Inhibition of mushroom tyrosinase by a newly synthesized ligand: Inhibition kinetics and computational simulations

Article abstract of DOI:10.1080/07391102.2012.682210

Alterations in the synthesis of melanin contribute to a number of diseases; therefore, the design of new tyrosinase inhibitors is very important. Mushroom tyrosinase (MT) is a metalloenzyme, which plays an important role in melanin biosynthesis. In this study, the inhibitory effect of a novel designed compound, i.e. 2-((1Z)-(2-(2,4-dinitrophenyl)hydrazin-1-ylidene)methyl) phenol, as a specific ligand which can bind to the copper ion of MT, has been assessed. The ligand was found to competitively inhibit both the cresolase and catecholase activities of MT, with small inhibition constants of 2.8 and 2.6 μM, respectively. Intrinsic fluorescence studies were performed to gain more information on the binding constants. Docking results indicated that the ligand binds to copper ions in the active site of MT via the OH group of the ligand. The ligand makes four hydrogen bonds with aspartic acid and one hydrogen bond with the histidine residue in the active site. Molecular dynamics results show that ligand binds to the MT via both electrostatic and hydrophobic interactions with its different parts. Copyright

Full text of DOI:10.1080/07391102.2012.682210

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Inhibition of mushroom tyrosinase by a newly
synthesized ligand: inhibition kinetics and
computational simulations
a
a
b
Mahdi Alijanianzadeh , Ali Akbar Saboury , Mohammad Reza Ganjali , Hamid Hadi-
a
a
Alijanvand & Ali Akbar Moosavi-Movahedi
a
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
b
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran,
Tehran, Iran
Version of record first published: 11 Jun 2012.
To cite this article: Mahdi Alijanianzadeh, Ali Akbar Saboury, Mohammad Reza Ganjali, Hamid Hadi-Alijanvand & Ali
Akbar Moosavi-Movahedi (2012): Inhibition of mushroom tyrosinase by a newly synthesized ligand: inhibition kinetics and
computational simulations, Journal of Biomolecular Structure and Dynamics, 30:4, 448-459
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Journal of Biomolecular Structure and Dynamics
Vol. 30, No. 4, 2012, 448–459
Inhibition of mushroom tyrosinase by a newly synthesized ligand: inhibition kinetics and
computational simulations
a
a
b
a
Mahdi Alijanianzadeh , Ali Akbar Saboury *, Mohammad Reza Ganjali , Hamid Hadi-Alijanvand and Ali Akbar
Moosavi-Movahedi
a
a
b
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; Center of Excellence in Electrochemistry, Faculty of
Chemistry, University of Tehran, Tehran, Iran
Communicated by Ramaswamy H. Sarma
(
Received 25 November 2011; final version received 2 March 2012)
Alterations in the synthesis of melanin contribute to a number of diseases; therefore, the design of new tyrosinase inhibitors
is very important. Mushroom tyrosinase (MT) is a metalloenzyme, which plays an important role in melanin biosynthesis. In
this study, the inhibitory effect of a novel designed compound, i.e. 2-((1Z)-(2-(2,4-dinitrophenyl)hydrazin-1-ylidene)methyl)
phenol, as a specific ligand which can bind to the copper ion of MT, has been assessed. The ligand was found to competi-
tively inhibit both the cresolase and catecholase activities of MT, with small inhibition constants of 2.8 and 2.6 μM, respec-
tively. Intrinsic fluorescence studies were performed to gain more information on the binding constants. Docking results
indicated that the ligand binds to copper ions in the active site of MT via the OH group of the ligand. The ligand makes four
hydrogen bonds with aspartic acid and one hydrogen bond with the histidine residue in the active site. Molecular dynamics
results show that ligand binds to the MT via both electrostatic and hydrophobic interactions with its different parts.
Keywords: mushroom tyrosinase; inhibition; cresolase; catecholase
Introduction
Mason (1948) elucidated the biosynthetic pathway of the
formation of melanin, and Cooksey et al. (1997) and
Schallreuter, Kothari, Chavan, & Spencer (2008) modi-
fied this pathway.
Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme
that is widely distributed in microorganisms, animals,
and plants (Robb, 1984; Whitaker, 1995; Zawistowski,
During melanogenesis, firstly tyrosinase oxidizes
tyrosine to dopaquinone, which is the rate-determining
step because the remainder of the reaction sequence can
spontaneously proceed at the physiological pH values
1
991). The enzyme catalyzes both the cresolase activity
(
(
monophenolase activity) that hydroxylates monophenol
L-tyrosine) to diphenol (L-Dopa) and the catecholase
activity (diphenolase activity) that oxidizes diphenol (L-
Dopa) to o-quinone (Sanchez-Ferrer, Rodriguez-Lopez,
Garcia-Canovas, & Garcia-Carmona, 1995). Tyrosinase
is responsible for skin pigmentation (Cho et al., 2009).
The enzymatic oxidation of L-tyrosine to melanin is
very important because melanin has many functions, and
alterations in the synthesis of melanin contribute to some
diseases (Prezioso, Epperly, Wang, & Bloomer, 1992).
(Halaban et al., 2002). Melanin protects human skin
from the harmful effects of UV from the sun (Artés &
Gil, 1998). Since hyperpigmentation in human skin is
not desirable, researchers are interested in finding new
potent tyrosinase inhibitors for the use in anti-browning
and skin whitening.
The active site of tyrosinase has a di-copper center
that is similar but not identical to hemocyanins (Eicken,
Krebs, & Sacchettini, 1999; Orlow et al., 1994). Each
copper ion in the active site is coordinated by three
nitrogen atoms from the three adjacent histidine residues
Finding new tyrosinase inhibitors with low K values is
i
very important, because the enzyme has a major role in
both mammalian melanogenesis and enzymatic browning
of fruits or fungi.
(
Cooksey, Garratt, Land, Ramsden, & Riley, 1998; Resc-
In melanogenesis, some pigments, like melanin, are
produced through the combination of enzymatically
catalyzed and chemical reactions. Raper (1928) and
igno, Sollai, Pisu, Rinaldi, & Sanjust, 2002). Mushroom
tyrosinase (MT) is the commercially available tyrosinase.
*
Corresponding author. Email: saboury@ut.ac.ir
ISSN 0739-1102 print/ISSN 1538-0254 online
Copyright Ó 2012 Taylor & Francis
http://dx.doi.org/10.1080/07391102.2012.682210
http://www.tandfonline.com

Inhibition of mushroom tyrosinase by a new synthesized ligand
449
The control of tyrosinase activity has been of special
interest among investigators due to its potential use in
medicinal, cosmetic, and agricultural products (Parvez,
Kang, Chung, & Bae, 2007). Therefore, understanding
the catalysis mechanism of tyrosinase and its regulation,
including its inhibition, is important. To understand the
mechanism of the enzymatic and inhibition reactions,
many studies have been performed to obtain additional
information about the function of MT (Gheibi, Saboury,
Haghbeen, & Moosavi-Movahedi, 2005; Haghbeen,
Saboury, & Karbassi, 2004; Karbassi, Saboury, Ranjbar,
2010; Ramalho, Rocha, da Cunha, Oliveira, & Carvalho,
2010; Tao, Rao, & Liu, 2010; Yang et al., 2010).
Due to our interest in designing an inhibitor for tyrosi-
+
2
nase, we expected that by blocking the Cu in the active
site, the enzyme activity would be inhibited. Recently, the
three-dimensional structure of MT has been determined
(Ismaya, Rozeboom, Schurink et al., 2011; Ismaya, Roze-
boom, Weijn et al., 2011). For gaining more insights into
the binding model, a docking study was also performed in
this study and the inhibitory effect of the newly synthe-
sized ligand was examined besides evaluating kinetics
analysis of it in both cresolase and catecholase activities.
&
Moosavi-Movahedi, 2003; Parvez et al., 2007;
Saboury et al., 2004; Shareefi Borojerdi, Haghbeen, Asg-
har Karkhane, Fazli, & Saboury, 2004).
Materials and methods
After introducing two new bi-pyridine synthetic com-
pounds as potent uncompetitive MT inhibitors (Saboury
et al., 2004), the inhibitory effects of three synthetic
n-alkyl dithiocarbamates (Gheibi, Saboury, Mansuri-Tors-
hizi, Haghbeen, & Moosavi-Movahedi, 2005), n-alkyl
xanthates (Alijanianzadeh, Saboury, Mansuri-Torshizi,
Haghbeen, & Moosavi-Movahedi, 2007; Saboury &
MT (MT; EC 1.14.18.1), specific activity 5340 units/mg,
L-Dopa, and L-tyrosine were purchased from Sigma Co.
(Madrid, Spain) 2-((1Z)-(2-(2,4-dinitrophenyl)hydrazin-1-
ylidene)methyl)phenol was synthesized as described
1
below (Figure 1). H NMR spectra were recorded using
a Brucker DRX-500 Avance Spectrophotometer at
500 MHz in DMSO-d and CDCl using TMS as internal
Alijanianzadeh, 2008; Saboury, Alijanianzadeh,
&
6
3
1
Mansoori-Torshizi, 2007), and benzyl and p-xylidine-
bisdithiocarbamate (Amin, Saboury, Mansuri-Torshizi, &
Moosavi-Movahedi, 2010) were elucidated. Also recently,
the inhibitory effects of isophthalic acid, dithioglycerine,
reference. H NMR data are expressed in parts per mil-
lion (ppm) and are reported as chemical shift position
(δH), multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, m = multiple), and assignment. A phosphate
buffer (10 mM, pH 6.8) was used throughout this
research and the corresponding salts were obtained from
Merck Co. All experiments were carried out at 20°C.
Ultrapure water was used throughout this research.
ꢀ
Cl , and thiobarbituric acid on MT have been studied
(
2
Han et al., 2007; Park, Lyou, Hahn, Hahn, & Yang,
006; Si et al., 2011; Yin et al., 2011). We hypothesized
that the ligand’s hydroxyl group may block MT activity
by binding to tyrosinase’s Cu atoms in the active site
based on the fact that previous findings had shown the
role of hydroxyl groups in tyrosinase inhibition (Arung,
Shimizu, & Kondo, 2006; Fu, Li, Wang, Lee, & Cui,
Synthesis of the ligand
2,4-Dinitro phenyl hydrazine (.01 mol), salicylaldehyde
(.01 mol), and a catalytic amount of toluene-4-sulfonic
2
005; Gao, Nishida, Saito, & Kawabata, 2007; Hyun,
Lee, Jeong da, Kim, & Choi, 2008; Jeong et al., 2009;
Kanade, Suhas, Chandra, & Gowda, 2007; Kim et al.,
2
006; Kim, Son, Chang, Kang, & Kim, 2003; Kubo
et al., 2000; Kubo & Kinst-Hori, 1999; Lee et al., 2002;
Shiino, Watanabe, & Umezawa, 2001, 2003; Shimizu,
Kondo, & Sakai, 2000; Shin et al., 1998; Yamazaki,
Kawano, Yamanaka, & Maruyama, 2009; Yan et al.,
2
009; Yokota, Nishio, Kubota, & Mizoguchi, 1998).
Currently, there is a great deal of interest in drug
design investigations involving ligand and protein inter-
actions and different theoretical methodologies have been
applied to the drug design in a variety of biological sys-
tems and there have been extensive publications in this
journal (Banappagari, Ronald, & Satyanarayanajois,
2
010; Cambria, Di Marino, Falconi, Garavaglia, &
Cambria, 2010; Chang et al., 2010; da Cunha, Barbosa,
Oliveira, & Ramalho, 2010; Huang et al., 2010; Kahlon,
Roy,
& Sharma, 2010; Nasiri, Bahrami, Zahedi,
Figure 1. The chemical structure of 2-((1Z)-(2-(2,4-
dinitrophenyl)hydrazin-1-ylidene)methyl)phenol as the newly
synthesized ligand.
Moosavi-Movahedi, & Sattarahmady, 2010; Nekrasov &
Zinchenko, 2010; Ompraba, Velmurugan, Louis, & Rafi,

4
50
M. Alijanianzadeh et al.
acid were refluxed in benzene (50 mL) for 5.5 h. Then
receptor. A blind docking was run with a pre-generated
ligand structure. The docking processes were performed
under shape and electrostatic correlation types in HEX
(Ritchie, 2003). A set of docking result was generated
by HEX and the complex with the lowest energy was
chosen as the best. The post-processing refinements were
Newton-like minimizations based on optimized potentials
for liquid simulations force field parameters. The final
structure was saved as a pdb file. To study the dynamics
of tyrosinase–ligand interaction, NAMD 2.8 engine was
used (Phillips et al., 2005). Generalized born implicit
solvent was used to reach an accurate and fast molecular
dynamics (MD). Constant temperature (300 K) MD was
performed by using langevin thermostat. Non-bonded
interaction cutoff sets to 13.5 A. Integration time step
sets to 2 fs. Before starting the production MD, protein–
ligand system was minimized for 2 ps and final produc-
tion run continued for 12 ns. To build the ligand
CHARMM force field, starting parameters were obtained
from ParamChem server before further routine justifica-
tions.
the solvent was evaporated to 20 mL to obtain the red
1
crystals of the product with a yield of 90%. H NMR
(
500 MHz, DMSO-d , ppm): δH = 5.53 (1H, s, OH),
6
7
.18–7.98 (9H, m, H ), 8.79 (1H, s, = CH), and 12.31
Ar
(
1H, s, NH).
Kinetic measurements
Catecholase and cresolase activities of MT were deter-
mined in the reaction medium for 1 and 2 min with
enzyme concentrations of 11.11 and 112.68 μg/ml by
spectrophotometrically measuring the rate of dopachrome
ꢀ1
ꢀ1
formation at 475 nm (ɛ = 3700 M cm ) in the first two
minutes, using a Cary spectrophotometer, 100 Biomodel,
with jacketed cell holders (Cabanes, Garcia-Canovas,
Lozano, & Garcia-Carmona, 1987). The assay was per-
formed with slight modifications as previously described
(
Chen & Kubo, 2002). Freshly dissolved enzyme, sub-
strate, and ligand were used in this work. All enzymatic
reactions were performed in a phosphate buffer (10 mM)
at pH 6.8 in a conventional thermostated quartz cell.
Substrate addition followed after incubation of the
enzyme with different concentrations of the ligand. The
initial rate was measured in each assay and all assays
were repeated three times.
Results
The effect of the ligand on both activities of MT was
examined at pH 6.8 and a temperature of 20°C. To assess
the reversibility of the ligand-mediated inhibition, plots
of the activity (catecholase activity) vs. the enzyme con-
centration ([E]) at different fixed concentrations of ligand
were used (Figure 2). The results showed straight lines
passing through the origin, indicating that the inhibition
by ligand was reversible.
Fluorescence quenching study
The fluorescence intensities were recorded using a Carry
Eclipse Spectrofluorophotometer (Varian, Australia) at an
excitation wavelength (λ ) of 290 nm. The maximum
ex
emission wavelength (λ ) for the MT was 340 nm. MT
em
(
1 μM) was chosen as the concentration for the fluores-
Kinetic parameters of cresolase activity of MT in the
presence of ligand
cence-quenching experiments. Series of the ligand
solutions with different concentrations of 2.7, 6, 7.5, 9.5,
and 14 μM were prepared in the phosphate buffer.
According to a previous report (Xie, Xu, & Wang,
The effect of the ligand on the cresolase activity of MT
was examined and it was found to inhibit the cresolase
2
005), when small molecules are bound to equivalent
sites on a macromolecule, the equilibrium between free
and bound molecules is given by Equation (1) to evaluate
the binding constant (K) and number of binding sites (n):
F
0
=(F
0
F) ¼ 1=n þ 1=K ꢁ 1=½Qꢂ
(1)
where F and F are the relative steady-state fluores-
0
cence intensities in the absence and presence of quencher,
respectively. [Q] is the quencher (ligand) concentration
and values for K and n can be derived from the intercept
and slope of a plot based on the above equation.
In silico docking of the tyrosinase structure and ligand
and molecular dynamics simulation
Figure 2. Plots of remaining activity, V (μM/min) (catecholase
activity) vs. (E) (μg/ml). The ligand concentrations are 3 μM
The minimized 3D structure of tyrosinase was the input
structure of HEX 5.1 protein docking software as the
(
r), 4 μM (N), 6 μM (d), and 7 μM (+). The final concen-
tration of L-Dopa was 50 μM.

Inhibition of mushroom tyrosinase by a new synthesized ligand
451
Figure 3. Double reciprocal Lineweaver–Burk plots of MT
Figure 5. Double reciprocal Lineweaver-Burk plots of MT
kinetic assay for catecholase reactions. L-Dopa was a substrate.
The reaction was done in 10 mM sodium phosphate buffer,
pH 6.8, at 20°C and 11.11 μg/ml enzyme, in the presence of
different concentrations of ligand, 0 mM (j), 3 μM (ꢀ), 4 μM
kinetic assay for cresolase reactions. L-tyrosine was a substrate.
The reaction was done in 10 mM sodium phosphate buffer,
pH 6.8, at 20°C and 112.68 μg/ml enzyme, in the presence of
different concentrations of ligand,0 mM (j), 3 μM (ꢀ), 7 μM
(
N), 11 μM (ꢃ), and 15 μM (d).
(
N), 6 μM (ꢃ), and 7 μM (d).
activity. Double reciprocal Lineweaver–Burk plot for the
cresolase activity of MT assayed as hydroxylation of
L-tyrosine in the presence of different fixed concentra-
tions of ligand is shown in Figure 3. These plots show a
set of straight lines, which intersect exactly on the verti-
cal axis. As it is seen the value of maximum velocity
(
Vmax) is unchanged by the inhibitor while the apparent
Michaelis–Menten (K ′) values are increased, indicating
m
that ligand induces a competitive inhibition. Figure 4
shows the secondary plot, the K ′ value at a given con-
m
centration of ligand vs. the concentration of inhibitor,
which gives the inhibition constants (K ) from the
i
abscissa intercepts (ꢀK ). The K value for ligand is
i
i
2
.8 μM, the small value of which shows that this ligand
tightly binds to the enzyme.
m
Figure 6. Secondary plot for the catecholase activity, the K ´
at given concentration of ligand vs. the concentration of ligand.
Kinetic parameters of catecholase activity of MT in the
presence of ligand
are shown in Figure 5. These plots give a set of straight
lines intersecting exactly on the vertical axis. It is
observed that the Vmax value is unchanged by the inhibi-
Double reciprocal Lineweaver–Burk plots for the cat-
echolase activity of MT assayed as oxidation of L-Dopa
in the presence of different fixed concentrations of ligand
tor while the K ´ value is increased, revealing that the
m
Figure 7. Plots of relative cresolase activity vs. [ligand]. The
ligand concentrations are 0, 1.5, 2.8, 3.6, 4.4, 4.9, 5.6, 6.3, 7,
8.3, 9.3, 10.2, and 11.4 μM. The final L-tyrosine concentration
was 150 μM.
Figure 4. Secondary plot for the cresolase activity, the K ´ at
given concentration of ligand vs. the concentration of ligand.
m

4
52
M. Alijanianzadeh et al.
lase activities of the MT against ligand concentration
were plotted (Figures 7 and 8, respectively).
Ligand docking and molecular dynamics simulation
The results of docking the ligand with the enzyme are
shown in Figure 9. As it is clear, the ligand is bound in
the active site of MT and the OH group of the aromatic
ring is bound to the copper ion.
The current ligand rough structure is depicted in Fig-
ure 10. R1 and R2 note for ring-like structure of ligand
Figure 8. Plots of relative catecholase activity vs. [ligand].
The ligand concentrations are 0, 1.1, 1.9, 2.6, 3.6, 4.7, 5.2, 6.3,
7
.3, 8.3, 9.3, 10.3, 11.4, 12.3, and 13.2 μM. The final
L-tyrosine concentration was 100 μM.
ligand induces a competitive inhibition. Figure 6 shows
the secondary plot, the K ′ value at a given concentra-
m
tion of ligand vs. the concentration of inhibitor, which
gives the inhibition constants (ꢀK ) from the abscissa
i
intercepts. The K value for ligand is 2.6 μM. The ligand
i
binds to the substrate-binding site and inhibits the cat-
echolase activity in the competitive mode. The small
value of K for the ligand indicates that it tightly binds
i
to the enzyme. The docking result not only shows the
binding site of ligand, but also reveals the OH group of
the ligand which has bound to the copper ion. Further,
for calculating IC , the relative cresolase and catecho-
Figure 10. Schematic ligand rough structure. R1 and R2 note
for ring-like structure of ligand molecule and L denotes the
linker part. N notes for each nitro groups.
5
0
Figure 9. Computational docking results for tyrosinase and ligand. Yellow ones are copper ions of the active site and ligand is in
stick and ball model and the histidine and aspartic residues are in line models.

Inhibition of mushroom tyrosinase by a new synthesized ligand
453
Figure 11. Molecular dynamics simulation between MT and ligand. The global ligand’s structure distances from MT binding site
acidic residues (pink line) and hydrophobic residues (blue line). Left and right Y-axes stand for distance from hydrophobic and acidic
residues, respectively.
molecule, and L denotes the linker part. N notes for each
nitro groups. The docking results indicate the position of
ligand is stable and its neighbors are CYS 83, HIS 85,
GLU 256, SER 254, ASP 269, ASP 273, HIS 259, HIS
263, PRO 270, and PHE 264, which are far from the
ligand 5 Å at most.
The global ligand’s structure distances from its bind-
ing site are shown in Figure 11. This is appeared that the
Figure 12. Molecular dynamics simulation between MT and ligand. The R1 distances from MT binding site acidic residues (pink
line) and hydrophobic residues (blue line). Left and right Y-axes stand for distance from hydrophobic and acidic residues,
respectively.

4
54
M. Alijanianzadeh et al.
Figure 13. Molecular dynamics simulation between MT and ligand. The R2 distances from MT binding site acidic residues (pink
line) and hydrophobic residues (blue line). Left and right Y-axes stand for distance from hydrophobic and acidic residues,
respectively.
distance between ligand and hydrophobic parts of
binding site decreases during MD and then it becomes
constant. It indicates that the ligand is a hydrophobic
molecule in which its binding site’s hydrophobic interac-
tion attracts it to the enzyme surface. The Global
distance of ligand did not change from acidic residues of
Figure 14. Molecular dynamics simulation between MT and ligand. The L distances from MT binding site acidic residues (pink
line) and hydrophobic residues (blue line). Left and right Y-axes stand for distance from hydrophobic and acidic residues,
respectively.

Inhibition of mushroom tyrosinase by a new synthesized ligand
455
Figure 15. Molecular dynamics simulation between MT and ligand. The two hanging N parts of ligand distances from MT binding
site acidic residues (pink line) and hydrophobic residues (blue line). Left and right Y-axes stand for distance from hydrophobic and
acidic residues, respectively.
binding surface despite of a small rearrangement along
the second to third nanosecond of simulation.
are hydrophobic interactions between ring 2 (R2) and
binding surface because of the obvious R2–hydrophobic
part of binding surface decreasing distance throughout
MD.
The linker part (L) which contained two nitrogen
atoms shows a pattern-like global distance from binding
site (Figure 14). Two hanging N parts of ligand (as in
Figure 10) clearly show an affinity toward acidic parts of
binding surface (Figure 15).
Figure 10 schemes the ligand in carton. More details
of ligand binding mode were available upon ligand
parts–binding surface distance studies. Figure 12 shows
that the ligand ring 1 (R1) has a tendency toward acidic
binding surface residues and it implies that this ring
globally interacts with binding surface via electrostatic
interactions. Figure 13 notes a fascinating result. There
Figure 16. Emission spectra of tyrosinase at λmax = 290 nm showing the quenching effect of increasing the concentration (2.7, 6, 7.5,
.5, and 14 μM) of ligand.
9

4
56
M. Alijanianzadeh et al.
in the fluorescence intensity was caused due to
quenching, there was no significant λem shift with the
accumulation ligand.
A plot of the maximum fluorescence intensity vs. the
concentration of the ligand revealed a linear relationship
(
Figure 17). From this data, we calculated the binding
ꢀ
1
constant as K = .061 μM
and the binding number as
n = 1.02 ± .6 according to Equation (1). These results
revealed that the newly synthesized ligand has a strong
binding affinity for tyrosinase in the absence of substrate
and that there is one possible binding site.
ꢀ
1
0 0
Figure 17. The secondary re-plot of F /(F – F) vs. [Q]
according to Equation (1). Data were obtained from Figure 10
The final concentration of the enzyme was 1 μM.
Discussion
The newly synthesized ligand competitively inhibited
both the cresolase and catecholase activities of MT, with
a small inhibition constant. The ligand competitively
inhibited the enzyme by binding to its active site, which
is also confirmed by docking results (Figure 9). The
ligand could form a stable complex with copper ion and
block the active site of MT and also make four hydrogen
bonds with aspartic acid and one hydrogen bond with
histidine residue in the active site. By comparing the Ki
values of different inhibitors, it was found that the ligand
is a powerful inhibitor (see Table 1).
Copper ions in the active site of MT play a major
role in tyrosinase activity by catalyzing electron trans-
fer to substrates. Formation of a complex between a
ligand and the copper ion in the tyrosinase active site
can prevent electron transfer by the metal ion, so that
the enzyme cannot oxidize substrates and tyrosinase
Fluorescence quenching
Tryptophan fluorescence has been frequently examined
among the intrinsic aromatic fluorophores in tyrosinase
molecules to obtain information about conformational
changes. The interaction of the ligand with tyrosinase
and the conformational alterations in the enzyme were
evaluated by measuring the intrinsic fluorescence intensi-
ties of the protein before and after the addition of the
ligand. The fluorescence emission spectra of MT were
collected at different concentrations of ligand. Figure 16
shows that the ligand causes a dramatic change in the
fluorescence emission spectra.
In this study, the fluorescence intensities of the emis-
sion peaks were found to be inversely decreased by
increasing the ligand concentration. Although the decline
Table 1. Comparing IC50 values of different inhibitors of MT (compounds with OH group) for both cresolase and catecholase
activities.
Reaction type
Ligands
IC50 (μM)
References
Cresolase activity
Catecholase activity
2-((1Z)-(2-(2,4-dinitrophenyl)hydrazine-1-ylidene)methyl)phenol
2-((1Z)-(2-(2,4-dinitrophenyl)hydrazine-1-ylidene)methyl)phenol
4.9
4.7
50
–
–
5
-benzylidene(thio)barbiturate-b-D-glycosides
Yan et al. (2009)
N-caffeoylserotonin
N-protocatechuoylserotonin
N-cyclopentyl-N-nitrosohydroxylamine
Dopastin
Neocupferron
Sophoraflavone
Kuraridin
Kurarinone
Artocarpanone
Kurarinol
Kuraridinol
Trifulirhizin
15.3
111
.6
20
46
6.6
.6
6.2
80.8
8.6
.8
506.77
1
155
252
490
500
Yamazaki et al. (2009)
Yamazaki (et al., 2009)
Shiino et al. (2001)
Shiino et al. (2001)
Shiino et al. (2001)
Kim et al. (2003)
Kim et al. (2003)
Kim et al. (2003)
Arung et al. (2006)
Hyun et al. (2008)
Hyun et al. (2008)
Hyun et al. (2008)
Shin et al. (1998)
Shin et al. (1998)
Shin et al. (1998)
Shin et al. (1998)
Shin et al. (1998)
Oxyresveratrol
Resveratrol
3
3
,5-Dihydroxy-4-methoxystilbene
,4-Dimethoxy-5-hydroxystilbene
Trimethylresveratrol

Inhibition of mushroom tyrosinase by a new synthesized ligand
457
activity is inhibited. As Figure 9 shows, the synthe-
sized ligand bound to the MT copper ion via an OH
group further making four hydrogen bonds with the
Asp and one hydrogen bond with the His residues in
the active site. As it has been mentioned before, the
histidine residues in the active site have an important
role in the enzyme activity (Guo et al., 2008), so the
ligand interrupted the MT activity by forming hydro-
gen bonds with it. Tyrosinase substrates (L-Dopa and
L-tyrosine) also bind to the copper ion of tyrosinase
via their OH group. However, the OH group of the
new ligand is more acidic than the OH group of
tyrosinase substrates, as its OH group was bound to
an aromatic ring connected to an imine group. There
is a resonance binding between two imine groups and
the aromatic ring; therefore, it is expected that ionized
OH group of the synthesized ligand is a powerful
acidic group, which can bind to the copper ion of
tyrosinase more strongly than other ligands. Tyrosinas-
es have similar binuclear copper sites (Halaouli,
Asther, Sigoillot, Hamdi, & Lomascolo, 2006), so MT
can be used as a model for human tyrosinase and
hence, we expect that human tyrosinase will also be
competitively inhibited by our newly synthesized
ligand.
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At the end, from MD results, we conclude that this
ligand binds to binding surface via electrostatic
interaction that sourced in ring 1 (R1) and hydrophobic
interactions which originated from ring 2 (R2). Figures
11–15 remind that the ligand never has gone far away
from the binding site when compared with docking
results structure. So, the binding mode seems good in
this manner.
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Acknowledgements
The financial support given by the University of Tehran is
gratefully acknowledged.
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