Binding Thermodynamics of Reaction Intermediate Analogues as
Potent Inhibitors of GH20 Exo-β-N-Acetylglucosaminidases
Piyanat Meekrathok1, Keith A. Stubbs2 and Wipa Suginta1,3*
1Biochemistry-Electrochemistry Research Unit, Institute of
Science, Suranaree University of Technology, Nakhon Ratchasima
30000, Thailand
2School of Molecular Sciences, The University of Western
Australia, 35 Stirling Highway, Crawley, WA, Australia, 6009
3Center of Excellence in Advanced Functional Materials,
Suranaree University of Technology, Nakhon Ratchasima 30000,
Thailand
Correspondence: Wipa Suginta, Biochemistry-Electrochemistry
Research Group, Institute of Science, Suranaree University of
Technology, Nakhon Ratchasima 30000, Thailand. E-mail:
[email protected]
Running title: Potent inhibitors of GH20
-N-acetylglucosaminidases
Keywords: β-N-acetylglucosaminidase; chitin recycling; GH20
glycoside hydrolases; marine Vibrios; reaction intermediate
analogues; isothermal titration microcalorimetry
Abstract
Exo--N-acetylglucosaminidases (GlcNAcases) are hydrolytic
enzymes involved in the metabolism of chitin in bacteria and in
eukaryotic glycosphingolipid metabolism, with genetic defects in
human GlcNAcases (HexA and HexB) resulting in Tay-Sachs and
Sandhoff diseases, respectively. Here, we determined the effects of
three known inhibitors of exo--N-acetylglucosaminidases (PUGNAc,
NHAcCAS and NHAcDNJ) on a GH20 exo--N-GlcNAcase (VhGlcNAcase) from
the pathogenic bacterium Vibrio harveyi, in dose-response
experiments. Molecular interactions between the inhibitors and the
enzyme were investigated by isothermal calorimetry (ITC), and were
confirmed using molecular docking. VhGlcNAcase was strongly
inhibited by these compounds, with PUGNAc having the lowest IC50
value, of 1.2 μM. Molecular docking suggested that the inhibitors
mimicked reaction intermediates, with enzyme-inhibitor interactions
being similar to those of the enzyme with the substrate diNAG. The
equilibrium dissociation constants (Kd) obtained from ITC were 0.19
μM for PUGNAc, 12.9 μM for NHAcCAS and 25.6 μM for NHAcDNJ,
confirming that PUGNAc was the most potent inhibitor. The ITC data
indicated that the binding of the inhibitors to the enzyme was
driven by enthalpy. The negative heat capacity change (ΔCp) of
-0.34 ± 0.05 kcal·mol−1·K−1 indicates that hydrophobic interactions
make a substantial contribution to the molecular interactions
between PUGNAc and the enzyme. Our results suggest that PUGNAc is a
highly potent inhibitor, and suggest its usefulness as a scaffold
for potential drugs targeting GlcNAcase-related metabolic
diseases.
1. Introduction
Bacterial GH20 -N-acetylglucosaminidases (GlcNAcases) are
exolytic enzymes that play a crucial role in the biomass recycling
of chitin, a homopolysaccharide of β-1,4-linked N-acetylglucosamine
(NAG) units [1-3]. Chitin is one of the most abundant biopolymers
on earth, and is the primary structural component of fungal cell
walls and the exoskeletons of crustaceans and insects. GlcNAcases
that act on chitin cleave the glycosidic bond between adjacent
GlcNAc units in the chitooligosaccharide chain, resulting in either
retention or inversion at the anomeric centre of the resultant
hemiacetal, relative to the starting material [4,5]. These enzymes
are sub-divided into glycoside hydrolase families 3 (GH3), 20
(GH20) and 84 (GH84), each exhibiting unique substrate recognition.
GH3 enzymes use a typical retaining mechanism involving the
formation and breakdown of a covalent glycosyl-enzyme intermediate
[6,7]. In contrast, GH20 and GH84 enzymes employ a
substrate-assisted retaining mechanism [8-10], which involves the
carbonyl oxygen of the C2-acetamido group acting as a nucleophile
to displace the aglycone leaving group, with the formation of an
oxazolinium intermediate.
Bacterial GlcNAcases and human β-hexosaminidases A and B (HexA
and HexB) are notable members of GH20. Bacterial GlcNAcases in
particular recognize a linear oligosaccharide, removing the
N-acetyl-β-D-glucosamine/N-acetyl-β-D-galactosamine units from the
non-reducing end of the oligosaccharide chain [11,12]. On the other
hand, HexA and HexB hydrolyse β-GalNAc and β-GlcNAc residues from
glycosphingolipids, such as the ganglioside GM2 [13]. Mutations in
HexA or HexB lead to lysosomal accumulation of gangliosides,
resulting in the neurodegenerative disorders known as Tay-Sachs and
Sandhoff diseases, respectively [13,14]. For GH84, rigorous
analysis has been conducted for human O-GlcNAcase, which removes
β-GlcNAc from serine and threonine residues of O-GlcNAcylated
proteins, this enzyme being implicated in many diseases including
Alzheimer’s disease [15], diabetes [16,17] and cancer [18,19]. As a
result of the biological importance of GlcNAcases in the modeling
of glycoconjugates in cells, various chemical tools and the design
of inhibitory small molecules [20-24] have been considered.
1,2-dideoxy-2ʹ-methyl-α-D-glucopyranoso-[2,1-d]-Δ2ʹ-thiazoline
(NAG-thiazoline) [22], which mimics the geometry of the oxazolinium
intermediate in the catalytic reaction sequence, has been
characterized as a highly potent inhibitor of GlcNAcases from both
GH20 and GH84 [9,22,25]. Recently,
O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino
N-phenylcarbamate (PUGNAc) has also been shown to potently inhibit
the GlcNAcases from all three families [25-28]. Iminosugars, such
as 2-acetamino-1,2-dideoxynojirimycin (NHAcDNJ) [21] and
6-acetamido-6-deoxy-castanospermine (NHAcCAS) [20] have been also
found to be the potent inhibitors of GlcNAcases [29]. Work on the
use of this type of molecules as drugs targeting GlcNAcase-related
metabolic diseases has resulted in NAG-thiazoline being suggested
as a treatment for Tay-Sachs and Sandhoff diseases, by acting as a
pharmacological chaperone that stabilizes the mutant form of HexA
and HexB, resulting in an increase of the amount of HexA and HexB
in lysosomes [24,30].
We have previously reported the recombinant expression and
characterization of a novel GH20 exo-β-N-acetylglucosaminidase from
V. harveyi (VhGlcNAcase) [1,31,32], the primary and opportunistic
pathogen that causes luminous Vibriosis, which devastates
commercial shrimp- and fish-farms worldwide [33]. The amino acid
sequence of VhGlcNAcase is closest to that of the α-chain of HexA,
with 30% sequence identity [34], and it is a member of GH20. The
enzyme was identified in the periplasm of the bacterium and is
thought to play an important role in the degradation of
chitooligosaccharides and transport of degradation products through
the outer membrane. Preliminary crystallographic data of
VhGlcNAcase in the absence and the presence of substrate was
previously reported [35] and kinetic modelling of the enzymic
reaction suggests that VhGlcNAcase contains four GlcNAc binding
subsites [1]. Here, we report that three reaction intermediate
analogues act as highly potent inhibitors of VhGlcNAcase. Our study
focuses on investigation of the thermodynamic parameters that
underlie the molecular interactions between these compounds and
VhGlcNAcase. The kinetic and thermodynamic data obtained from our
study may help to elucidate the further development of rationally
designed drugs based on potent inhibitors of the GH20 glycoside
hydrolases.
2. Materials and methods
2.1. Recombinant expression and purification of VhGlcNAcase
Recombinant wild-type VhGlcNAcase was expressed in E. coli M15
(pREP) cells as a C-terminally His6-tagged polypeptide as described
by Meekrathok and Suginta [31]. Briefly, cells expressing
recombinant VhGlcNAcase were harvested by centrifugation at 4,500
×g for 30 min and resuspended in lysis buffer containing 20 mM
Tris-HCl buffer, pH 8.0, 150 mM NaCl, 1 mM phenylmethylsulphonyl
fluoride (PMSF), 5% (v/v) glycerol and 1 mg mL-1 lysozyme, and then
lysed on ice using a Sonopuls ultrasonic homogenizer with a 6-mm
diameter probe. For purification, the crude enzyme obtained after
centrifugation was immediately applied to a gravity-fed TALON®
SuperflowTM metal affinity resin column (Clontech Laboratories,
Inc., USA) operated at 4 °C, followed by the addition of 20 column
volumes of equilibration buffer (20 mM Tris-HCl buffer, pH 8.0
containing 150 mM NaCl), and then 7 column volumes of equilibration
buffer containing 5 mM imidazole. The protein was then eluted
with 50 mM potassium phosphate buffer, pH 7.0 containing
150 mM imidazole. Fractions with GlcNAcase activity were then
further purified on a HiPrep 26/60 Sephacryl S-300 prepacked column
connected to an ӒKTAprime purification system (Amersham Bioscience,
Piscataway, New Jersey, USA), using the running buffer of
50 mM potassium phosphate, pH 7.0. The eluted fractions were
pooled and subjected to several rounds of Vivaspin-20
ultrafiltration membrane concentration (10 kDa molecular-weight
cutoff, Vivascience AG, Hannover, Germany). The final protein
concentration was determined by measuring absorbance at 280 nm
[36].
2.2. GlcNAcase activity assay
The activity of the enzyme was determined in 96-well microtiter
plates, using pNP-GlcNAc as substrate. PUGNAc, NHAcCAS, and NHAcDNJ
were prepared by published methods [20,37,38]. The assay mixture
(200 µL) contained 125 µM pNP-GlcNAc, 0.1 µg of VhGlcNAcase and 0,
1, 10 or 100 μM of the appropriate compound, dissolved in 100 mM
phosphate buffer, pH 7.0. The reaction mixtures were incubated at
37 °C for 10 min with constant agitation in an Eppendorf
ThermoMixer® Comfort (Eppendorf AG, Hamburg, Germany), and the
reaction terminated by the addition of 100 µL of 3 M Na2CO3. The
amount of p-nitrophenol (pNP) released was determined
spectrophotometrically at 405 nm in a Benchmark PlusTM microplate
spectrophotometer (Bio-Rad Laboratories, Hercules, California,
USA). The molar concentrations of the liberated pNP were calculated
from a calibration curve constructed with pNP standards of 0 to 20
nmol per well. The hydrolytic activity of the enzyme was defined as
one unit of enzyme producing 1 nmol of pNP in 1 min at 37 °C.
2.3. Determination of dose-response curves
The half maximal inhibitory concentration (IC50) of each
compound against VhGlcNAcase was determined from a dose-response
curve using pNP-GlcNAc as substrate. A 200 µL reaction mixture was
prepared in a 96-well microtiter plate containing 125 µM
pNP-GlcNAc, 0.1 µg of VhGlcNAcase and varying concentrations of the
compound (a ten-fold dilution series with a concentration range of
10-10 to 10-2 M) in 100 mM phosphate buffer, pH 7.0. The reaction
mixture was incubated at 37 °C for 10 min with constant agitation,
and terminated by the addition of 100 µl 3 M Na2CO3. The enzyme
activity was estimated from the liberated pNP, quantitated as
described previously. The IC50 value was obtained from a plot of
the logarithm of inhibitor concentration versus fractional activity
of the enzyme, in GraphPad Prism version 6.0. (GraphPad Software,
California, USA).
2.4. Isothermal titration microcalorimetry (ITC)
ITC studies of binding of the compounds to VhGlcNAcase were
carried out in triplicate, using the MicroCal-PEAQTM ITC system
(Malvern Instruments Ltd.) at 25 °C with a stirring speed of 600
rpm. The ligand solution, containing 2.3 μL of 0.15 mM PUGNAc, 0.4
mM NHAcCAS or 0.4 mM NHAcDNJ, was injected into the 300-μl sample
cell containing 10 μM purified VhGlcNAcase in 50 mm potassium
phosphate buffer, pH 7.0. The control measurement was made by
injecting the corresponding ligand into the sample cell containing
only buffer. Each injection was repeated 18 times over 120-s
intervals and the ITC data were collected and analyzed using the
MicroCal-PEAQ ITC software. The ITC profile, obtained by titrating
the ligand into VhGlcNAcase, was then subtracted from the profile
of ligand to buffer, before the titration curve was fitted by a
single-site binding model in the nonlinear least square algorithm.
Direct measurements of binding affinity and thermodynamics in a
single experiment allowed determination of the equilibrium binding
association constant (Ka), reaction stoichiometry (n) and enthalpy
change (ΔH°). The Gibb’s free energy change (ΔG°) and the entropy
change (ΔS°) were calculated from the relationship ΔG° = ΔH° – TΔS°
= -RT ln(Ka) where R is the gas constant (1.98 cal·K−1·mol−1) and T
is the absolute temperature in kelvin. Values presented are means ±
S.D. obtained from experiments carried out in triplicate.
Temperature-dependence experiments were carried out in
triplicate using the MicroCal-PEAQTM ITC system as described above
at 15, 20, 25, and 30 °C with a stirring speed of 600 rpm. PUGNAc
(2.3 μL, 0.15 mM) was injected into the 300-μl sample cell
containing 10 μM purified VhGlcNAcase in 50 mm potassium phosphate
buffer, pH 7.0 as described previously. The heat capacity changes
(ΔCp) of the binding reactions were obtained by examining the
temperature-dependent change in reaction enthalpy: ΔG° = ΔH° – TΔS
= -RT ln(Ka) and the ΔCp is equal to ∂ΔH°/∂T, the slope of the
linear plot of the enthalpy change (ΔH°) versus temperature (T)
[39]. Each parameter of the entropy change (ΔS°) was calculated as
follows [39,40]: Mixing entropy change (ΔS°mix) = R ln (1/55.5) =
−33 J·mol−1·K−1 = -8 cal·mol−1·K−1 = 0.0078 kcal·mol−1·K−1, which
is a statistical correction that reflects mixing of solute and
solvent molecules. Solvent entropy change (ΔS°solv) = ΔCp ln
(298.15/385.15). Note that the entropy of solvation (ΔS°solv) is
close to zero for proteins near 385 K; Cp can be correlated to the
solvation entropy change (ΔS°solv) of the binding reaction at T =
25 °C (298 K). Conformational entropy change ΔS°conf = ΔS° − ΔS°mix
− ΔS°solv. Finally, the reaction entropy change ΔS° = ΔS°solv +
ΔS°mix + ΔS°conf.
2.5. Molecular Docking
All hydrogen atoms were added into the atomic coordinates of the
VhGlcNAcase structure in complex with NAG (PDB id 6EZS) using the
Protonation and Tautomers function in the GOLD 5.3 program [41].
The other configuration parameters were set up following the
default values. The three-dimensional coordinates of the ligand,
water and ion molecules were removed from the structure of the
enzyme by the Protein Setup wizard in the program. The chemical
structures of PUGNAc, NHAcCAS and NHAcDNJ were taken from the
crystal structures of GlcNAcase from Paenibacillus sp. TS12 [29]
deposited in the Protein Data Bank. Automatic GA parameter settings
were used in all GOLD docking calculations, with 100 GA operations
per ligand. The binding site was defined so as to include all amino
acid residues within a radius of 7 Å from individual atoms of
N-acetylglucosamine. The ChemPLP scoring function embedded in the
GOLD package was applied in all docking calculations. This scoring
function includes the contributions of hydrogen bonds, which are
the predominant type found in GlcNAcases.
3. Results
3.1. Evaluation of the inhibitory effects of the compounds on
VhGlcNAcase
We first examined the effects of the putative inhibitors on the
hydrolytic activity of VhGlcNAcase. The chemical structures of
PUGNAc, NHAcCAS and NHAcDNJ, are presented in Fig. 1. The residual
activity of VhGlcNAcase was determined after the enzyme was exposed
to different concentrations of each compound. As seen in Fig. 2,
the activity of VhGlcNAcase, measured using the synthetic substrate
pNP-GlcNAc, was suppressed in a concentration-dependent manner.
PUGNAc showed the greatest effect, with the activity of the enzyme
almost completely abolished at 100 μM. Similar results were
obtained with the other two compounds, though the inhibitory
effects were less pronounced, with residual activities of 23% and
35% being observed with 100 μM NHAcCAS and NHAcDNJ, respectively
(Fig. 2). To quantify the inhibitory effects of these compounds,
dose-response experiments were performed. The IC50 value for each
compound was determined from the dose-response plot of the
fractional residual activity of the enzyme (vo/vi) against the
logarithm of the inhibitor concentration. The IC50 values for
PUGNAc (Fig. 3A), NHAcCAS (Fig. 3B) and NHAcDNJ (Fig. 3C) were
estimated to be 1.2 ± 0.04, 70.7 ± 2.7, 96.2 ± 4.2 μM,
respectively, confirming that PUGNAc was the most potent inhibitor
of VhGlcNAcase.
3.2. Molecular docking and interaction analysis
Molecular docking was used to examine specific interactions of
the natural substrate chitobiose (diNAG) with the inhibitors, using
the crystal structure of VhGlcNAcase in complex with NAG (PDB id
6EZS; unpublished data) as a template. Docking diNAG into the
active site of VhGlcNAcase gave a Piecewise Linear Potential (PLP)
docking score of 71.8 ± 2.1 [41], indicating that the substrate
fitted well into the substrate-binding pocket of the enzyme, with
the substrate located within the active site (Fig. 4). The pyranose
ring of the NAG in the -1 subsite adopts a 4E/boat conformation
whereas the NAG in the +1 subsite adopts a 1C4/chair conformation,
which is similar to what was observed with diNAG in complex with
the chitobiase from Serratia marcescens (SmChb, PDB id: 1QBB) [42].
In Fig. 4 (upper left panel), the polar and charged residues R274,
Q398, Y530, D532, E584 and W546 are located within a distance of
3.8-Å. These residues make hydrogen bonds with various hydroxyl
groups of NAG in the -1 subsite (assigned as -1NAG). In Fig. 4
(lower panel), five aromatic residues, W487, W505, Y530, W546 and
W582, form the hydrophobic wall of the substrate-binding pocket of
the enzyme, with W582 stacked against the pyranose ring of -1NAG,
whereas the side chain of W546 makes a hydrogen bond with 6-OH of
-1NAG and also stacks against the planar face of NAG in the +1
subsite (assigned as +1NAG). All the inhibitors fitted well within
subsite -1, with no steric clash against the surface of VhGlcNAcase
(Fig. 4, lower panels). PUGNAc showed the highest PLP fit score of
62.7 ± 1.2, followed by NHAcCAS (score = 53.1 ± 2.1), and NHAcDNJ
(score = 46.9 ± 0.2). PUGNAc, for which the enzyme had the greatest
affinity, based on activity measurements, was found to form a
H-bond network with the binding residues, similar to that of diNAG.
In addition, three key aromatic residues that bind to diNAG, Y530,
W546 and W582, were also found to interact with PUGNAc. On the
other hand, fewer interactions were observed with NHAcCAS and
NHAcDNJ. Notably, the stacking interaction of W546 and the phenyl
carbamate moiety of PUGNAc is missing in the other two inhibitors.
The interactions between the substrate/inhibitor and the enzyme are
summarized in Table 1.
3.3. Binding thermodynamics of VhGlcNAcase with reaction
intermediate analogues
To gain further insight into the modes of binding of the
inhibitors with VhGlcNAcase, ITC was performed to determine the
equilibrium dissociation binding constant (Kd), stoichiometry (n),
enthalpy change (ΔH°), entropy change (ΔS°) and Gibb’s free energy
change (ΔG°). The heat release profiles of the wild-type enzyme
(Fig. 5, upper panels) were measured during titration with
different concentrations of each inhibitor (Fig. 5A for PUGNAc, 5B
for NHAcCAS and 5C for NHAcDNJ). The individual troughs of the
thermogram were integrated, yielding the enthalpy change as kcal
per mol of injectant plotted against the molar ratio,
inhibitor:VhGlcNAcase (Fig. 5A-C, lower panels). This analysis gave
Kd values for PUGNAc, NHAcCAS and NHAcDNJ of 0.19, 12.9, and 25.6
μM, respectively (Table 2), an order of values that agreed with
those of the IC50 values determined using dose-response
experiments. Theoretical curve fitting of single-site binding gave
a stoichiometry (n) of 0.9 for PUGNAc and 1.0 for NHAcCAS and
NHAcDNJ (Table 2), confirming one molecule of inhibitor bound per
molecule VhGlcNAcase.
Thermodynamic parameters obtained from the ITC experiments,
including the enthalpy change (ΔH°), the entropy change (−TΔS°) and
the free energy change (ΔG°), were further analyzed (inset, Fig. 5,
lower panels). Plots of the free energy change (ΔG°) as the sum of
negative ΔH° and positive −TΔS° terms demonstrate how the enthalpy
and the entropy changes contribute to binding of the inhibitors by
the enzyme. The results which are similar for all three compounds,
indicate that the bindings are exergonic (-ΔG°) and are driven by
enthalpy (Table 2), with ΔG° values ranging from -6.3 to -9.2
kcal·mol−1.
3.4. Temperature effects on inhibitor-enzyme binding
Since PUGNAc was the most potent inhibitor of VhGlcNAcase
identified in this study, we performed further thermodynamic
analysis of this compound. ITC experiments were carried out at
different temperatures (Fig. 6A-D, upper panels), allowing heat
capacity changes (Cp) to be calculated from the secondary plots of
binding isotherms (Fig. 6A-D, lower panels). The equilibrium
binding constants (Kd) for PUGNAc at 15, 20, 25, and 30 oC were
0.11, 0.13, 0.19, and 0.13 μM, respectively (Table 3). The
theoretical fit with a single-site binding model gave the
stoichiometry (n) close to 1.0 over the entire range of the tested
temperatures, confirming a one-to-one binding mode. The heat
capacity change (Cp) linked to hydration of the system was obtained
from the slope of a linear plot of the enthalpy change (ΔH°) versus
temperature (Fig. 7A). The Cp of PUGNAc was calculated to be -0.34
± 0.05 kcal·mol−1·K−1. The reaction entropy change (ΔS°) for the
binding of PUGNAc by VhGlcNAcase reveals a large increase in the
solvent-entropy change (ΔS°solv) and a large unfavorable
conformational entropy change (ΔS°conf) in the enzyme (Fig. 7B).
The results indicate that PUGNAc binds to VhGlcNAcase rigidly and
the binding results in a large favorable ΔS°solv, compensating a
large unfavorable ΔS°conf.
4. Discussion
The exo-β-N-acetylglucosaminidase (VhGlcNAcase) from V. harveyi
is an exolytic chitin-degrading enzyme that belongs to family 20 of
the glycoside hydrolases [1,31]. VhGlcNAcase plays a crucial role
in the recycling of chitin biomass by the marine Vibrio, as it
helps to completely degrade chitin degradation products, generated
by secreted chitinases [43,44] and transported through outer
membrane of the bacterium through chitoporin [45,46], to GlcNAc
monomers that are readily metabolized further in the chitin
catabolic pathway and finally serve as nitrogen and carbon sources
for the cells.
VhGlcNAcase uses a substrate-assisted catalytic mechanism,
involving the formation of an oxazolinium intermediate that is then
broken down to give a hemiacetal, retaining the anomeric
conformation of the substrate. This mechanism requires the amino
acids D437 and E438 for catalysis [31]. An understanding of the
reaction mechanism and the development of inhibitors of GlcNAcases
has led to efforts to develop these types of molecules as drugs
targeting GlcNAcase-related metabolic diseases. For example,
NAG-thiazoline has been suggested as a treatment for Tay-Sachs and
Sandhoff diseases, acting as a pharmacological chaperone that acts
to stabilize the mutant form of HexA and HexB, resulting in an
increased amount of HexA and HexB in the lysosome [24,30]. The
compounds studied here have been shown to lower the enzymatic
activity of GlcNAcases in general, as demonstrated previously with
GH20 and GH84 GlcNAcases [21,25,29,47], but NHAcDNJ, for example,
has also been used therapeutically and reported to increase the
impaired GlcNAcase activity in adult Tay-Sachs and infantile
Sandhoff cell lines [48]. Since the compounds studied here have
been employed as potent inhibitors of GH20 enzymes, in this study
we carried out a series of experiments to examine the effects of
the three compounds on the activity of VhGlcNAcase. Our results
showed that all compounds strongly inhibited VhGlcNAcase activity
at concentrations in the low μM range. PUGNAc was found to be the
most potent inhibitor of VhGlcNAcase with an IC50 value of 1.2 μM,
whereas NHAcCAS and NHAcDNJ showed moderate effects. The inhibitory
effect of PUGNAc on VhGlcNAcase was similar to that on human HexA
[29], while its C4-epimer, Gal-PUGNAc was found to be more
effective than PUGNAc against Paenibacillus sp. TS12 β-Hex1 [29]
and human HexB [29,49].
Our molecular docking analysis indicates that the
substrate-binding pocket of VhGlcNAcase can accommodate a short
chain of chitooligosaccharides, agreeing with the previously
reported kinetic data, which showed that VhGlcNAcase preferred a
chitooligosaccharide chain of 2-4 GlcNAc units. The active sites of
human HexA and HexB also have a small pocket in which the
C2-acetamido group of GalNAc-isofagomine nestles between three
tryptophan residues, W405, W424 and W489 (equivalent to W487, W505
and W582 in VhGlcNAcase) [50,51]. In the VhGlcNAcase structure
modelled with diNAG, five aromatic residues, W487, W505, W546, W582
and Y530, form the wall in the active site pocket, which is
stabilized by hydrogen bonding interactions. W582 was found to
stack directly with the plane of the pyranose ring of the -1NAG
moiety, and these interactions are expected to contribute to
binding. All aromatic residues are completely conserved in other
GH20 enzymes and play an important role in substrate binding
[9,51]. The pyranose ring of PUGNAc appears to be in a 4H3
conformation, similar to that observed for this compound with other
GH20 bacterial chitinolytic β-N-acetyl-D-hexosaminidases [52], and
thought to be the conformation of the transition state for enzymes
that use substrate-assisted catalysis [53]. PUGNAc forms hydrogen
bonds with polar and charged residues at both -1 and +1 subsites.
The pyranose ring of PUGNAc makes stacking interactions with W582
and the extended phenylcarbamate moiety stacks against W546 in the
+1 subsite to facilitate binding (Fig. 4). Additionally, a trigonal
sp2-hybridization at the pseudoanomeric position (C1) potentially
enhances the binding, through partial mimicry of the transition
state of these types of enzymes [53]. This is presumably why PUGNAc
shows the strongest binding and is the most potent inhibitor of
VhGlcNAcase. On the other hand, the piperidine rings of NHAcCAS and
NHAcDNJ can only make stacking interactions with W582 and the amine
nitrogen of both analogues does not interact with other residues in
the active site.
The enthalpic changes produced by increasing concentrations of
the inhibitors were measured using ITC, allowing binding affinities
and thermodynamic parameters [54,55] for VhGlcNAcase to be
determined. The study revealed that PUGNAc had the highest affinity
for VhGlcNAcase, supporting the results of the inhibition
experiments. From the ITC experiments, VhGlcNAcase binds to all
compounds through enthalpy-driven reactions, indicating that
electrostatic interactions dominate the binding. It was also found
that the stoichiometry (n) of binding of the inhibitor to
VhGlcNAcase was 1:1 for all compounds tested. Large increases in
enthalpy with increasing temperature are often compensated by an
entropy decrease, resulting in only a small change in free energy,
the so called ‘entropy-enthalpy compensation’. This thermodynamic
compensation is a natural consequence of finite Cp values and in
general can take place due to multiple weak interactions and
limited free energy windows [56]. The effect is particularly
apparent in aqueous systems, in which non-covalent interactions
dominate binding. In a GH19 chitinase, Ohnuma et al. [57] found Cp
for binding of (GlcNAc)6 to BcChi-A-E61A to be -0.11 ± 0.008
kcal·mol−1·K−1, suggesting that an aromatic residue (W103) of
BcChi-A may contribute to binding through a hydrophobic interaction
with (GlcNAc)6. Regarding the binding of PUGNAc to VhGlcNAcase, the
large negative value of Cp (-0.34 ± 0.05 kcal·mol−1·K−1) reflects
stronger hydrophobic interactions and a larger accessible area of
nonpolar surface than in BcChi-A, with W582 and W546 stacking
against the aromatic rings of the bound inhibitor. The entropy
change (ΔS°) of the reaction with PUGNAc reveals a large favourable
increase in the solution (ΔS°solv), which compensates for a large
unfavorable conformational entropy change (ΔS°conf) (Fig. 7B). It
is likely that a large change in ΔS°solv results from the greater
dehydration of the enzyme at the binding interface.
In conclusion, kinetic and thermodynamic characterization of the
interaction of VhGlcNAcase with some known inhibitors of
exo-β-N-acetylglucosaminidases suggested that PUGNAc is the most
potent inhibitor for VhGlcNAcase, and the data are well supported
by number of interactions observed by docking simulation. The other
compounds tested, NHAcCAS and NHAcDNJ, showed moderate inhibitory
effects on the enzyme. The binding affinities obtained from ITC
analysis suggested that VhGlcNAcase bound to the compounds with Kd
values in the nM - µM range and in a 1:1 ratio. Analysis of the
thermodynamic parameters indicates that binding of all compounds to
VhGlcNAcase is driven mainly by enthalpy, with the degree of
inhibition depending on the functional moiety and the chain length
of the compounds.
Conflicts of interest
All authors declare no conflict.
Acknowledgements
This research was supported by Suranaree University of
Technology (SUT) and by Office of the Higher Education Commission
under the National Research University (NRU) Project of Thailand
(FtR.33/2559). WS was funded by the Thailand Research Fund and
Suranaree University of Technology through a Basic Research Grant
(Grant no. BRG578001) and an SUT grant (SUT1-102-60-24-12). The
authors gratefully acknowledge the Biochemistry-Electrochemistry
Research Unit and Biochemistry Laboratory, and the Centre for
Scientific and Technological Equipment, Suranaree University of
Technology for providing the facilities for carrying out this
research and thank Dr. Kiattawee Choowongkomon, Department of
Biochemistry, Faculty of Science, Kasetsart University, Bangkok,
Thailand for use of the software GOLD 5.3. KAS also thanks the
Australian Research Council for funding (FT100100291).
Author contributions
WS was the grant holder and supervised the entire project. PM
designed and performed all the experiments. WS also provided
reagents and materials used in this study. KAS synthesized the
inhibitors tested. PM and WS analyzed the data and wrote the paper
with support from KAS.
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Figure legends
Fig. 1. Chemical structures of PUGNAc, NHAcCAS and NHAcDNJ used
in this study and the structure of the presumed intermediate in
GlcNAcase substrate-assisted catalytic mechanisms.
Fig. 2. Relative activity of WT VhGlcNAcase against pNP-GlcNAc
in the absence and presence of various concentrations of
inhibitors, in 100 mM sodium phosphate buffer, pH 7.0. The values
shown are mean ± S.D. obtained from experiments carried out in
triplicate.
Fig. 3. Dose-response plot of the fractional activity of WT
VhGlcNAcase as a function of various concentrations of inhibitors.
The value of IC50 for each inhibitor was determined from this graph
with the error bars representing the standard deviation (S.D.) from
triplicate experiments.
Fig. 4. Analysis of VhGlcNAcase interaction with the natural
substrate and inhibitors by the use of molecular docking. Details
of the hydrogen-bonding network (upper panels) and hydrophobic
interactions (lower panels) show the positions of the regularly
surface-exposed residues in the active site of diNAG, PUGNAc,
NHAcCAS and NHAcDNJ complexed with VhGlcNAcase.
Fig. 5. Inhibitor binding to WT VhGlcNAcase. Microcalorimetric
titrations for the binding of PUGNAc (A), NHAcCAS (B) and NHAcDNJ
(C) to VhGlcNAcase and its control traces for ligand to buffer
(upper panels) and thermographic binding isotherms with theoretical
fits (lower panels) obtained from ITC experiments. Thermodynamic
parameter values of analogue binding (inset) were plotted as bar
graphs with means ± S.D. obtained from experiments carried out in
triplicate.
Fig. 6. Microcalorimetric titrations and control traces for
ligand to buffer (upper panels) and thermographic binding isotherms
with theoretical fits (one set of sites) (lower panels) for the
binding of PUGNAc to VhGlcNAcase, obtained from ITC experiments at
15 °C - 30 °C (A-D).
Fig. 7. ΔCp, ΔSsolv, and ΔSconf. PUGNAc binding to VhGlcNAcase
was tested at 15, 20, 25 and 30 °C. (A) The thermodynamic
parameters ΔG° (black), ΔH° (blue) and -TΔS° (green): values were
obtained as described in Table 3 and plotted against the
temperature. (B) The entropy change (ΔS°) of VhGlcNAcase-PUGNAc
reaction. ΔS°solv and ΔS°conf were calculated according to the
equations quoted in the Materials and Methods.
Table 1 Summary of the interactions of the natural substrate and
inhibitors observed in the substrate binding pocket of
VhGlcNAcase.
Subsite
Ligand
diNAG
PUGNAc
NHAcCAS
NHAcDNJ
R274, Q398, W487,
R274, Q398, D532,
R274, Q398, Y530,
R274, Q398, D532,
-1
W505, Y530, D532,
W582, E584
W582, E584
W582, E584
W582, E584
+1
W546
W546
W546
Normal-and-underlined: hydrogen bonding residues
Bold: hydrophobic interaction residues
Bold-and-underlined: hydrogen bonding and hydrophobic
interaction residues
Table 2 Binding parameters obtained from ITC.
Inhibitor
Isothermal microcalorimetric (ITC) parameter
IC50
Kd
ΔG°
ΔH°
ΔS°
-TΔS°
n
(μM)
(μM)
(kcal/mol)
(kcal/mol)
(kcal/mol/K)
(kcal/mol)
PUGNAc
1.2 ± 0.04
0.19 ± 0.04
-9.18 ± 0.12
-9.44 ± 0.09
-0.001 ± 0.0
0.26 ± 0.03
0.9 ± 0.1
NHAcCAS
70.7 ± 2.7
12.9 ± 0.87
-6.67 ± 0.04
-7.15 ± 0.18
-0.002 ± 0.001
0.48 ± 0.20
1.0 ± 0.0
NHAcDNJ
96.2 ± 4.2
25.6 ± 0.95
-6.27 ± 0.02
-7.82 ± 0.73
-0.005 ± 0.002
1.55 ± 0.72
1.0 ± 0.1
Table 3 Effects of temperature on the binding of PUGNAc to
VhGlcNAcase.
Temperature
(°C)
Isothermal microcalorimetric (ITC) parameter
Kd
ΔG°
ΔH°
ΔS°
-TΔS°
n
(μM)
(kcal/mol)
(kcal/mol)
(kcal/mol/K)
(kcal/mol)
15
0.11 ± 0.04
-9.20 ± 0.23
-6.19 ± 0.22
0.012 ± 0.002
-3.01 ± 0.38
0.9 ± 0.1
20
0.13 ± 0.04
-9.23 ± 0.17
-9.71 ± 0.31
-0.002 ± 0.00
0.47 ± 0.18
0.7 ± 0.1
25
0.19 ± 0.02
-9.17 ± 0.07
-9.27 ± 0.25
-0.0003 ± 0.00
0.10 ± 0.21
0.9 ± 0.0
30
0.13 ± 0.02
-9.55 ± 0.09
-12.07 ± 0.38
-0.008 ± 0.001
2.55 ± 0.45
0.9 ± 0.1
Fig. 1.
2
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.