Two-Dimensional Metal-Organic Framework Nanosheets as an Enzyme Inhibitor: Modulation of the α-Chymotrypsin Activity

Article abstract of DOI:10.1021/jacs.7b03450

Two-dimensional metal-organic framework (MOF) nanosheets are utilized as effective enzyme inhibitors, providing an inspiring means to enhance the control of cellular processes as well as improve our understanding of the surface chemistry between MOFs and enzymes. In this paper, we demonstrated that the activity of α-chymotrypsin (ChT) can be effectively inhibited with 96.9% inhibition by 2-D Cu(bpy)₂(OTf)₂ nanosheets, while Zn₂(bim)₄ nanosheets show no significant inhibition effect toward ChT. Kinetic studies revealed that the material acts as a competitive inhibitor toward ChT. Furthermore, fluorescence and circular dichroism spectroscopy reveal that the 2-D MOF nanosheets do not change the secondary structure of the enzyme. The Cu(II) center of the 2-D nanosheets binds the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) molecules in the buffer, leading to an electrostatic interaction between the nanosheets and the enzyme. In addition, the irreversible coordination interactions between Cu(II) center and His-57 played an important role during the inhibition process, as supported by ionic strength experiments and UV absorbance changes of Cu(II) d-d transitions. As a result, the substrate is prevented from reaching the active sites of the enzyme causing enzyme inhibition. The modulation of enzyme activity by 2-D MOF nanosheets opens up a new direction for the exploration of the MOF-bio interface in physiological and catalytic systems.

Full text of DOI:10.1021/jacs.7b03450

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Article
Two-Dimensional Metal-Organic Framework Nanosheets as an
Enzyme Inhibitor: Modulation of the #-Chymotrypsin Activity
Ming Xu, Shuai Yuan, Xin-Yu Chen, Yu-Jie Chang, Gregory Steven Day, Zhi-Yuan Gu, and Hong-Cai Zhou
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 24 May 2017
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Two-Dimensional Metal-Organic Framework Nanosheets as an
Enzyme Inhibitor: Modulation of the α-Chymotrypsin Activity
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Ming Xu,† Shuai Yuan,‡ Xin-Yu Chen,† Yu-Jie Chang,† Gregory Day,‡ Zhi-Yuan Gu*† and Hong-Cai Zhou*‡§
†Jiangsu Key Laboratory of Biofunctional Materials, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials,
College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, China
‡Department of Chemistry, Texas A&M University, College Station, TX 77843-3255, United States
§Department of Materials Science and Engineering, Texas A&M University, College Station, Texas 77842, United States
Two-dimensional nanosheets, α-chymotrypsin, inhibitor, metal-organic frameworks, biocompatibility.
ABSTRACT: Two-dimensional metal-organic framework (MOF) nanosheets are utilized as effective enzyme inhibitors, providing an inspiring
means to enhance the control of cellular processes as well as improve our understanding of the surface chemistry between MOFs and enzymes.
In this paper, we demonstrated that the activity of α-Chymotrypsin (ChT) can be effectively inhibited with 96.9% inhibition by the 2-D
Cu(bpy)2(OTf)2 nanosheets, while the other Zn2(bim)4 nanosheets show no significant inhibition effect towards ChT. Kinetic studies revealed
the material acts as a competitive inhibitor towards ChT. Furthermore, fluorescence and circular dichroism spectroscopy reveal that the 2-D
MOF nanosheets do not change the secondary structure of the enzyme. The Cu (II) center of the 2-D nanosheets binds the 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES) molecules in the buffer, leading to an electrostatic interaction between the nanosheets and the
enzyme. In addition, the irreversible coordination interactions between Cu(II) center and His-57 played an important role during the inhibition
process, as supported by ionic strength experiments and UV absorbance changes of Cu(II) d-d transitions. As a result, the substrate is prevented
from reaching the active sites of the enzyme causing enzyme inhibition. The modulation of enzyme activity by 2-D MOF nanosheets opens up
a new direction for the exploration of the MOF–bio interface in physiological and catalytic systems.
dispersion in water while nanoscale MOFs easily form aggregates in
INTRODUCTION
solution, which hinders the analysis.26-28 Besides, the pore apertures
Metal-organic frameworks (MOFs) with high specific surface areas,
of most MOFs are not large enough for the enzymes. Recently,
tunable pores and diverse functionalities have been explored
several examples of two dimensional (2-D) MOF nanosheets have
extensively for gas adsorption and separation, energy storage,
been synthetized.29-35 Although 2-D MOF nanosheets have highly
catalyst, and carbon dioxide capture.1-7 Recently, MOFs were
accessible active sites and homogeneous feature, their successful
utilized in the stabilization of enzymes for catalysis in extreme
applications were limited to membrane separation, energy storage,
conditions as well as in biomedical applications, such as drug
iodine capture, MALDI-TOF matrices and fluorescent sensing.9,36-39
delivery, bioimaging and photodynamic therapy.8-17 Although the
2-D MOF nanosheets have good dispersibility and large surface
applications are in diverse number of fields, understanding the
areas for adequate protein binding. In contrast to some small
fundamentals of MOF-protein interactions will be key in developing
molecules and biological molecules, MOF nanosheets can easily
these technologies.18 At the same time, few researchers are focused
enter cells. Due to the advantages of 2-D MOF nanosheets, we
on the modulation of enzyme activity within the MOF–bio interface;
although general metal-based coordination compounds have been
employed such a material to form a MOF-bio interface, enabling us
to study the modulation of enzyme activity. Alpha-Chymotrypsin
extensively studied in the biomedical research.19
(ChT, EC 3.4.21.1) was selected as model enzyme because it is an
Modulation of enzyme activity provides an infusive means to
important serine protease in the alimentary canal and its structure
enhance the control of cellular processes such as DNA replication,
has been well characterized (Figure 1c, d).40 Investigating the
signal transduction and metabolism.11 Gold nanoparticles, carbon
modulation of ChT is of great importance in the field of biology, as
nanotubes, graphene oxide, functionalized MoS2, dendrimers, small
immoderate ChT activity may leads to cystic fibrosis, inflammatory
organic molecules, and metal-organic complexes have all been used
arthritis, and other medical disorders.41,42 ChT can be inhibited by
to alter the activity of biomolecules.20-25 Compared with other solid
several materials, such as gold nanoparticles, due to the interactions
state materials, MOFs contain precisely designed structure and
between ChT and the inhibitive materials, such as hydrophobic
surface properties that are ordered on the molecular level through
interactions, electrostatic interactions, hydrogen bonding, and π−π
the judicious selection of ligand and metal nodes. This provides
MOF structures with a high degree of tunability, enabling the
modulation of enzymatic activity to rigorously controlled. However,
stacking.24,43-45 The ChT has a hydrophobic “hot spot” surrounded
by cations on the surface which allows for both hydrophobic and
electrostatic interactions. The surface charges and organic
MOFs have seldom been investigated for enzyme inhibition to the
compositions of 2-D MOF nanosheets offer great opportunities to
best of our knowledge. In order to be applicable to enzyme
take advantage of these interactions, possibly allowing for the tuning
inhibition, MOFs are usually required to be stable and dispersive in
water. However, bulk crystals of MOFs give inhomogeneous
of the enzyme activity.
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Scheme 1. The enzymatic hydrolysis reaction.
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EXPERIMENTAL METHODS
The 2-D MOF nanosheets were obtained by physical exfoliating
from their bulk MOFs. Bulk Cu-MOF were prepared according to
the reported literature procedure with only a few changes.34,47,48
Typically, an aqueous solution of Cu(OTf)2 (30.0 mM, 10.0 mL)
was added to a 22-mL glass vial upon which 1 mL of pure ethanol
was layered meticulously. Then, an ethanol solution of bpy (60.0
mM, 10.0 mL) was layered onto the ethanol layer to obtain a three-
layers system, which was left to stand for three weeks at room
temperature. The dark blue crystals were collected by centrifugation,
and washed with 20 mL acetone and ethanol five times, respectively.
To obtain 2-D Cu-MOF nanosheets, single crystals of Cu-MOF
were placed in distilled water and stirred for 9 h. After removal of the
sediment, a dispersed solution of nanosheets was obtained. The wet
2-D Cu-MOF nanosheets were collected by centrifugation at 12000
rpm for 20 min and then dried at room temperature. Synthesis
method for 2-D Zn2(bim)4 nanosheets are described in Supporting
Information.
Figure 1. (a) Schematic representation of
a single-layered 2-D
nanosheet of Cu-MOF. The C, O, S and F atoms are shown in grey, red,
yellow and green respectively. The coordination polyhedra of the Cu
ions are displayed in blue. H atoms are omitted for clarity. (b) The
Schematic representation of a Cu(II) center in the nanosheets. (c)
Molecular structure of α-chymotrypsin. The polychrome sections in this
structure are the active site with residues of Asp-102, His-57 and Ser-195.
To facilitate the interactions at the MOF-bio interface without
interference from a polymer coating, a top-down synthesis strategy
was chosen, although macromolecule-assisted bottom-up synthesis
could also result in a well-controlled morphology for the 2-D MOF
nanosheets.36,46 Herein, we synthetized two MOF nanosheets with
different metals. One is 2-D Zn2(bim)4 (bim=benzimidazole)
nanosheets, while the other is Cu-based 2-D MOF nanosheets
(Figure 1a, b), namely Cu(bpy)2(OTf)2 (Cu-MOF, bpy = 4,4-
bipyridine, OTf = trifluoromethanesulfonate).34,35,47,48 Both MOFs
have similar layered structures and large surface area with divalent
metal ions and nitrogen-containing organic ligands, showing good
water stability. However, the isostructural bpy/OTf based
nanosheets with other metal centers is highly moisture sensitive,
which implements its applications as enzyme inhibitors.49 To
investigate the possible interactions between ChT and the 2-D MOF
nanosheets, we used N-succinyl-L-phenylalanine-p-nitroanilide
(SPNA) as a substrate which can be hydrolyzed by ChT forming a
reaction product with a characteristic UV-Vis absorption at 410 nm
(Scheme 1). By observing the rate of the reaction before and after
incubating ChT with 2-D MOF nanosheets, the enzyme activity and
modulation effect can be well evaluated.
X-ray diffraction (XRD) patterns were obtained from a Rigaku
D/MAX-2500 diffractometer with a CuKα radiation (1.54056Å),
with data recorded from 2o to 50o. Transmission electron
microscope (TEM) images were made on a JEOL JEM-2100F
transmission electron microscope operated at an accelerating
voltage of 200 kV. To prepare samples for TEM analysis, 2-D Cu-
MOF nanosheets were dispersed in water and ultrasounded for 1 h.
The dispersed solution (0.5 mg∙mL-1) was then dropped onto a
copper-supported ultrathin carbon film (Beijing Zhongjingkeyi
Technology Co., Ltd, China). Scanning electron microscope(SEM)
images and energy disperse spectroscopy (EDS) spectra were
collected on
microscope.
a JSM-7600F (JEOL Ltd) scanning electron
All enzyme experiments were performed in HEPES buffer (100
o
mM, pH 7.4) at 30 C. The concentration of ChT was 80 µg∙mL-1,
while the concentration of 2-D MOF nanosheets ranged from 0 to
100 µg∙mL-1 ChT was incubated with those 2-D MOF nanosheets
solution from 0 h to 4 h. To obtain the activity of ChT, an enzymatic
hydrolysis substrate (SPNA, 160 µL) was added to the MOF-ChT
solution (1840 µL). The concentration of SPNA was 2 mM in the
solvent mixture of ethanol/DMSO (90%/10%). Enzyme activity
was calculated by monitoring the absorbance of p-nitroaniline (PNA)
every 2 min for a total of 20 min at 410 nm with a Hitachi UH5300
spectrophotometer. Different concentration of NaCl solution was
added to 80 µg∙mL-1 ChT either before or after incubation to study
the ionic strength effect. The concentration of NaCl solution was
varied from 0 to 600 mM in 100 mM HEPES buffer (pH 7.4). For
comparison, the NaCl solution at the same concentrations were
added to an 80 µg∙mL-1 ChT solution without 2-D Cu-MOF
nanosheets. To demonstrate the advantage of 2-D Cu-MOF
nanosheets, the inhibition effects of Cu(OTf)2py4 complex and
Cu(OTf)2 were evaluated under different ionic strength. To
In this paper, we demonstrated that the activity of ChT (80
µg∙mL-1) can be effectively inhibited with 96.9% inhibition by the 2-
D Cu-MOF nanosheets at a concentration of 100 µg∙mL-1, while
Zn2(bim)4 shows no significant inhibition effects towards ChT.
From the kinetic data, we found that the material acted in
competitive inhibition mode. Furthermore, with the fluorescence
spectroscopy and circular dichroism patterns, it can be concluded
that the material does not change the secondary structure of the
enzyme. Besides the electrostatic interaction, an irreversible
coordination interaction between a Cu(II) center and the His-57
fragment were also observed during the incubation process, strongly
inhibiting the enzyme.
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investigate the function of HEPES molecule, two more Good’s Tyndall scattering of this system was unchanged for 18 h indicating
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zwitterionic buffers, 4-morpholinoethanesulfonic acid (MES) and
N-(2-hydroxyethyl) piperazine-N′-(2-hydroxypropanesulfonic acid)
monohydrate (HEPPSO) were also used instead of HEPES at the
same concentration (100 mM).50
good water stability of this 2-D material (Figure S5). The UV
absorbance of 2-D Cu-MOF nanosheets dispersed in HEPES buffer
over 4 h showed no difference indicating good stability of the
nanosheets in this buffer (Figure S6). The successful synthesis of
Zn2(bim)4 and 2-D Zn2(bim)4 nanosheets were also confirmed by
the XRD and TEM (Figure S7,8).
In order to calculate inhibition constant, the concentration of
SPNA was varied from 0.5 mM to 4 mM with 80 µg∙mL-1 ChT and
40 µg∙mL-1 2-D Cu-MOF. kcat and Km were calculated according to
the Michaelis-Menten equation in the control experiment in which
the rate of ChT with different SPNA concentrations were measured
in the absence of inhibitor. The inhibition constants Ki and  were
calculated using the mixed-model inhibition equation. Furthermore,
dissociation constant (Kd) and molar binding radios (n, the number
of ChT bonding to single Cu(II) center site) were evaluated by
nonlinear least-squares curve-fitting analysis of activity assay.51,52
Activity Assays. In order to investigate the inhibition effect between
two MOF nanosheets and ChT, SPNA was chosen as a substrate and
catalytically hydrolyzed by ChT in a HEPES buffer (pH 7.4) at 30oC.
The reaction rate of the hydrolysis was used to determine the
enzyme activity. To make a fair evaluation of the capacity of the
inhibitor, the inhibited enzyme activity was normalized to the ChT
activity by the same incubation time without any inhibitors (Figure
S9-14). The ChT (80 µg∙mL-1) was respectively incubated with 2-D
MOF nanosheets at different concentrations ranging from 0 to 100
µg∙mL-1 for 1 h. The ChT activity shows no significant decrease with
the incubation of 2-D Zn2(bim)4 nanosheets (Figure 3a, S13). In
contrast, ChT activity performance remarkably decreased with
increasing concentration of 2-D Cu-MOF nanosheets. 96.9% of
ChT activity was inhibited with 2-D Cu-MOF nanosheets at a
concentration of 100 µg∙mL-1 (Figure 3a, S12). Compared to other
materials, 2-D Cu-MOF nanosheets showed a superior inhibition
effect, while the Zn2(bim)4 nanosheets did not exhibit noticeable
levels of inhibition for ChT (Figure S15).
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Different incubation times between ChT and 2-D Cu-MOF
nanosheets were investigated with both fluorescence spectroscopy
(Hitachi
F-4600)
and
circular
dichroism
(CD,
AppliedPhotophysics Chirascan) to evaluate the possible secondary
structure changes of ChT. The mixture of ChT (80 µg∙mL-1) and 2-
D Cu-MOF nanosheets (40 µg∙mL-1) were excited at 295 nm, and
the emission spectra were recorded from 300 to 450 nm with ChT
and denatured ChT as control experiments. The denatured ChT was
obtained by heating ChT at 100 oC for 30 min. The CD spectra were
measured on a CD Spectrophotometer with quartz cuvettes of 1 mm
path length at room temperature. The spectra were recorded from
190 to 250 nm. The ChT concentration was 80 µg∙mL-1 and the 2-D
Cu-MOF nanosheets concentration was 40 µg∙mL-1. NaOH (100
mM) and HCl (100 mM) were added into water (pH = 7.4) to
replace the HEPES buffer in order to get clear CD background. The
CD spectra of 2-D Cu-MOF nanosheets was measured to eliminate
any background effects.
The coordination interactions between Cu(II) center and His-57
were confirmed through the UV spectra of 2-D Cu-MOF
nanosheets in water, in HEPES buffer, and incubated with and
without ChT in HEPES buffer from 0 to 4 h. The UV spectra were
recorded from 300-900 nm on
a
Hitachi UH5300
spectrophotometer.50,53 In order to get the significant d-d transition
band in UV absorbance from 400 to 800 nm, the concentration of 2-
D Cu-MOF nanosheets was 200 µg∙mL-1. To confirm the active site,
different concentrations of histidine solution from 0 to 590 µM were
added into 80 µg∙mL-1 ChT after incubation with 40 µg∙mL-1 Cu-
MOF nanosheets. For comparison, the catalytic activity of histidine
and the activity of ChT with 590 µM histidine without MOF were
recorded as well.
RESULT AND DISCUSSION
Materials Characterization. The MOF nanosheets were fully
characterized and their water stability was tested before the MOF
nanosheets were utilized for enzyme inhibition. The XRD pattern of
the prepared bulk Cu-MOF is consistent with the simulated pattern
47,48
(Figure 2a).
The bulk Cu-MOF showed layered structures in
SEM images (Figure S1a). After exfoliation, 2-D Cu-MOF
nanosheets were successfully obtained and confirmed by SEM, EDS
and TEM (Figure S1-S3). Under TEM analysis conditions the
nanosheets preferentially roll up to form nanorolls (Figure 2b).
Similar nanoroll structure were also observed in AFM (Figure S4).
This phenomenon is attributed to the different exfoliation method
compared to the previous paper which has also been supported by
Kondo et al.34 To confirm their water stability, the 2-D Cu-MOF
nanosheets were dispersed in water at room temperature. The
Figure 2. (a) The XRD patterns of as-synthesized Cu-MOF (blue), Cu-
MOF soaked in water for 18 h (red) and the simulated XRD pattern
(black). (b)TEM image of the 2-D Cu-MOF nanosheets.
We propose that the different inhibition effects with the two
kinds of 2-D MOF nanosheets might suggest different metal
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configurations in different coordination environment. According to extent. The successful bonding between ChT and 2-D Cu-MOF
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previous publications, the main driving force for an electrostatic
interaction between ChT and an inhibitor resulted from the
positively charged residues surrounding the active sites of ChT and
the negative charged functional groups on inhibitors.24,51 The stable
fully coordinated Zn(II) in the 2-D Zn2(bim)4 nanosheets would be
unable to bind HEPES molecules, leaving the overall Zn2(bim)4
nanosheets positively charged like its ZIF precursor and thus
preventing Zn2(bim)4 from inhibiting ChT.54 On the other hand, the
six coordinate Cu engaged in John-Teller distortional effects in the
2-D Cu-MOF nanosheets could easily exchange OTf ligands with
HEPES and form a negatively charged surface, leading to an
electrostatic interaction towards ChT.
nanosheets was supported by the particle size distribution result, as
after incubating with the 2-D Cu-MOF nanosheets the average size
of ChT is much larger than the natural one (Figure S16).
2-D Cu- MOF Nanosheets-ChT Inhibition Kinetics. The inhibition
mode was investigated to explore the potential bonding sites of ChT
for 2-D Cu-MOF nanosheets. The Michaelis-Menten equation and
the mixed-model inhibition equation were plotted with different
concentrations of substrate (S) and inhibitor (I) to calculate the
maximum reaction rate (vmax), the Michaelis-Menten constant (Km),
and the inhibition constants (Ki) and α.23 The mixed-model
inhibition equation is given as: :
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[ ]
(
[ ]
[ ]
)
푖
1
푣
1 + 퐼 /훼퐾 퐾푚 1 + 퐼 /퐾
푖
=
+
푣푚푎푥
푣푚푎푥
푆
The value of α is used to describe the type of inhibition, whether
the inhibition is competitive, noncompetitive or uncompetitive.
When α = 1, the inhibitor does not affect the substrate combining
with the enzyme, which is noncompetitive inhibition. When α ≫ 1,
the inhibitor binds with the enzyme at its active site preventing the
binding of the substrate, in a process termed competitive inhibition.
When α ≪ 1, the inhibitor binds with the enzyme causing an
enhancement of substrate binding, preventing the catalytic process,
in this case hydrolysis, from occurring, in a process termed
uncompetitive inhibition.55
From the slope and intercept of the Lineweaver-Burk plot, we can
determine kcat = 1.687×10-2 s-1 and Km = 1681.4 µg∙mL-1 (Figure 4),
typical values for ChT. We obtained Ki = 12.25 µg∙mL-1 as well as α
= 12.05, implying that the 2-D Cu-MOF nanosheets inhibits ChT
through competitive inhibition. In other words, 2-D Cu-MOF
nanosheets bind with ChT at its active sites and prevent any further
interaction of the substrate towards the enzyme.
Figure 3. (a) The activity of ChT (80 µg∙mL-1) with different
concentrations of 2-D Cu-MOF and Zn2(bim)4 nanosheets. (b) The
activity of ChT (80 µg∙mL-1) with different incubation times between
the enzyme and 2-D Cu-MOF nanosheets (40 μg·mL-1). The inhibited
enzyme activity was normalized to the non-inhibited ChT activity at the
same incubation time.
Figure 4. Lineweaver-Burk plot without inhibitors (black) and mixed-
model inhibition equation plot with 40 µg∙mL-1 2-D Cu-MOF
nanosheets as an inhibitor (red).
Because of the poor inhibition effect of 2-D Zn2(bim)4
nanosheets, we chose 2-D Cu-MOF nanosheets with moderate
concentrations of 40 μg·mL-1 for the following investigation. The
incubation time was evaluated to show the bonding kinetics between
Investigation of ChT Secondary Structure Changes Induced by 2-D
Cu-MOF Nanosheets. The fluorescence and CD spectroscopy were
used to investigate whether the competitive inhibition between 2-D
Cu-MOF nanosheets and ChT would change the secondary
structure of the enzyme. In our research, natural ChT in HEPES
buffer (pH 7.4) has its characteristic fluorescence emission peak at
331 nm under excitation of 295 nm, while the weak fluorescence of
2-D Cu-MOF nanosheets would not affect the measurements
(Figure S17). The denatured ChT shows a significant red shift with
ChT and 2-D Cu-MOF nanosheets (Figure 3b).
A longer
incubation time resulted in better inhibition effect in first 1 h while
the time of incubation exhibited no significant influence on
inhibition effect after 1 h. The incubation time of 1 h was selected for
all following experiments to make sure the electrostatic bonding
between ChT and 2-D Cu-MOF nanosheets formed to their fullest
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its fluorescence emission peak at 353 nm due to the exposure of Trp in order to determine if the electrostatic interaction can be weakened
by high ionic strength.51 Different concentrations of NaCl, from 0 to
600 mM, were used to change the ionic strength. For each NaCl
concentration, the activity of ChT (80 μg∙mL-1) before and after
incubation with 2-D Cu-MOF nanosheets (40 μg∙mL-1) for 1 h was
normalized by the ChT activity without inhibitor at the same salt
concentration. When the NaCl was added before incubation, the
enzyme activity showed a significant recovery of activity dependent
on the concentration of NaCl (Figure 6). Specifically, when the
concentration of NaCl was increased to 400 mM, 2-D Cu-MOF
nanosheets were unable to inhibit ChT activity. However, when
NaCl was added after incubation, the activity of ChT showed almost
no recovery. These results indicate that ChT could not bind to 2-D
Cu-MOF nanosheets under high ionic strength conditions,
confirming the importance of electrostatic interactions between the
ChT and inhibitor for binding. However, once ChT has bound to
the material after incubation, the enzymatic activity cannot be
restored even at high NaCl concentrations. A purely electrostatic
interaction should see recovery of ChT activity as the NaCl
concentration increases, as it should destabilize the 2-D Cu-MOF-
ChT interaction as it does in the pre-incubation experiments. In
other words, the electrostatic interactions provide a kinetic rational
for the inhibition of ChT by 2-D Cu-MOF nanosheets but other
thermodynamic driving forces might be available to maintain
inhibitor binding once the 2-D Cu-MOF nanosheets have begun
interacting with the ChT active site.
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residues. After incubation with 2-D Cu-MOF nanosheets at a
concentration of 40 µg∙mL-1 for 0 h, 4 h and 24 h, the peak of ChT
shows no shift which indicates that ChT retained its natural
secondary structure (Figure 5a). To authenticate these results, CD
spectra were recorded as a secondary source for information
regarding possible changes to the secondary structure or ChT during
2-D Cu-MOF nanosheets inhibition. Uninhibited ChT shows two
characteristic minima at 230 nm and 202 nm, while denatured ChT
has one distinctive minimum at 200 nm (Figure 5b). Almost no
change in the CD data of ChT was observed with preincubated with
2-D Cu-MOF nanosheets from 0 h to 24 h, which corroborated that
the conformation of ChT exhibits no change in the presence of 2-D
Cu-MOF nanosheets. The retention of the ChT structure during the
incubation indicates that the competitive inhibition from 2-D Cu-
MOF nanosheets through electrostatic interaction does not change
the secondary structure of the enzyme.
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Figure 6. The activity of ChT (80 µg∙mL-1) before and after incubation
with 2-D Cu-MOF nanosheets (40 µg∙mL-1) in the presence of NaCl at
different concentrations.
The Coordination Interactions. Herein, we propose that this
possible force is a coordination interaction between the Lewis acidic
copper metal site and the ChT His-57 residue. The coordination
interactions were not only evidenced by the ionic strength
experiments but also by the strong electrostatic interactions between
2-D Cu(OTf)2(bpy)2 nanosheets and ChT in HEPES buffer. The
strong Lewis acid of Cu(II) center in Cu-MOF nanosheets resulting
from the dissociation of OTf are likely to be attacked by the
nucleophilic nitrogen of deprotonated HEPES (pKa = 7.5).56
Although in electroneutral status, the Cu-MOF nanosheets might
also bind to positively charged residues of ChT through the
electrostatic interaction with the negatively charged surface of the
nanosheet-HEPES compounds. Two more Good’s zwitterionic
buffers MES and HEPPSO were used instead of HEPES to support
this conclusion. MES molecule could hardly bind with Cu2+ while
Figure 5. (a) Tryptophan fluorescence and (b) CD spectra of ChT (80
µg∙mL-1) with and without 2-D Cu-MOF nanosheets (40 µg∙mL-1) as
inhibitor for 0 h, 4 h and 24 h. The fluorescence and CD spectra of
denatured ChT (80 µg∙mL-1) were also recorded.
Effect of Ionic Strength. To further prove the driving force for the
interaction between ChT and 2-D Cu-MOF nanosheets is
electrostatic interaction we changed the ionic strength of the
solution before and after incubation with 2-D Cu-MOF nanosheets,
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HEPPSO reveals significant interaction with Cu2+ which is similar to
kinetic factor that controls the accessibility of inhibitor. Meanwhile,
HEPES.50 As shown in Figure S18, HEPPSO performs almost the
same inhibition effect as HEPES, but MES shows lower inhibition
towards ChT with the incubation of 2-D Cu-MOF nanosheets. The
buffer molecules play a significant role in the binding with the Cu-
MOF nanosheets. The inhibition effect would be further strongly
regulated by this binding.
the formation of a coordination bond between His-57 and the Cu(II)
center represents a thermodynamic sink, which explains the
irreversible inhibition of enzymatic activity. For comparison, the
inhibition effect of Cu(OTf)2 salt and the Cu(OTf)2py4 complex
towards ChT could hardly be regulated by changing the ionic
strength of the solution (Figure S25).
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The UV patterns in Figure 7 also give evidence to support the
binding between HEPES and Cu(II) centers. As shown in the insert
pattern in Figure 7a, the UV absorbance of MOF samples show an
obvious change around 600 nm when dispersed in HEPES buffer as
compared with spectra performed in unbuffered water. These
changes correspond to the change of d−d transition around the
Cu(II) centers. An obvious color change was also observed upon the
addition of buffer which, in principle, reflects the changes in the
chemical environment around the Cu(II) centers. Although the
absorbance suggested the aggregation of nanosheets in HEPES
buffer, no precipitation was observed throughout the test and the
nanosheets in HEPES still performed as an effective inhibitor
towards ChT.
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The structure of the catalytic triad in uninhibited ChT (Asp-102,
His-57 and Ser-195) has been well established. The carboxylate
group of Asp-102 forms a compressed hydrogen bond with His 57,
increasing the alkalinity of the imidazole nitrogen with the pKa going
from 7.0 to 12.0 (Figure S19). The coordinative interaction between
the copper metal site and ChT His-57 residue was elucidated by UV
spectra. The pKa of His-57 is larger than HEPES, the nitrogen of
His-57 might easily replace the nitrogen of HEPES, attacking the
Cu(II) center of the 2-D Cu-MOF nanosheets (Figure S20). Upon
the addition of ChT, the UV absorbance also change, especially in
the region from 400 to 800 nm, which in consistent with our
arguments regarding the Cu(II) center’s interaction with the
HEPES molecule through coordination bonds. In addition, the
decrease of absorbance baseline reveals the disaggregation of 2-D
Cu-MOF nanosheets. Other than baseline drift, the absorbance of d-
d transition around 600 nm exhibited almost no change after 1-h
incubation of ChT with 2-D Cu-MOF nanosheets which is
consistent with the enzyme activity data (Figure 3b).
Different concentrations of histidine solution were used to
confirm the binding of His-57 towards 2-D Cu-MOF nanosheets.
With the increasing of histidine concentration, the activity of ChT
recovered (Figure S21-23). The influence of histidine indicated that
the Cu-MOF nanosheets bind with His-57 on the enzyme. The
strong affinity between the enzyme and Cu-MOF nanosheets is
demonstrated by the significant low Kd of 1.0410-7 M which is
smaller than the Kd between ChT and other materials (Figure
S24).51,52,57 It consists with the data that the interaction between ChT
and Cu-MOF nanosheets would be eliminated only when the molar
ratio of histidine-to-ChT reaches as high as 184:1.
Figure 7. (a) The UV absorbance of the 2-D Cu-MOF nanosheets (200
µg∙mL-1) in water, in HEPES, and incubated with ChT in HEPES buffer
for 4 h. (b) The UV absorbance of 2-D Cu-MOF nanosheets (200
µg∙mL-1) incubated with ChT in HEPES buffer from 0 h to 4 h.
CONCLUSIONS
In summary, we have demonstrated 2-D Cu-MOF nanosheets
can act as an effective enzyme inhibitor through competitive binding
of the active site of ChT, instead of changing the enzyme’s native
conformation. The competitive inhibition resulted from not only
the electrostatic interaction between the nanosheets and the enzyme
but also irreversible coordination interactions. Ionic strength
experiments suggest that the electrostatic interaction is primarily a
kinetic effect and the binding of the Cu(II) center by His-57
provides the thermodynamic sink that fully inhibits the enzyme. The
modulation of enzyme activity by 2-D MOF nanosheets will open a
new direction for exploration of the MOF–bio interface in
physiological and catalytic systems, providing infusive means to
enhance the control of cellular processes as well as our
understanding of the surface chemistry between MOF and enzymes.
The coordination interaction could occur only when the Cu(II)
center and His-57 were at a suitable distance which was highly reliant
on the electrostatic interaction between 2-D Cu-MOF nanosheets
and ChT. It is evidenced by the ionic strength experiment that
coordination interactions could not occur if high concentrations of
NaCl were added before incubation, in those instances the enzyme
activity could be recovered. On the other hand, coordination
interactions already occurred in the incubation samples and these
coordination interactions changed little with increasing
concentration of NaCl added after incubation, leading to no
recovery of enzyme activity. Therefore, the electrostatics act as a
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AUTHOR INFORMATION
Corresponding Author
E-mail: guzhiyuan@njnu.edu.cn
E-mail: zhou@chem.tamu.edu
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AUTHOR CONTRIBUTIONS
M. Xu and S. Yuan contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by NSFC (No. 21505076), Young Elite
Scientist Sponsorship Program by CAST (No. 2015QNRC001), the
program of Jiangsu Specially-Appointed Professor, the NSF of Jiangsu
Province of China (No. BK20150967), the program of Jiangsu Province
Innovation Team, the Priority Academic Program Development of
Jiangsu Higher Education Institutions. This research was supported by
the Center for Gas Separations Relevant to Clean Energy Technologies,
an Energy Frontier Research Center funded by the U.S. Department of
Energy, Office of Science, Office of Basic Energy Sciences under Award
Number DESC0001015. This study was supported as part of the
Hydrogen and Fuel Cell Program under Award Number DEEE-
0007049. We thank Dr. Jun Luo (State Key Laboratory of
Pharmaceutical Biotechnology in Nanjing University) for his help in
affinity constant measurement.
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Scheme 1. The enzymatic hydrolysis reaction.
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Figure 1. (a) Schematic representation of a singleꢀlayered 2ꢀD nanosheet of CuꢀMOF. The C, O, S and F
atoms are shown in grey, red, yellow and green respectively. The coordination polyhedra of the Cu ions are
displayed in blue. H atoms are omitted for clarity. (b) The Schematic representation of a Cu(II) center in the
nanosheets. (c) Molecular structure of αꢀchymotrypsin. The polychrome sections in this structure are the
active site with residues of Aspꢀ102, Hisꢀ57 and Serꢀ195.
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Figure 2. (a) The XRD patterns of as-synthesized Cu-MOF (blue), Cu-MOF soaked in water for 18 h (red) and
the simulated XRD pat-tern (black). (b)TEM image of the 2-D Cu-MOF nanosheets.
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Figure 3. (a) The activity of ChT (80 µg∙mLꢀ1) with different concentrations of 2ꢀD CuꢀMOF and Zn2(bim)4
nanosheets. (b) The activity of ChT (80 µg∙mLꢀ1) with different incubation times between the enzyme and 2ꢀ
D CuꢀMOF nanosheets (40 ꢁgꢂmLꢀ1). The inhibited enzyme activity was normalized to the nonꢀinhibited ChT
activity at the same incubation time.
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Figure 4. LineweaverꢀBurk plot without inhibitors (black) and mixedꢀmodel inhibition equation plot with 40
µg∙mLꢀ1 2ꢀD CuꢀMOF nanosheets as an inhibitor (red).
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Figure 5. (a) Tryptophan fluorescence and (b) CD spectra of ChT (80 µg∙mLꢀ1) with and without 2ꢀD CuꢀMOF
nanosheets (40 µg∙mLꢀ1) as inhibitor for 0 h, 4 h and 24 h. The fluorescence and CD spectra of denatured
ChT (80 µg∙mLꢀ1) were also recorded.
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Figure 6. The activity of ChT (80 µg∙mLꢀ1) before and after incubation with 2ꢀD CuꢀMOF nanosheets (40
µg∙mLꢀ1) in the presence of NaCl at different concentrations.
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Page 17 of 17
Journal of the American Chemical Society
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Figure 7. (a) The UV absorbance of the 2ꢀD CuꢀMOF nanosheets (200 µg∙mLꢀ1) in water, in HEPES, and
incubated with ChT in HEPES buffer for 4 h. (b) The UV absorbance of 2ꢀD CuꢀMOF nanosheets (200 µg∙mLꢀ
1) incubated with ChT in HEPES buffer from 0 h to 4 h.
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