G Model
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the folding of ␣-glucosidase using Zn2+ and Ca2+ [24,25], and
several denaturants such as urea, guanidine hydrochloride, and
trifluoroethanol have also focused on conformational and activ-
ity changes [26–28]. The previous studies revealed characteristics
of the ␣-glucosidase active site and the presence of several
unfolded states and transient intermediate structures, reflect-
ing that ␣-glucosidase has a stable tertiary structure. However,
the relationship between ␣-glucosidase and Co2+ has not been
reported, and a direct ligand binding mechanism between ␣-
glucosidase and Co2+ has not been found. In this study, Co2+, as
an inhibitor of ␣-glucosidase, was applied to alter the inhibition
kinetics. In addition, a molecular dynamics simulation revealed
the ligand binding mechanism between Co2+ and ␣-glucosidase in
detail. Results of the Co2+-induced inhibitory effect, including struc-
tural changes of ␣-glucosidase, provide new insights into inhibition
of this important enzyme and suggest possible clinical applications
0.0
1.0
2.0
3.0
4.0
5.0
Co2+ (mM)
of Co2+
.
Fig. 1. Effect of Co2+ on the activity of ␣-glucosidase. The enzyme was incubated
with Co2+ for 1 h at 25 ◦C. The activity was then measured by comparing with a
native control enzyme and measuring the change in absorbance at 400 nm at 37 ◦C.
The reaction mixture was composed of 2.5 mM pNPG and 8.0 l of enzyme solution
5.0 M. Data are presented as the mean values (n = 3).
2. Materials and methods
2.1. Materials
␣-Glucosidase (Saccharomyces cerevisiae), 4-nitrophenyl-
-d-glucuronic acid (pNPG), cobalt dichloride, 8-anilino-
1-naphthalenesulfonic acid (ANS), and 2-(N-morpholino)
ethanesulfonic acid (MES) buffer were obtained from
Sigma–Aldrich (USA).
Restraint Simulated Annealing (PQR-SA) protocol [29]. The same
methods were used to generate the homology model, so the
developed structure was similar to one from previous work [28].
Using a generated homology model, we searched for plausible
active sites using the Dockable Pocket Site Prediction (DPSP)
software. Five nanosecond (ns) molecular dynamics (MD) simu-
lations were performed in simulation conditions with and without
Co2+. We counted twenty Co2+ satisfying the experimental con-
dition of a 10 mM concentration. A periodic boundary box size of
2.2. Enzyme assay
Co2+ (0–5.0 mM) in 50 mM MES buffer (pH 6.5) at 25 ◦C. The activity
(v) was determined at 37 ◦C by measuring the change in absorbance
at 400 nm using a U-3900 spectrophotometer (Hitachi, Japan),
which accompanies the hydrolysis of pNPG to generate pNP accord-
ing to a previously described method [27,28]. The substrate reaction
mixture contained 2.5 mM pNPG substrate and 8.0 l of enzyme
solution in 1.0 ml 50 mM phosphate buffer (pH 7.2), with or with-
out Co2+ at various concentrations. One unit of enzyme activity was
defined as the amount of enzyme that liberated 1.0 M of d-glucose
from pNPG per minute at 37 ◦C (pH 7.2).
˚
˚
˚
118 A × 118 A × 118 A was used to prevent ions from moving away
from the protein. A Generalized Born model of simple SWitching
function (GBSW) was used to consider implicit water molecules
[30]. For trajectory analyses, the Root Mean Square Deviation
(RMSF) as a function of residue number, and the number of bound
Co2+ were calculated for every picosecond trajectory. All compu-
tational calculations were conducted using the CHARMM program
[31]. The ratio of secondary structure (alpha/beta; units shown in
percentage) was measured using the DSSP program [32].
2.3. Tertiary structure measurements via intrinsic and
ANS-binding fluorescence monitoring
Samples were treated for 1 h at 25 ◦C in incubation solutions
containing different concentrations of Co2+. Intrinsic fluorescence
spectra were measured using an excitation wavelength of 280 nm
and emission wavelengths ranging from 300 to 400 nm that
were recorded using an F-2500 fluorescence spectrophotometer
(Hitachi, Japan), using a 1.0 cm path length cuvette. Monitoring
hydrophobic surface via ANS-binding fluorescence was performed
by labeling the enzyme samples with 63 M ANS for 40 min prior
to measurement. An excitation wavelength of 380 nm was used for
ANS-binding fluorescence, and emission wavelengths ranged from
400 to 600 nm. After the enzyme sample was incubated with Co2+
for 1 h, ANS was added to each sample. The final enzyme concen-
tration was 2.0 M. All reactions and measurements were carried
out in 50 mM MES buffer (pH 6.5) at 25 ◦C.
3. Results
We found that ␣-glucosidase activity was inhibited in
a
dose-dependent manner following incubation with varying con-
centrations of Co2+ (Fig. 1). The enzyme activity was completely
inactivated at a concentration of 5.0 mM Co2+, and the IC50
(inhibitor concentration leading to a 50% loss of activity) of Co2+
was measured for ␣-glucosidase as 0.345 0.01 mM (n = 3). This
inhibitor for ␣-glucosidase.
In a subsequent experiment, we investigated the reversibility of
Co2+-induced inhibition by evaluating the plots of v (␣-glucosidase
activity) vs. various enzyme concentrations in the presence of dif-
ferent Co2+ concentrations (Fig. 2). The results revealed that all
straight lines passed through the origin of point zero; these data
indicate that the inhibition by Co2+ is reversible.
2.4. Computational simulations
Since the structure of ␣-glucosidase from yeast (NCBI accession:
NP 011808.1) has not been characterized by any experimenta-
tion, homology modeling was conducted using a Pseudo-Quadratic
Please cite this article in press as: Li X, et al. The inhibitory role of Co2+ on ␣-glucosidase: Inhibition kinetics and molecular dynamics