molar ratio resulted
in X-ray quality crystals at rt. after 24 h. The isolated crystals
were subjected to various physicochemical characterizations
including SXRD. The space group assigned to the mounted
crystal for SXRD was P4/nmm (tetragonal, No. 129). Analyses
revealed that it was a 2D CP having a formulae {[Cu0.5(µ-
AIA)0.5(H
carboxylate moiety of AIA formed paddle-wheel SBU with Cu(II)
the axial positions of which were occupied by coordinated
2 2 α
O)].2H O} (CP1). In the crystal structure of CP1, the
–
water molecules; the supramolecular architecture may best be
described as corrugated 2D square grid that further packed in
parallel fashion along the c-axis. 4-fold symmetry axis was
located at the center of each grid and the alternating grids were
occupied by solvate water and pendent azido moiety
(
rotationally disordered around the 4-fold axis). While one of the
solvate water molecules was found to be interacting with the
metal bound water of the 2D CP via O-H O hydrogen bonding,
Scheme 3
…
the other solvate water molecule formed a discrete hydrogen
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linkage adjacent to phenylalanine of the colourless SPNA
releasing light yellow 4-nitroaniline (Scheme 3). Thus, the
progress of the catalytic reaction can be monitored by UV-vis
spectroscopy.
substrate recognition by the addition of free histidine; the driving
force behind such release of ChT in presence of free histidine is
in most likelyhood the entropy gain (Figure 3).
To get more insight into the mode of binding of the substrate
(
SPNA) and inhibitor ENS(CP1) with ChT, enzyme kinetic
studies were undertaken. Both Michaelis−Menten and mixed
model kinetics (Lineweaver−Burk plot) were invoked for this
[
28]
purpose.
Figure 2: Normalized activity of ChT a) in presence of varying concentration
of ENS(CP1) and copper nitrate; b) in presence of 100 M ENS(CP1) as a
function of time.
Figure 3: Normalized activity of ChT a) with varying ionic strength; b) with
The rate of the catalytic reaction can be used to determine the
extent of enzyme inhibition. The activity of free ChT is
considered unity (100%) thereby normalizing the inhibited
enzyme activity in the presence of nano-sheet keeping the
incubation period of 1 h in all experiments. Prior monitoring of
ChT activity in presence of 100 M ENS(CP1) suggested that
the inhibition of ChT reached a saturation level after 1 h. The
enzyme activity was found to be reduced with the increase in
concentration of the nano-sheet. Maximum inhibition of ~97 %
was observed at a concentration of 100 M of the nano-sheet.
As a control experiment, we have carried out the assay in
varying concentration of free histidine.
Enzyme velocity data as a function of substrate concentration at
a fixed inhibitor concentration (100 M) provided important
kinetic parameters such as Michaelis−Menten constant (K
m
),
inhibition constants (K and α), catalytic constant (Kcat) and
i
maximum reaction rate (Vmax) from Lineweaver−Burk plot (Eqn.
1) (Supporting information).
3 2
presence Cu(NO ) (100 M) that could only inhibit up to ~37 %
under identical conditions. These data clearly indicated that
ENS(CP1) was able to inhibit ChT activity more efficiently than
[29]
The mode of enzyme catalysis depends on the value of .
When  = 1, it is a noncompetitive inhibition process wherein
inhibitor does not influence enzyme-substrate binding. If  << 1,
the inhibition process is via uncompetitive mode wherein
inhibitor–enzyme binding causes enhanced substrate binding. In
competitive mode,  >> 1 wherein enzyme-inhibitor binding
significantly prevents enzyme-substrate binding. In the present
study, we obtained  = 32.9 indicating competitive mode of
inhibition i.e. the inhibitor-enzyme binding reduces enzyme-
substrate binding. We attributed such competitive mode of
inhibition to effective coordination between Cu(II) metal center of
the nano-sheet with His-57 of ChT active site via imidazole N-
Cu(II) coordination thereby preventing substrate binding (Figure
simple Cu(NO
based on which the current design was undertaken (see above)
Figure 2).
To provide further support that the coordinative interactions
between His-57 of ChT and Cu(II) metal center of the nano-
sheet was responsible for such inhibition of the enzyme, we
carried out ChT activity assay in presence of varying
concentration of NaCl. Since the active site of ChT is cationic
3 2
) that corroborated well with the hypothesis
(
(
see above), NaCl is likely to interact strongly with the enzyme
driven by electrostatic interactions. Thus, under high ionic
strength, the enzyme activity is expected to be recovered.
Indeed, the enzyme pre-incubated with the increasing
concentration of NaCl (0-300 mM) showed a recovery of its
activity from ~2% (0 mM NaCl) to ~60% (300 mM NaCl) in
presence of fixed concentration of ENS(CP1) (100 M).
Interestingly, when the same experiments were performed with
ChT pre-incubated with ENS(CP1) (100 M), the recovery of the
enzyme activity was negligible (from ~3% (0 mM NaCl) to ~15%
4).
The fact that the secondary structure of ChT did not change
significantly in the presence of ENS(CP1) was confirmed from
both the emission and CD spectra. Excitation of free ChT as well
as in presence of ENS(CP1) (100 M) in PBS at 295 nm
provided a peak at 334 nm whereas denatured ChT (obtained
by heating at 90 C for 1.5 h) resulted in a bathochromic shift to
(
300 mM NaCl)). Interestingly, the activity of ChT pre-incubated
347 nm under identical conditions. In CD, free ChT in PBS
with ENS(CP1) (100 M) with the increasing concentration of
free histidine (0-300 M) was found to be significantly recovered
showed two characteristic minima (232 and 204 nm) whereas
denatured ChT displayed a single minima at 200 nm. The CD
spectrum of ChT preincubated with ENS(CP1) for 1 h was found
to be nearly superimposable with that obtained from free ChT
(
from ~5% (0 M histidine) to ~64% (300 M histidine)). From
these data, it can be concluded that the inhibition of ChT was
predominantly driven by blocking the active site of the enzyme
via His-57-Cu(II) coordination which can be made free for
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inhibitor and provide valuable insights towards the
understanding of enzyme-coordination polymer interactions that
might lead to developing organic-inorganic hybrid materials as
enzyme inhibitors.
Acknowledgements
K.S thanks CSIR for research fellowship. P.D thanks DST
(
EMR/2016/000894) for financial support. Single crystal X-ray
diffraction facility at the Department of Organic Chemistry, IACS
supported by CEIB program of DBT (BT/01/CEIB/11/V/13) was
used for data collection. We thank Prof. Dilip Kr. Maity,
Department of Organic Chemistry, University of Calcutta for
helping in DLS data collection. We thank Ms. Ivy Ghosh,
Department of Inorganic Chemistry, IACS for helping in UV-Vis
data collection.
Figure 4: Competitive mode of inhibition: a) theoretical plot; b) experimental
plot.
indicating that the enzyme retained its secondary structure
during competitive inhibition (Figure S7, Supporting information).
The data presented thus far suggest that the ENS(CP1)
provided an array of coordinatively unsaturated Cu(II) metal
center on the 2D nano-sheets for the enzyme to bind via His-57-
Cu(II) coordination leading towards efficient inhibition. Such
understanding encouraged us to study the inhibition of ChT
using nanoparticles of CP1. Ball-mill grinding of CP1 crystals
resulted in nanoparticles (~170 nm) as revealed by TEM and
DLS (Figure S8, S9, supporting information). It may be noted
that the fits to the scattering data in DLS were not perfect as the
nanoparticles were assumed to be spherical in the fit and
therefore, the size of the nanoparticles inferred from DLS might
be prone to error. Interestingly, CP1 in its nanoparticle form was
found to be relatively less efficient in inhibiting ChT (~92 %) as
compared that of ENS(CP1) under identical conditions (Figure
S10, Supporting information).
So far the data obtained suggested that the exfoliated nano-
sheet of CP1 could indeed be utilized to regulate the ChT
activity. In order to translate such property of the nano-sheet in
real-life application, it is important to evaluate its cytotoxicity. For
this purpose, we carried out MTT assay on RAW 264.7 cells, a
murine macrophage cell line of mice. Data revealed that the
survival of the cells was >60 % at a concentration of 100 M and
Keywords: Coordination polymer • Exfoliation • -chymotrypsin
• Enzymatic modulation • Biocompatibility
Caution! Although we did not experience any problems with AIA
and CP1 reported in this work, azide containing compounds are
potentially explosive. Therefore it should be handled with care.
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of α-chymotrypsin was achieved by exfoliated nano-sheet of a
counter anion free neutral 2D Cu(II) coordination polymer CP1
which was designed based on structural consideration of the
active site of the enzyme as well as its mode of action. SXRD
revealed that CP1 was indeed a 2D neutral coordination polymer
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COMMUNICATION
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Entry for the Table of Contents
COMMUNICATION
As much as ~97 % inhibition of
the activity of a digestive
Koushik Sarkar and Parthasarathi
Dastidar*
enzyme -chymotrypsin was
achieved by 2D exfoliated nano-
sheets of a Cu(II) coordination
polymer (CP1) designed by
considering the structure of the
active site of the enzyme as well
as its mode of action.
Page No. – Page No.
A Cu(II) coordination polymer
based exfoliated nano-sheet
inhibits -chymotrypsin
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