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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, Thailand2School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA,
Australia, 60093Center 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
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AbstractExo--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
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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
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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),
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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
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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
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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. Results3.1. 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
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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.
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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. DiscussionThe 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
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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.
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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 interestAll authors declare no conflict.
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AcknowledgementsThis 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 contributionsWS 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.
References[1] W. Suginta, D. Chuenark, M. Mizuhara, T. Fukamizo, Novel β-N-acetylglucosaminidases from Vibrio
harveyi 650: cloning, expression, enzymatic properties, and subsite identification, BMC Biochem. 11
(2010) 40.
[2] N.N. Thi, W.A. Offen, F. Shareck, G.J. Davies, N. Doucet, Structure and activity of the Streptomyces
coelicolor A3(2) β-N-acetylhexosaminidase provides further insight into GH20 family catalysis and
inhibition, Biochemistry 53 (2014) 1789-1800.
[3] J. Zhou, Z. Song, R. Zhang, C. Chen, Q. Wu, J. Li, X. Tang, B. Xu, J. Ding, N. Han, Z. Huang, A
Shinella β-N-acetylglucosaminidase of glycoside hydrolase family 20 displays novel biochemical and
molecular characteristics, Extremophiles 21 (2017) 699-709.
[4] G. Davies, B. Henrissat, Structures and mechanisms of glycosyl hydrolases, Structure 3 (1995) 853-
859.
[5] J. Gebler, N.R. Gilkes, M. Claeyssens, D.B. Wilson, P. Beguin, W.W. Wakarchuk, D.G. Kilburn, R.C.
Miller, Jr., R.A. Warren, S.G. Withers, Stereoselective hydrolysis catalyzed by related β-1,4-glucanases
and β-1,4-xylanases, J. Biol. Chem. 267 (1992) 12559-12561.
13
[6] D.J. Vocadlo, C. Mayer, S. He, S.G. Withers, Mechanism of action and identification of Asp242 as
the catalytic nucleophile of Vibrio furnisii N-acetyl-β-D-glucosaminidase using 2-acetamido-2-deoxy-5-
fluoro-α-L-idopyranosyl fluoride, Biochemistry 39 (2000) 117-126.
[7] D.J. Vocadlo, S.G. Withers, Detailed comparative analysis of the catalytic mechanisms of β-N-
acetylglucosaminidases from families 3 and 20 of glycoside hydrolases, Biochemistry 44 (2005) 12809-
12818.
[8] S. Drouillard, S. Armand, G.J. Davies, C.E. Vorgias, B. Henrissat, Serratia marcescens chitobiase is a
retaining glycosidase utilizing substrate acetamido group participation, Biochem. J. 328 (1997) 945-949.
[9] B.L. Mark, D.J. Vocadlo, S. Knapp, B.L. Triggs-Raine, S.G. Withers, M.N. James, Crystallographic
evidence for substrate-assisted catalysis in a bacterial β-hexosaminidase, J. Biol. Chem. 276 (2001)
10330-10337.
[10] F.V. Rao, H.C. Dorfmueller, F. Villa, M. Allwood, I.M. Eggleston, D.M.F. van Aalten, Structural
insights into the mechanism and inhibition of eukaryotic O‐GlcNAc hydrolysis, EMBO J. 25 (2006)
1569-1578.
[11] P.W. Robbins, K. Overbye, C. Albright, B. Benfield, J. Pero, Cloning and high-level expression of
chitinase-encoding gene of Streptomyces plicatus, Gene 111 (1992) 69-76.
[12] K. Sandhoff, T. Kolter, Processing of sphingolipid activator proteins and the topology of lysosomal
digestion, Acta Biochim. Pol. 45 (1998) 373-384.
[13] R.A. Gravel, J.T.R. Clarke, M.M. Kaback, D. Mahuran, K. Sandhoff, K. Suzuki, The GM2
gangliosidoses, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic Basis of
Inherited Disease, McGraw-Hill, New York, 1995, pp. 1807-1839.
[14] R. Myerowitz, Tay-Sachs disease-causing mutations and neutral polymorphisms in the Hex A gene,
Hum. Mutat. 9 (1997) 195-208.
[15] S.A. Yuzwa, D.J. Vocadlo, O-GlcNAc and neurodegeneration: biochemical mechanisms and
potential roles in Alzheimer’s disease and beyond, Chem. Soc. Rev. 43 (2014) 6839-6858.
[16] J. Ma, G.W. Hart, Protein O-GlcNAcylation in diabetes and diabetic complications, Expert Rev.
Proteomics 10 (2013) 365-380.
[17] K. Vaidyanathan, L. Wells, Multiple tissue-specific roles for the O-GlcNAc post-translational
modification in the induction of and complications arising from type II diabetes, J. Biol. Chem. 289
(2014) 34466-34471.
[18] C.M. Ferrer, V.L. Sodi, M.J. Reginato, O-GlcNAcylation in cancer biology: linking metabolism and
signaling, J. Mol. Biol. 428 (2016) 3282-3294.
[19] J.P. Singh, K. Zhang, J. Wu, X. Yang, O-GlcNAc signaling in cancer metabolism and epigenetics,
Cancer Lett. 356 (2015) 244-250.
14
[20] R.H. Furneaux, G.J. Gainsford, J.M. Manson, P.C. Tyler, The chemistry of castanospermine, part I:
synthetic modifications at C-6, Tetrahedron 50 (1994) 2131-2160.
[21] E. Kappes, G. Legler, Synthesis and inhibitory properties of 2-acetamido-2-deoxynojirimycin (2-
acetamido-5-amino-2,5-dideoxy-D-glucopyranose, 1) and 2-acetamido-1,2-dideoxynojirimycin (2-
acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol, 2), J. Carbohyd. Chem. 8 (1989) 371-388.
[22] S. Knapp, D. Vocadlo, Z. Gao, B. Kirk, J. Lou, S.G. Withers, NAG-thiazoline, an N-acetyl-β-
hexosaminidase inhibitor that implicates acetamido participation, J. Am. Chem. Soc. 118 (1996) 6804-
6805.
[23] K.A. Stubbs, N. Zhang, D.J. Vocadlo, A divergent synthesis of 2-acyl derivatives of PUGNAc yields
selective inhibitors of O-GlcNAcase, Org. Biomol. Chem. 4 (2006) 839-845.
[24] M.B. Tropak, S.P. Reid, M. Guiral, S.G. Withers, D. Mahuran, Pharmacological enhancement of β-
hexosaminidase activity in fibroblasts from adult Tay-Sachs and Sandhoff patients, J. Biol. Chem. 279
(2004) 13478-13487.
[25] M.S. Macauley, G.E. Whitworth, A.W. Debowski, D. Chin, D.J. Vocadlo, O-GlcNAcase uses
substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired
inhibitors, J. Biol. Chem. 280 (2005) 25313-25322.
[26] K.A. Stubbs, M. Balcewich, B.L. Mark, D.J. Vocadlo, Small molecule inhibitors of a glycoside
hydrolase attenuate inducible AmpC-mediated β-lactam resistance, J. Biol. Chem. 282 (2007) 21382-
21391.
[27] R.J. Dennis, E.J. Taylor, M.S. Macauley, K.A. Stubbs, J.P. Turkenburg, S.J. Hart, G.N. Black, D.J.
Vocadlo, G.J. Davies, Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase
activity, Nat. Struct. Mol. Biol. 13 (2006) 365-371.
[28] D.L. Dong, G.W. Hart, Purification and characterization of an O-GlcNAc selective N-acetyl-β-D-
glucosaminidase from rat spleen cytosol, J. Biol. Chem. 269 (1994) 19321-19330.
[29] T. Sumida, K.A. Stubbs, M. Ito, S. Yokoyama, Gaining insight into the inhibition of glycoside
hydrolase family 20 exo-β-N-acetylhexosaminidases using a structural approach, Org. Biomol. Chem. 10
(2012) 2607-2612.
[30] K.S. Bateman, M.M. Cherney, D.J. Mahuran, M. Tropak, M.N. James, Crystal structure of β-
hexosaminidase B in complex with pyrimethamine, a potential pharmacological chaperone, J. Med.
Chem. 54 (2011) 1421-1429.
[31] P. Meekrathok, W. Suginta, Probing the catalytic mechanism of Vibrio harveyi GH20 β-N-
acetylglucosaminidase by chemical rescue, PLoS One 11 (2016) e0149228.
[32] P. Sirimontree, T. Fukamizo, W. Suginta, Azide anions inhibit GH-18 endochitinase and GH-20 exo
β-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi, J. Biochem. 159 (2016) 191-200.
15
[33] B. Austin, X.H. Zhang, Vibrio harveyi: a significant pathogen of marine vertebrates and
invertebrates, Lett. Appl. Microbiol. 43 (2006) 119-124.
[34] M.J. Lemieux, B.L. Mark, M.M. Cherney, S.G. Withers, D.J. Mahuran, M.N. James,
Crystallographic structure of human β-hexosaminidase A: interpretation of Tay-Sachs mutations and loss
of GM2 ganglioside hydrolysis, J. Mol. Biol. 359 (2006) 913-929.
[35] P. Meekrathok, M. Burger, A.T. Porfetye, I.R. Vetter, W. Suginta, Expression, purification,
crystallization and preliminary crystallographic analysis of a GH20 β-N-acetylglucosaminidase from the
marine bacterium Vibrio harveyi, Acta Crystallogr. F 71 (2015) 427-433.
[36] S.C. Gill, P.H. von Hippel, Calculation of protein extinction coefficients from amino acid sequence
data, Anal. Biochem. 182 (1989) 319-326.
[37] E.D. Goddard-Borger, K.A. Stubbs, An improved route to PUGNAc and its galacto-configured
congener, J. Org. Chem. 75 (2010) 3931-3934.
[38] K.A. Stubbs, J.P. Bacik, G.E. Perley-Robertson, G.E. Whitworth, T.M. Gloster, D.J. Vocadlo, B.L.
Mark, The development of selective inhibitors of NagZ: increased susceptibility of Gram-negative
bacteria to β-lactams, ChemBioChem 14 (2013) 1973-1981.
[39] H. Zakariassen, M. Sorlie, Heat capacity changes in heme protein-ligand interactions, Thermochim.
Acta 464 (2007) 24-28.
[40] K.P. Murphy, D. Xie, K.S. Thompson, L.M. Amzel, E. Freire, Entropy in biological binding
processes: estimation of translational entropy loss, Proteins 18 (1994) 63-67.
[41] M.L. Verdonk, J.C. Cole, M.J. Hartshorn, C.W. Murray, R.D. Taylor, Improved protein-ligand
docking using GOLD, Proteins 52 (2003) 609-623.
[42] I. Tews, A. Perrakis, A. Oppenheim, Z. Dauter, K.S. Wilson, C.E. Vorgias, Bacterial chitobiase
structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease, Nat. Struct. Biol.
3 (1996) 638-648.
[43] W. Suginta, A. Vongsuwan, C. Songsiriritthigul, H. Prinz, P. Estibeiro, R.R. Duncan, J. Svasti, L.A.
Fothergill-Gilmore, An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence
analyses, and chitin hydrolysis, Arch. Biochem. Biophys. 424 (2004) 171-180.
[44] W. Suginta, A. Vongsuwan, C. Songsiriritthigul, J. Svasti, H. Prinz, Enzymatic properties of wild-
type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MS, FEBS J.
272 (2005) 3376-3386.
[45] W. Suginta, W. Chumjan, K.R. Mahendran, P. Janning, A. Schulte, M. Winterhalter, Molecular
uptake of chitooligosaccharides through chitoporin from the marine bacterium Vibrio harveyi, PLoS One
8 (2013) e55126.
16
[46] W. Suginta, W. Chumjan, K.R. Mahendran, A. Schulte, M. Winterhalter, Chitoporin from Vibrio
harveyi, a channel with exceptional sugar specificity, J. Biol. Chem. 288 (2013) 11038-11046.
[47] M.S. Macauley, Y. He, T.M. Gloster, K.A. Stubbs, G.J. Davies, D.J. Vocadlo, Inhibition of O-
GlcNAcase using a potent and cell-permeable inhibitor does not induce insulin resistance in 3T3-L1
adipocytes, Chem. Biol. 17 (2010) 937-948.
[48] A.J. Steiner, G. Schitter, A.E. Stutz, T.M. Wrodnigg, C.A. Tarling, S.G. Withers, D.J. Mahuran,
M.B. Tropak, 2-Acetamino-1,2-dideoxynojirimycin-lysine hybrids as hexosaminidase inhibitors,
Tetrahedron Asymmetry 20 (2009) 832-835.
[49] K.A. Stubbs, M.S. Macauley, D.J. Vocadlo, A selective inhibitor Gal-PUGNAc of human lysosomal
β-hexosaminidases modulates levels of the ganglioside GM2 in neuroblastoma cells, Angew. Chem. Int.
Ed. 48 (2009) 1300-1303.
[50] T. Maier, N. Strater, C.G. Schuette, R. Klingenstein, K. Sandhoff, W. Saenger, The X-ray crystal
structure of human β-hexosaminidase B provides new insights into Sandhoff disease, J. Mol. Biol. 328
(2003) 669-681.
[51] B.L. Mark, D.J. Mahuran, M.M. Cherney, D. Zhao, S. Knapp, M.N. James, Crystal structure of
human β-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease, J.
Mol. Biol. 327 (2003) 1093-1109.
[52] T. Liu, H. Zhang, F. Liu, L. Chen, X. Shen, Q. Yang, Active-pocket size differentiating insectile
from bacterial chitinolytic β-N-acetyl-D-hexosaminidases, Biochem. J. 438 (2011) 467-474.
[53] G.E. Whitworth, M.S. Macauley, K.A. Stubbs, R.J. Dennis, E.J. Taylor, G.J. Davies, I.R. Greig, D.J.
Vocadlo, Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-
GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise, J. Am.
Chem. Soc. 129 (2007) 635-644.
[54] M.L. Doyle, Characterization of binding interactions by isothermal titration calorimetry, Curr. Opin.
Biotechnol. 8 (1997) 31-35.
[55] G.A. Holdgate, W.H. Ward, Measurements of binding thermodynamics in drug discovery, Drug
Discov. Today 10 (2005) 1543-1550.
[56] A. Cooper, C.M. Johnson, J.H. Lakey, M. Nöllmann, Heat does not come in different colours:
entropy-enthalpy compensation, free energy windows, quantum confinement, pressure perturbation
calorimetry, solvation and the multiple causes of heat capacity effects in biomolecular interactions,
Biophys. Chem. 93 (2001) 215-230.
[57] T. Ohnuma, M. Sorlie, T. Fukuda, N. Kawamoto, T. Taira, T. Fukamizo, Chitin oligosaccharide
binding to a family GH19 chitinase from the moss Bryum coronatum, FEBS J. 278 (2011) 3991-4001.
17
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.
18
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
19
(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.