1270 Protein & Peptide Letters, 2010, 17, 1270-1279

Inhibition Kinetics of Flavonoids on Yeast
-Glucosidase Merged with

Docking Simulations

Heng Xu*

College of Life Science, Jiaying University, Guangdong, Meizhou 514015, P.R. China

Abstract: Flavonoids, also called vitamin P, are widely distributed in plants fulfilling many functions. Yeast
-
glucosidase (YAGH; EC 3.2.1.20), as extensively used target protein for screening bioactive compounds from medicine
plants, was selected to explore the possible mechanisms of multiple biological function of flavonoids. The results in this
study indicated that flavonoids, as mixed-type inhibitors, quenched the intrinsic fluorescence of YAGH by a mixed fluo-
rescence quenching mechanism. The interaction information between flavonoids and YAGH was analyzed using a flexi-
ble docking method (AutoDock) and showed that 3’, 4’ dihydroxyl groups of B ring and 3-OH of C ring played a more
important role in the inhibition activity than other hydroxyl groups, because the 3’, 4’ dihydroxyl groups of B ring directly
interacted with the active-site residues of YAGH to inhibit enzyme activity and 3-OH of C ring seemed to be necessary to
maintain the proper binding orientation of flavonoid molecules, thereby making the hydroxyl groups of B ring interact
with active-site residues tightly in the hydrophobic pocket of YAGH. The results supply a basis for understanding the
mechanisms of multiple biological functions of flavonoids.
Keywords: AutoDock, flavonoid, fluorescence quenching, inhibition kinetics.

1. INTRODUCTION The primary structure of flavonoids consists of two moie-

ties: benzopyran (A and C rings) and phenyl (B ring) groups
Yeast alpha-glucosidase (YAGH, EC 3.2.1.20) catalyzes
Fig. (1). The inhibitory effect of flavonoids on YAGH has
the hydrolysis of the
-glucosidic bond from the non-
been extensively studied, including quercetin, luteolin, rutin
reducing end of a chain, as well as the
-glucosidic bond of
and other twenty one naturally occurring flavonoids [6, 11,
free disaccharides [1]. The enzyme, which belongs to Glyco-
21]. These reports indicated that flavonoids showed different
side Hydrolases family 13, has common specific structural
inhibition activity to YAGH and the structural factor corre-
features such as the catalytic (/
)8-barrel domain, which sponding to the inhibition activity including the unsaturated
acts specifically on
-1,4- and
-1,6-O-glucosidic linkages
C ring, 3-OH, 4-CO, the linkage of the B ring at the 3 posi-
[2-5]. YAGH has been extensively used to identify its inhibi-
tion, and the hydroxyl substitution on the B ring [21]. The
tors from traditional medicine plants [6, 7] and food items
polyhydroxyl groups in the B ring seem to play an important
[8] and these inhibitors from plants and foods have shown
role in the inhibition of YAGH, but few reports described the
potential biological function, such as antihyperglycemic po-
mechanism at structural level.
tential [9], antioxidant ability [10], etc. It is also useful to
enrich the chemical library that is used in screening assays
with molecules that are likely to have biological activities by
selecting YAGH as the target protein [11, 12].
Flavonoids, also collectively known as Vitamin P [13],
are a class of plant secondary metabolites, which are widely
distributed in plants carrying out many functions. The wide-
spread distribution of flavonoids, their variety and their rela-
tively low toxicity compared to other active plant com-
pounds mean that many animals, including humans, ingest
significant quantities in their diet. They show anti-allergic
[14], anti-inflammatory [15, 16], anti-microbial [17] and
anti-cancer activity [18]. Consumers and food manufacturers Figure 1. Chemical structures of quercetin, luteolin, myricetin and
have become interested in flavonoids for their medicinal kaempferol.
properties, especially their potential role in the prevention of
cancers and cardiovascular disease [19]. The beneficial ef- To examine the role of polyhydroxyl groups of flavon-
fects of fruit, vegetables, and tea have been attributed to fla- oids in the inhibition to YAGH, quercetin, myricetin, kaemp-
vonoids compounds rather than to known nutrients and vita- ferol and luteolin were selected to explore the inhibition
mins [20]. mechanism by inhibition kinetics, fluorescence quenching at
*Address correspondence to this author at the College of Life Science, Jiay- experimental level and docking simulation at structural level.
ing University, Guangdong, Meizhou 514015, P.R. China; Tel: +86 753 The interaction information was explored to elucidate the
2985433; E-mail: xhx@jyu.edu.cn possible inhibition mechanism between flavonoids and

0929-8665/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.

Interaction Between Flavonoids and
-Glucosidase
YAGH by molecular docking. Quercetin, myricetin and lute-
olin were used to dock into the YAGH molecule, to evaluate
the docking quality and explore the role of polyhydroxyl
groups in the inhibition activity.
2. MATERIALS AND METHODS
2.1. Reagents
Yeast -glucosidase, p-Nitrophenyl - D-glucopyranoside
(pNPG, Mr 301.25), Quercetin, myricetin, kaempferol and
luteolin were purchased from Sigma-Aldrich (USA). All
other reagents used were local products of analytical grade.
2.2. Sample Preparation and Enzyme Activity Assay
Yeast -glucosidase was incubated in solutions contain-
ing different concentrations of flavonoids in 0.1 M PBS
buffer (pH 6.8) for 2 h at 298 K. Flavonoids were dissolved
in dimethyl sulfoxide (DMSO) and used for the enzyme in-
cubation by diluting with 0.1 M PBS buffer (pH 6.8). The
final concentration of DMSO in the activity assay system is
0.25%. The YAGH activity was determined at 310 K by
measuring the change of absorbance at 400 nm accompany-
ing the hydrolysis of pNPG to generate pNP according to
Tremblay [22]. The assay system contained 495 μL 0.1 M
PBS buffer (pH 6.8) containing 5 mM pNPG and 5 μL 1.8
μM enzyme solution. The reaction was ended by adding 2
volumes of 1 M Na2 CO3 solution. The enzyme activity was
measured at 400 nm on a Heios  UV-Visible spectropho-
tometer (ThermoSpectronic). A calibration curve was con-
structed to correlate the absorption change with the genera-
tion of pNP. One unit of enzyme activity was defined as the
amount of enzyme that liberated 1.0
mole of pNP from
pNPG per min at pH 6.8 at 310 K.
The extent of inhibition by the flavonoids was expressed
as the concentration at a 50 % inhibition (IC50). The inhibi-
tion type was determined by the Lineweaver-Burk plot, and
the inhibition constant was determined by the secondary plot.
For the analysis of the mixed-type inhibition, the Line-
weaver-Burk equation in double reciprocal form can be writ-
ten as [23]:
1 Ks [I ] 1 1 [I ]
= [1 + ] + [1 + ]
í Vm K i [S] Vm
Ki (1)
Secondary plot can be constructed from the Eq. 1
Ks K The fluorescence quenching of proteins can also be de-
Slope = + s [I ]
Vm Vm K i
(2)
1 [I ]
Y -intercept = + fluorophores have identical absorptivities. The protein quan-
Vm Vm
K i (3)
where Ks is the dissociation constant, Vm is the maximum
reaction velocity, [S] is the substrate concentration, [I] is the
concentration of flavonoids, Ki is the inhibition constant for
enzyme and inhibitor, and Ki represents the inhibition con-
stant for enzyme-substrate complex and inhibitor. Ki and Ki
values can be yielded from the equations above. All the ki-
netic experiments were conducted at least three independent
experiments with three repetitions.
Protein & Peptide Letters, 2010, Vol. 17, No. 10 1271
2.3. Determination of the Binding Information by Fluo-
rescence Quenching
The enzyme samples were treated by different concentra-
tions of flavonoids (quercetin, myricetin, kaempferol and
luteolin) dissolved in 0.1 M PBS (pH 6.8) buffer for 2 h at
298 K. Then the intrinsic fluorescence spectra of these sam-
ples were measured with an excitation wavelength of 280 nm
and the emission spectra were over the range of 300-500 nm.
The data were recorded with an F-2500 fluorescence spec-
trophotometer with using a 1 cm path-length quartz cuvette.
There are two possible reasons for the fluorescence
quenching of YAGH, the static quenching and dynamic
quenching. The dynamic quenching results from encounters
of quencher and fluorophore during the excited state, and the
static quenching is due to the quenching by formation of a
complex between quencher and fluorophore predating the
excitation. The simple Stern-Volmer plot for fluorescence
quenching can be obtained with either dynamic quenching or
static quenching [24].
F0
= 1+ K Q
0 [Q] = 1+ K sv [Q]
F (4)
where F0 and F are the relative steady-state fluorescence
intensities in the absence and presence of quencher, respec-
tively. [Q] is the quencher concentration, KQ is the quench-
ing rate constant for a biomolecular reaction, Ksv is the Stern-
Volmer quenching constant and
0 is the average lifetime of
fluorophore in the absence of quencher.
In many instances, the fluorophore can be quenched both
by collision and by complex formation with the same
quencher (mixed fluorescence quenching). In this case, the
Stern-Volmer plot shows an upward curvature, concave to-
wards the y-axis at high [Q], and F0/F is related to [Q] by the
following form of the Stern-Volmer equation [25]:
F0
= (1 + K D [Q])(1 + KS [Q])
F (5)
where KD and KS are the dynamic and static quenching con-
stants, respectively. The first factor on the left-hand side
describes the dynamic quenching and the second factor de-
scribes the static quenching. This modified form of the
Stern-Volmer equation is second order with respect to [Q],
which accounts for the upward curvature observed at high
[Q] when both static and dynamic quenching occur for the
same fluorophore.
scribed by a modification of the Stern-Volmer equation [26],
in which it is assumed that energy transfer between accessi-
ble and inaccessible fluorophores is negligible and that all
tum yields in the presence and absence of quencher (F and
F0, respectively) are defined as:
1
F0 =
F0i
n i=1 (6)
1 1 F0i
F =
Fi =

n i =1 n i =1 1 + K Qi [Q]
(7)
where n is the number of fluorophores of a given class, F0i
and KQi are the quantum yield and quenching constant for the
1272 Protein & Peptide Letters, 2010, Vol. 17, No. 10
ith fluorophore. Assuming there are m accessible and n-m
inaccessible fluorophores (KQ = 0), then
F0 1 1
= +
F0
F ( f a )eff ( K Q )eff [Q] ( f a )eff
where (fa)eff is effective fractional maximum accessible fluo-
rescence and (KQ)eff is effective quenching constant. For low
quencher concentrations, a plot of F0/
F vs. 1/[Q] is linear
and values of (KQ)eff = intercept/slope were calculated sepa-
rately at 286 K and 291 K. In all cases straight lines were
obtained when F0/
F was plotted versus 1/[Q] according to
the modified Stern-Volmer plot above [26].
The apparent binding constant K and binding sites n can
be calculated from the following equation [27]:
F0
F 1 genetic algorithm in AutoDock4.0 was applied to search the
log( ) = n log K
nlog(
F [Q t ]
( F0
F )[Pt ] / F0
where K is the binding constant and n is the number of bind-
ing sites, [Qt] and [Pt] are the total quencher concentration
and the total protein concentration, respectively. For the fla-
vonoids-YAGH system, the values for K and n can be de-
rived from the intercept and slope of plots of log (F0-F)/F
versus log (1/([Qt]-(F0-F)[Pt]/F0)) based on Eqn. (9).
2.4. Molecular Modeling
2.4.1. Preparation of the Compound and Macromolecule
The structures of quercetin and myricetin were obtained
from NCBI website Fig. (1). The compounds were converted
to 3D structure using ChemBio3D ultra 11.0 software [Cam-
bridge Soft Corporation, USA (2007)] and energy minimiza-
tion was performed using MM2 with 10000 iterations and
minimum RMS gradient of 0.10. As the structures of quer-
cetin, myricetin and luteolin were rather rigid, six, seven and
five rotatable bonds were defined in the multi-conformation
docking, respectively.
Because the structural information for -glucosidase
from Saccharomyces cerevisiae is still unavailable, we car-
ried out the homology modeling of YAGH in Swiss-Model
by using the software Swiss-PdbViewer 4.0.1 [28] to obtain
its three-dimentional structure. This homology modeling
started with the retrieval of the amino acid sequence of -
glucosidase MAL12 from baker’s yeast that comprises 584
amino acid residues from the SWISS-PROT protein se-
quence data bank (Accession No. P53341) [29, 30]. We
searched the Protein Data Bank (PDB) using BLAST with
the amino acid sequence as input and the results showed
oligo-1,6-glucosidase from Bacillus cereus revealed the hig-
hest sequence identity (38%) with YAGH. Therefore, its X-
ray crystal structure (PDB ID: 1UOK) [31] was selected as
the template for homology modeling. The constructed PDB
file AGS.pdb was used in molecular docking experiment.
The Root Mean Square Deviation (RMSD) between the
AGS.pdb and the template structure was calculated by using
TopMatch-web [32, 33] to evaluate the quality of the mod-
eled structure AGS.pdb.
2.4.2. Molecular Docking
AutoDock 4.0 [34] was used for the docking study com-
bined with the Lamarckian genetic algorithm (LGA) to
Heng Xu
search for the globally optimized conformation. The LGA
was applied to model the binding between flavonoids (quer-
cetin, myricetin and luteolin) and YAGH and describes the
relationship between the ligands and the macromolecule by
the translation, orientation, and conformation of the ligands.
(8)
The AGS.pdb file was checked for polar hydrogens and
merged non-polar hydrogens. At the same time, the number
of torsional bonds of the ligands was defined with the Auto-
DockTools program so that they could be explored during
molecular docking. Then the 3D grid was created by the
Autogrid Algorithm to generate the grid parameter file. The
grid spacing was 0.0375 nm in each dimension, and each
grid map consisted of a 100106100 grid point. For every
snapshot of protein, the center of the grid was set to the posi-
tion in the gridcenter 56.768 36.235 11.928. The Lamarckian
) conformational and orientational space of the quercetin,
(9) myricetin and luteolin. The global optimization was started
with a population of 150 randomly positioned individuals,
with a maximum of 2.5106 energy evaluations and a maxi-
mum of 2.7104 generations. During each docking experi-
ment, 50 runs were carried out. Docking solutions with a
ligand all-atom root mean square deviation (RMSD) within
2.0 nm of each other were clustered together and ranked by
the lowest docking energy. The lowest energy conformation
of all docking results was selected for further analysis.
3. RESULTS AND DISCUSSION
3.1. Inhibitory Effects of Flavonoids on the Activity of
YAGH
The inhibitory effect of flavonoids on the activity of
YAGH was investigated. 0.25% DMSO in activity assay
system has no effect on the activity of -glucosidase. Fig. (2)
showed that the inhibition of YAGH by quercetin was con-
centration-dependent. In addition, the concentration leading
to 50% activity lost (IC50) of the quercetin was determined to
be 7.94 μM according to the inset of Fig. (2).
Figure 2. Effects of quercetin on the activity of -glucosidase at
310 K. The final concentration of enzyme was 1.8 μM. The concen-
trations of quercetin were 0, 1, 2.5, 5, 7.5, 10, 15, 20, 25 μM respec-
tively. To determine IC50 value precisely, this figure was replotted
(inset) using a Log10 scale for inhibitor concentration to get a
straight line.
Interaction Between Flavonoids and
-Glucosidase
The Lineweaver-Burk plot for quercetin was constructed
Fig. (3A). The results showed that quercetin was a mixed-
type inhibitor, since increasing quercetin concentrations re-
sulted in series of lines with different slopes and intersected
in the second quadrant. This is well matched to the model of
Scheme 1. Quercetin can directly interact with YAGH and
YAGH-pNPG complex by weak bonds at, near, or inside the
active site, which inactivates the enzyme but does not affect
the binding of substrate pNPG.
Scheme 1. A reaction scheme.
The inhibition constants for quercetin binding with
YAGH were evaluated from the double reciprocal plot of
slope and intercept versus the concentrations of quercetin
Fig. (3B and C) according to the Eqns. (1) to (3).
The inhibition kinetic parameters of other flavonoids
(myricetin, kaempferol and luteolin) were also calculated
using the same method and listed in Table 1. Compared with
other flavonoids, quercetin and myricetin showed higher
inhibition activity. The difference of inhibition activity
showed that the hydroxyl substitution of B ring and 3-OH of
C ring were important to the inhibition activity.
Figure 3. Inhibition kinetics of quercetin on the activity of
-glucosidase. (A) Lineweaver-Burk plots for inhibition of quercetin on
-
glucosidase at 310 K, pH 6.8. The concentrations of the quercetin for curves 1-5 were 0, 1.0, 2.5, 5.0, 7.5 μM, respectively. (B) The secon-
dary plot of slope versus concentrations of quercetin to determine the inhibition constant Ki. (C) The secondary plot of Y-intercept versus
concentrations of quercetin to determine the inhibition constant
Ki.
Protein & Peptide Letters, 2010, Vol. 17, No. 10 1273
3.2. Determination of the Binding Information by Fluo-
rescence Quenching
To elucidate the binding between YAGH and flavonoids,
fluorescence quenching technique was used at different tem-
peratures. Fluorescence spectra were obtained by keeping the
YAGH concentration constant while varying the concentra-
tion of flavonoids. Fig. (4) showed that the fluorescence in-
tensity of YAGH at 334 nm gradually dropped with increas-
ing concentrations of the quercetin, while the peak shapes
did not change. The results indicated that quercetin was a
fluorescence quencher of YAGH, not only an inhibitor.
Myricetin, kaempferol and luteolin also showed the fluores-
cence quencher characteristic.
However, the fluorescence quenching of the enzyme did
not seem to follow the simple Stern-Volmer relationship as
judged by the nonlinear plot of Fig. (5A). The Stern-Volmer
plot showed an upward curvature, concave towards the y-
axis and F0/F is related to [Q] following the form of the Eqn.
(4) [25]. The results suggested that the fluorescence quench-
ing of YAGH by quercetin might consist of dynamic and
static quenching. Plotting the data according to the modified
relationship Eqn. (8) resulted in a linear dependence as seen
in Fig. (5B). The results showed that the values of KQ (slope)
decreased with increasing temperatures, which indicated that
the fluorescence quenching mechanism of YAGH by quer-
cetin also appeared to be a static quenching process, because
dynamic quenching results from the diffusion of molecules
and diffusion rate would increase with increasing tempera-
tures. Therefore, all the results indicated that the fluores-
cence quenching process of YAGH existed two kind of que-
nching mechanism (static quenching and dynamic quench-
ing) but the process was mainly driven by a static quenching
mechanism. That meant quercetin and YAGH existed in a
complex form in the quenching system mainly. Myricetin,
kaempferol and luteolin were also the complicated quench-
ing mechanism. In order to explore the complex information
further, quercetin-YAGH system was selected to study in
depth.
1274 Protein & Peptide Letters, 2010, Vol. 17, No. 10 Heng Xu

Table 1. Inhibitory Activity of Flavonoids Against Yeast
-Glucosidase

Flavonoid IC50 (μM) Ki (μM)
Ki (μM) Type of inhibition

Quercetin 7.94, 7a, 8b 4.12, 3.4a 9.15, 14a Mixed type

a b a a
Myricetin 5.15, 5 , 4 3.21, 3.0 8.31, 7.6 Mixed type

Kaempferol 19.1, 20b 23.3 35.4 Mixed type

Luteolin 18.6, 21a, 21.2, 29a 33.5, 38 a Mixed type

The enzyme activity was estimated by measuring p-nitrophenol liberated. The reaction conditions were described in “Materials and methods”. Ki and Ki values were determined
from the slope and intercept on the vertical axis in double reciprocal plots. All values are averages of three independent experiments.
a
Tadera et al. [21]
b
Iio et al. [35]

and binding sites n were calculated for quercetin associated

Figure 4. Fluorescence quenching spectra of YAGH by quercetin at
286 K. From the top to the bottom, the concentrations of quercetin
were 0, 1, 2, 3, 4, 5, 6, 7 and 8
M, respectively. The dashed line at
the bottom is the fluorescence spectra of quercetin.
The plot of log (F0-F)/F versus log(1/([Qt]-(F0-F)[Pt]
/F0)) for the quercetin-YAGH system at 286 K and 291 K
was obtained Fig. (6), from which the binding constants K
with YAGH (Table 2). The results showed that the binding
constants K decreased with the temperatures, which indi-
cated the formation of an unstable complex [36]. The stabil-
ity of quercetin-YAGH complex and the n value decreased
with the increasing temperatures, which were below the
physiological temperature, so the system is more instable and
complicated than the rutin-AK system which obtained the
values of K and n successfully using the same method [37]
and the calculation of binding constant K and binding sites n
were uncertain using the fluorescence quenching technology.
At low temperature, there may be one binding site between
YAGH and quercetin. But the value increased with the in-
creasing temperature, which also showed the complex was
unstable and unpredictable.
There are 20 tryptophan residues in YAGH, and most of
them are located in the hydrophobic surface, which contrib-
ute a lot to the fluorescence intensity of YAGH. Effective
fluorescence quenching occurred, even at low concentrations
of quercetin which did not cause the visible changes in
YAGH activity and conformation Fig. (2 and. 4). According
to the difference of quenching mechanism, the fluorescence
quenching was most likely dynamic quenching at low con-
centration of quercetin. With increasing concentrations of
quercetin, the compound inserts to the hydrophobic pocket
and binds to the active sites to affect the activity and form

Figure 5. Stern-Volmer plot for the fluorescence quenching of YAGH by quercetin under different temperatures. The experiment was per-
formed at pH 6.8, and the excitation wavelength was 280 nm. The data were taken from Figure 4. (A) Simple Stern-Volmer plot of the
quenching of YAGH fluorescence by quercetin at () 286K and () 291 K, respectively. (B) Modified Stern-Volmer plot of the quenching
of YAGH fluorescence by quercetin.
Interaction Between Flavonoids and
-Glucosidase
non-fluorescence complex (static quenching mechanism).
The binding analysis between quercetin and YAGH also
supplies a possible binding mode between flavonoids and
YAGH. But the detailed structural analysis needs to be dis-
cussed by the methods of molecular docking.
Figure 6. Plots of log(F0-F)/F versus log(1/([Qt]-(F0-F)[Pt]/F0)) for
the Quercetin-YAGH system at pH 6.8 under different tempera-
tures: (), 286 K; (), 291 K.
Table 2. Binding Constant K and Binding Sites n of The Sys-
tem of Quercetin-YAGH
T(K) K(
104 M-1) n R2 a
286 2.21 1.384 0.9976
291 2.12 1.525 0.9963
All values are averages of three independent experiments.
a
Coefficient of determination
3.3. Molecular Docking Study
Molecular docking studies can provide useful informa-
tion for in-depth understanding some subtle action mecha-
nisms at the molecular level [38, 39], such as the marvelous
allosteric mechanism revealed recently by the NMR observa-
tions on the M2 proton channel of influenza A virus [40, 41].
They can also provide useful insights to screen potential drug
candidates and accelerate drug developments [42-51].
Therefore, this computer-assisted method was employed
to improve our understanding of the interaction between fla-
vonoids and YAGH. The sequence alignment between YA-
GH and oligo-1,6-glucosidase from Bacillus cereus (BOGH)
was shown in Fig. (7). According to this alignment, the se-
quence identity and the similarity amount to 38% and 59%,
respectively. Judging from such a high sequence homology,
a high-quality 3D structure of YAGH can be expected in the
homology modeling. It is indeed well known that a homol-
ogy-modeled structure of a target protein can be accurate
enough to be used in docking studies once the sequence
identity between target and template approaches 40% [52].
Protein & Peptide Letters, 2010, Vol. 17, No. 10 1275
The predicted high quality PDB file AGS.pdb (RMSD:
0.5Å.) was obtained. The active site of YAGH (Asp214,
Glu276, Asp349) [30, 53] was located in the hydrophobic
pocket Fig. (8A).
The information of hydrophobic pocket or binding pocket
of a protein receptor to its ligand is very important for drug
design, particularly for conducting mutagenesis studies to
find key active-site residues [42]. The binding pocket is usu-
ally defined by those residues that have at least one heavy
atom, i.e., an atom other than hydrogen, with a distance
5Å
from a heavy atom of the ligand. Such a criterion was origi-
nally used to define the binding pocket of ATP in the Cdk5-
Nck5a* complex [55], which had later proved quite useful in
identifying functional domains and stimulating the relevant
truncation experiments [56]. The similar approach has also
been used to define the binding pockets of many other recep-
tor-ligand interactions which are important for drug design
[38, 43, 47, 48, 57-61].
The AutoDock 4.0 program was used to calculate the
possible conformation of the ligand that binds to the protein
and the hydrophobic pocket was found. As shown in Fig.
(8A) quercetin, myricetin and luteolin are located within the
hydrophobic pocket of the enzyme. The hydrophobic pocket
defined consists of 14 residues which are Phe157, Phe158,
Phe 177, Asp214, Thr215, Glu276, Ala278, Phe300, Glu304,
Phe311, Arg312, Asp349, Asp408, and Arg439. It would be
an effective way to find out which of the 14 residues are the
most sensitive for the catalytic activity by site-directed
mutagenisis. The opening of the hydrophobic pocket is rela-
tively small and this may be an important factor that influ-
ences the binding between flavonoids and YAGH active
sites. The interactions between flavonoids (quercetin, myri-
cetin and luteolin) and YAGH were shown in detail Fig. (8B,
C and D) and the major interaction was involved the -1 and
+1 binding subsites [62]. Quercetin bound to the YAGH by
hydrogen bonds with Asp214, Glu276 and Glu304. Myri-
cetin, which has one more 5-OH, interacted with Asp214,
Glu276, Glu304, Asp408 and Arg439 of YAGH. The two
docking results were almost identical and it also indicated
the docking results were relatively credible. However, the
orientation of luteolin in the hydrophobic pocket showed a
different situation compared with quercetin and myricetin,
interacting with Asp214, Glu304, Arg312, Asp349 and
Arg349 by hydrogen bonds. The B ring of luteolin almost
overlapped with the B rings of quercetin and myricetin while
the plane of A and C rings rotates due to the lack of 3-OH of
C ring, leading to the different binding mode between fla-
vonoids and enzyme active sites to cause decreasing inhibi-
tory activity obviously. This docking result suggested that 3-
OH of C ring seemed to play a pivotal role in maintaining
the effective binding position of flavonoids which can inhibit
YAGH activity effectively.
In the active-site pocket, the flavonoids inserted into the
catalytic domain pocket and occupied -1 and +1 sugar sub-
sites. The active site residues Asp214 and Glu276 are impor-
tant to enzyme activity. Asp214 acts as the nucleophile in the
catalytic reaction and Glu276 acts as a proton donor [53].
The 3’, 4’-dihydroxyl groups interact with Glu276 and
Asp214 by hydrogen bonds respectively, so the two hy-
droxyl groups are important factors of enzyme activity inhi-
1276 Protein & Peptide Letters, 2010, Vol. 17, No. 10
Figure 7. Sequence alignment between yeast -glucosidase (YAGH) and oligo-1,6-glucosidase (BOGH). '*' indicates positions which have a
single, fully conserved residue. ':' indicates that one of strong groups (strong score > 0.5) is fully conserved. '.' indicates that one of weaker
groups (weak score
0.5) is fully conserved [54].
bition. The difference of inhibition effect during myricetin,
quercetin and kaempferol also confirm the results (Table 1).
The inhibitory activity of luteolin was almost identical to
quercetin and myricetin [21], but the IC50 values and inhibi-
tion constants of luteolin was bigger than the values of quer-
cetin and myri-cetin. Due to the lack of 3-OH of C ring, the
conformation of luteolin changed and 3’, 4’-dihydroxyl
groups did not interact with Asp214 and Glu276 like quer-
cetin and myricetin, which may be the main reason for sud-
den decrease in inhibitory activity. The results proved that
3’, 4’-dihydroxyl grou-ps are the main factors of enzyme
inhibition activity again and showed the existence of 3-OH is
necessary to maintain the “correct” binding orientation of
flavonoids in the binding pocket of YAGH and high inhibi-
tory activity.
The inhibitory power of flavonoids to YAGH has been
extensively investigated [6, 11, 21], which found that the
hydroxyl groups of the B-ring played an important role in the
activation inhibition of YAGH, but it lacked evidence and
further explanation at structural level. As revealed by the
molecular docking experiment in this article, the possible
reasons could be: (i) the oxygen of the three hydroxyl groups
of B ring can form hydrogen bonds with Asp214, Glu276
and Arg439 respectively Fig. (8B, C) and inhibit the YAGH
activity by binding to its active sites. According to the differ-
ent inhibitory activity of different flavonoids, the different
role of the hydroxyl groups was shown. 3’-OH and 4’-OH
were the most important to the inhibitory activity and the
Heng Xu
role of the other hydroxyl groups was relatively little. (ii)
The opening of hydrophobic pocket of YAGH was relative
narrow, and the free rotary B-ring of flavonoids is smaller
than the A-ring and C-ring, which made the B-ring insert to
the hydrophobic pocket more easily and bond the active sites
more smoothly. As expected, the molecular docking results
suggested that the B-ring of flavonoids actually interacts
with the active sites, which might be the principal mecha-
nism of inhibition. Furthermore, the role of 3-OH was shown
to direct the binding between enzyme and flavonoids. Ana-
lyzing from the docking result, 3-OH maybe can orientate
the flavonoids and form static electric attraction or hydrogen
bond with Asp408 to make the flavonoids molecules bind to
the hydrophobic pocket properly and tightly.
4. CONCLUSIONS
The inhibitory effect of flavonoids on YAGH was inves-
tigated by fluorescence spectroscopy and molecular docking.
This study showed that the quercetin, myricetin, kaempferol
and luteolin are mixed-type inhibitors of YAGH, which is
identical to the result reported before [6, 21]. The intrinsic
fluorescence of YAGH was quenched through a combining
quenching mechanism including dynamic and static quench-
ing mechanism by flavonoids. In addition, the interaction
between flavonoids and YAGH was shown in the docking
results Fig. (8). The two hydroxyl groups in B-ring of quer-
cetin and myricetin were shown to play an important role in
the catalytic activity of enzyme, which well explained the
Interaction Between Flavonoids and
-Glucosidase Protein & Peptide Letters, 2010, Vol. 17, No. 10 1277

Figure 8. The results of docking simulation between YAGH and flavonoids. (A) Quercetin (diagonal), myricetin (dots) and luteolin (grid)
are in the hydrophobic pocket of YAGH. Their structures are represented using balls and sticks. (B) The interaction between quercetin and
YAGH. The hydrogen bonds are represented using dashed lines. The residues of YAGH are represented using sticks and the structure of
quercetin is represented using balls and sticks. (C) The interaction between myricetin and YAGH. The display parameters are the same as B.
(D) The interaction between luteolin and YAGH. The display parameters are also the same as B.

important role of the two hydroxyl groups of flavonoids on pNP = P-Nitrophenol

the activity of YAGH. Moreover, 3-OH of C ring seems to
have the ability to make flavonoid molecules bind into the
binding pocket of YAGH properly to maintain high inhibi-
tory activity. The other hydroxyl groups also affect the inhi-
bition power of flavonoids, but their influence is relatively
limited. These results show that the B ring of flavonoids
plays a more important role than A and C ring in the inhibi-
tion of YAGH. The flexibly and relatively small volume of B
ring may be the main factors.
Accurate measurements of binding properties between
flavonoids and key enzymes and knowledge of its structural
factors are important to understand the common mechanism
of multiple biological functions of flavonoids. That provides
a basis for systematically predicting and artificially synthe-
sizing effective drugs.
ABBREVIATIONS USED
YAGH = Yeast alpha-glucosidase
DMSO = Dimethyl sulfoxide
pNPG = P-Nitrophenyl
-D-glucopyranoside
REFERENCES
[1] Noguchi, A.; Yano, M.; Ohshima, Y.; Hemmi, H.; Inohara-Ochiai,
M.; Okada, M.; Min, K. S.; Nakayama, T.; Nishino, T. Deciphering
themolecular basis of the broad substrate specificity of alpha-
glucosidase from Bacillus sp SAM1606. J. Biochem., 2003, 134,
543-550.
[2] Henrissat, B. A classification of glycosyl hydrolases based on
amino acid sequence similarities. Biochem. J., 1991, 280, 309-316.
[3] Jespersen, H. M.; MacGregor, E. A.; Sierks, M. R.; Svensson, B.
Comparison of the domain-level organization of starch hydrolases
and related enzymes. Biochem. J., 1991, 280, 51-55.
[4] Jespersen, H. M.; MacGregor, E. A.; Henrissat, B.; Sierkes, M. R.;
Svensson, B. Starch and glycogen-debranching and branching en-
zymes: Prediction of structural features of the catalytic (/
)8-
barrel domain and evolutionary relationship to other amylolytic en-
zymes. J. Protein Chem., 1993, 12, 791-805.
[5] Svensson, B. Protein engineering in the
-amylase family: catalytic
mechanism, substrate specificity, and stability. Plant Mol. Biol.,
1994, 25, 141-157.
[6] Watanabe, J.; Kawabata, J.; Kurihara, H.; Niki, R. Isolation and
identification of alpha-glucosidase inhibitors from Tochu-cha (Eu-
commia ulmoides). Biosci., Biotechnol., Biochem., 1997, 61(1),
177-178.
1278 Protein & Peptide Letters, 2010, Vol. 17, No. 10
[7] Suresh Babu, K.; Tiwari, A. K.; Srinivas, P. V.; Ali, A. Z.; China
Raju, B.; Rao, J. M. Yeast and mammalian
-glucosidase inhibitory
constituents from Himalayan rhubarb Rheum emodi Wall. ex Meis-
son. Bioorg. Med. Chem. Lett., 2004, 14, 3841-3845.
[8] Matsui, T.; Yoshimoto, C.; Osajima, K.; Oki, T.; Osajima, Y. In
vitro survey of
-glucosidase inhibitory food components. Biosci.,
Biotechnol., Biochem., 1996, 60(12), 2019-2022.
[9] Subramanian, R.; Asmawi, M. Z.; Sadikun, A. In vitro alpha gluco-
sidase and alpha amylase enzyme inhibitory effects of Andro-
graphis paniculata extract and andrographolide. Acta Biochim. Pol.,
2008, 55, 391-398.
[10] Chen, H. M.; Zheng, L.; Yan, X. J. The preparation and bioactivity
research of agaro-oligosaccharides. Food Technol. Biotechnol.,
2005, 43(1), 29-36.
[11] Kim, J. S.; Kwon, C. S.; Son, K. H. Inhibition of Alpha-glucosidase
and Amylase by Luteolin, a Flavonoid. Biosci., Biotechnol., Bio-
chem., 2000, 64(11), 2458-2461.
[12] Park, H.; Hwang, K. Y.; Oh, K. H.; Kim, Y. H.; Lee, J. Y.; Kim, K.
Discovery of novel
-glucosidase inhibitors based on the virtual
screening with the homology-modeled protein structure. Bioorg.
Med. Chem., 2008, 16(1), 284-292.
[13] Rusznyak, S.; Szent-Györgyi, A. Vitamin P: flavonols as vitamins.
Nature, 1936, 138, 27-27.
[14] Cheong, H.; Ryu, S. Y.; Oak, M. H.; Cheon, S. H.; Yoo, G. S.;
Kim, K. M. Studies of structure activity relationship of flavonoids
for the anti-allergic actions. Arch. Pharmacal Res., 1998, 21(4),
478-480.
[15] Yamamoto, Y.; Gaynor, R. B. Therapeutic potential of inhibition of
the NF-B pathway in the treatment of inflammation and cancer. J.
Clin. Invest., 2001, 107(2), 135-142.
[16] Gábor, M. Anti-inflammatory and anti-allergic properties of fla-
vonoids. Prog. Clin. Biol. Res., 1986, 213, 471-480.
[17] Cushnie, T. P. T.; Lamb, A. J. Antimicrobial activity of flavonoids.
Int. J. Antimicrob. Agents, 2005, 26(5), 343-356.
[18] Birt, D. F.; Hendrich, S.; Wang, W. Dietary agents in cancer pre-
vention: flavonoids and isoflavonoids. Pharmacol. Ther., 2001,
90(2-3), 157-177.
[19] Yochum, L.; Kushi, L. H.; Meyer, K.; Folsom, A. R. Dietary fla-
vonoid intake and risk of cardiovascular disease in postmenopausal
women. Climacteric, 1999, 2(3), 237-238.
[20] Yao, L. H.; Jiang, Y. M.; Shi, J.; TomÁS-BarberÁN, F. A.; Datta,
N.; Singanusong, R.; Chen, S. S. Flavonoids in Food and Their
Health Benefits. Qual. Plant. - Plant Foods Hum. Nutr, 2004,
59(3), 113-122.
[21] Tadera, K.; Minami, Y.; Takamatsu, K.; Matsuoka, T. Inhibition of

-glucosidase and
-amylase by flavonoids. J. Nutr. Sci. Vitami-
nol., 2006, 52(2), 149-153.
[22] Chapdelaine, P.; Tremblay, R. R.; Dube, J. Y. p-Nitrophenol-alpha-
D-glucopyranoside as substrate for measurement of maltase activ-
ity in human semen. Clin. Chem. (Washington, DC, U. S.), 1978,
24(2), 208-211.
[23] Wu, X. Q.; Wang, J.; Lü, Z. R.; Tang, H. M.; Park, D.; Oh, S. H.;
Bhak, J.; Shi, L.; Park, Y. D.; Zou, F. Alpha-Glucosidase Folding
During Urea Denaturation: Enzyme Kinetics and Computational
Prediction. Appl. Biochem. Biotechnol., 2009, 160(5), 1341-1355.
[24] Lakowicz, J. R. Principles of Fluorescence Spectroscopy. Plenum
Press: New York, 1983.
[25] Sharma, A.; Schulman, S. G. Introduction to Fluorescence Spec-
troscopy. Wiley-Interscience: New York, 1999.
[26] Lehrer, S. S. Solute perturbation of protein fluorescence. The
quenching of the tryptophyl fluorescence of model compounds and
of lysozyme by iodide ion. Biochemistry, 1971, 10(17), 3254-3263.
[27] Bi, S.; Song, D.; Tian, Y.; Zhou, X.; Liu, Z.; Zhang, H. Molecular
spectroscopic study on the interaction of tetracyclines with serum
albumins. Spectrochim. Acta, Part A, 2005, 61(4), 629-636.
[28] Guex, N.; Peitsch, M. C. SWISS-MODEL and the Swiss-
PdbViewer: an environment for comparative protein modeling.
Electrophoresis, 1997, 18(15), 2714-2723.
[29] Bairoch, A.; Apweiler, R. The SWISS-PROT protein sequence data
bank and its supplement TrEMBL in 1999. Nucleic Acids Res.,
1999, 27(1), 49-54.
[30] Ghaemmaghami, S.; Huh, W. K.; Bower, K.; Howson, R. W.;
Belle, A.; Dephoure, N.; O'Shea, E. K.; Weissman, J. S. Global
analysis of protein expression in yeast. Nature, 2003, 425(6959),
737-741.
Heng Xu
[31] Watanabe, K.; Hata, Y.; Kizaki, H.; Katsube, Y.; Suzuki, Y. The
refined crystal structure of Bacillus cereus oligo-1, 6-glucosidase at
2.0 Å resolution: structural characterization of proline-substitution
sites for protein thermostabilization. J. Mol. Biol., 1997, 269(1),
142-153.
[32] Ware, W. R. Oxygen quenching of fluorescence in solution: an
experimental study of the diffusion process. J. Phys. Chem., 1962,
66 (3), 455-458.
[33] Tian, J.; Liu, J.; He, W.; Hu, Z.; Yao, X.; Chen, X. Probing the
binding of scutellarin to human serum albumin by circular dichro-
ism, fluorescence spectroscopy, FTIR, and molecular modeling
method. Biomacromolecules, 2004, 5(5), 1956-1961.
[34] Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W.
E.; Belew, R. K.; Olson, A. J. Automated docking using a La-
marckian genetic algorithm and an empirical binding free energy
function. J. Comput. Chem., 1998, 19(14), 1639-1662.
[35] Iio, M.; Yoshioka, A.; Imayoshi, Y.; Koriyama, C.; Moriyama, A.
Effect of flavonoids on
-glucosidase and -fructosidase from
yeast. Agric. Biol. Chem., 1984, 48, 1559-1563.
[36] Hu, Y.; Liu, Y.; Shen, X.; Fang, X.; Qu, S. Studies on the interac-
tion between 1-hexylcarbamoyl-5-fluorouracil and bovine serum
albumin. J. Mol. Struct., 2005, 738(1), 143-147.
[37] Wu, X. Q.; Zhu, W. J.; Lü, Z. R.; Xia, Y.; Yang, J. M.; Zou, F.;
Wang, X. Y. The effect of rutin on arginine kinase: Inhibition ki-
netics and thermodynamics merging with docking simulation. Int.
J. Biol. Macromol., 2009, 44(2), 149-155.
[38] Huang, R. B.; Du, Q. S.; Wang, C. H.; Chou, K. C. An in-depth
analysis of the biological functional studies based on the NMR M2
channel structure of influenza A virus. Biochem. Biophys. Res.
Commun., 2008, 377(4), 1243-1247.
[39] Du, Q. S.; Huang, R. B.; Wang, C. H.; Li, X. M.; Chou, K. C. En-
ergetic analysis of the two controversial drug binding sites of the
M2 proton channel in influenza A virus. J. Theor. Biol., 2009,
259(1), 159-164.
[40] Schnell, J. R.; Chou, J. J. Structure and mechanism of the M2 pro-
ton channel of influenza A virus. Nature, 2008, 451(7178), 591-
595.
[41] Pielak, R. M.; Schnell, J. R.; Chou, J. J. Mechanism of drug inhibi-
tion and drug resistance of influenza A M2 channel. Proc. Natl.
Acad. Sci. U. S. A., 2009, 106(18), 7379.
[42] Chou, K. C. Review: Structural bioinformatics and its impact to
biomedical science. Curr. Med. Chem., 2004, 11(16), 2105-2134.
[43] Chou, K. C. Molecular therapeutic target for type-2 diabetes. J.
Proteome Res., 2004, 3(6), 1284-1288.
[44] Wei, D. Q.; Wang, J. F.; Chen, C.; Li, Y.; Chou, K. C. Molecular
modeling of two CYP2C19 SNPs and its implications for personal-
ized drug design. Protein Pept. Lett., 2008, 15(1), 27-32.
[45] Du, Q. S.; Huang, R. B.; Wang, S. Q.; Chou, K. C. Designing In-
hibitors of M2 Proton Channel against H1N1 Swine Influenza Vi-
rus. PLoS One, 2010, 5(2), e9388.
[46] Wang, J. F.; Yan, J. Y.; Wei, D. Q.; Chou, K. C. Binding of
CYP2C9 with diverse drugs and its implications for metabolic
mechanism. Med. Chem., 2009, 5(3), 263-270.
[47] Chou, K. C.; Wei, D. Q.; Zhong, W. Z. Binding mechanism of
coronavirus main proteinase with ligands and its implication to
drug design against SARS. Biochem. Biophys. Res. Commun.,
2003, 308(1), 148-151.
[48] Chou, K. C.; Wei, D. Q.; Du, Q. S.; Sirois, S.; Zhong, W. Z. Re-
view: Progress in computational approach to drug development
against SARS. Curr. Med. Chem., 2006, 13(27), 3263-3270.
[49] Wei, D. Q.; Sirois, S.; Du, Q. S.; Arias, H. R.; Chou, K. C. Theo-
retical studies of Alzheimer’s disease drug candidate 3-[(2, 4-
dimethoxy) benzylidene]-anabaseine (GTS-21) and its derivatives.
Biochem. Biophys. Res. Commun., 2005, 338(2), 1059-1064.
[50] Zhang, R.; Wei, D. Q.; Du, Q. S.; Chou, K. C. Molecular modeling
studies of peptide drug candidates against SARS. Med. Chem.,
2006, 2(3), 309-314.
[51] Wang, J. F.; Chou, K. C. Insight into the molecular switch mecha-
nism of human Rab5a from molecular dynamics simulations. Bio-
chem. Biophys. Res. Commun., 2009, 390(3), 608-612.
[52] Baker, D.; Sali, A. Protein structure prediction and structural ge-
nomics. Science, 2001, 294(5540), 93-96.
[53] Kimura, A. Molecular Anatomy of
-Glucosidase. Trends Glyco-
sci. Glycotechnol., 2000, 12, 373-380.
[54] Thompson, J. D.; Gibson, T. J.; Plewniak, F.; Jeanmougin, F.;
Higgins, D. G. The CLUSTAL_X windows interface: flexible
Interaction Between Flavonoids and
-Glucosidase
strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res., 1997, 25(24), 4876-4882.
[55] Chou, K. C.; Watenpaugh, K. D.; Heinrikson, R. L. A model of the
complex between cyclin-dependent kinase 5 and the activation do-
main of neuronal Cdk5 activator. Biochem. Biophys. Res. Com-
mun., 1999, 259(2), 420-428.
[56] Zhang, J.; Luan, C. H.; Chou, K. C.; Johnson, G. V. W. Identifica-
tion of the N-terminal functional domains of Cdk5 by molecular
truncation and computer modeling. Proteins: Struct. Funct. Bioinf.,
2002, 48(3), 447-453.
[57] Chou, K. C. Insights from modelling the 3D structure of the ex-
tracellular domain of alpha 7 nicotinic acetylcholine receptor. Bio-
chem. Biophys. Res. Commun., 2004, 319(2), 433-438.
[58] Wei, D. Q.; Du, Q. S.; Sun, H.; Chou, K. C. Insights from modeling
the 3D structure of H5N1 influenza virus neuraminidase and its
Received: April 07, 2010 Revised: May 16, 2010 Accepted: May 17, 2010
Protein & Peptide Letters, 2010, Vol. 17, No. 10 1279
binding interactions with ligands. Biochem. Biophys. Res. Com-
mun., 2006, 344(3), 1048-1055.
[59] Wang, S. Q.; Du, Q. S.; Chou, K. C. Study of drug resistance of
chicken influenza A virus (H5N1) from homology-modeled 3D
structures of neuraminidases. Biochem. Biophys. Res. Commun.,
2007, 354(3), 634-640.
[60] Du, Q. S.; Sun, H.; Chou, K. C. Inhibitor Design for SARS
Coronavirus Main Protease Based on Distorted Key Theory. Med.
Chem., 2007, 3(1), 1-6.
[61] Housaindokht, M. R.; Bozorgmehr, M. R.; Bahrololoom, M.
Analysis of ligand binding to proteins using molecular dynamics
simulations. J. Theor. Biol., 2008, 254(2), 294-300.
[62] Chiba, S. Molecular mechanism in alpha-glucosidase and glu-
coamylase. Biosci., Biotechnol., Biochem., 1997, 61(8), 1233-1239.