Determining mushroom tyrosinase inhibition by imidazolium ionic liquids: A spectroscopic and molecular docking study

Article abstract of DOI:10.1016/j.ijbiomac.2017.10.066

The inhibition effects of imidazolium ionic liquids (ILs) on the enzyme kinetics of mushroom tyrosinase is reported. A simple UV-VIS spectrophotometric assay was used to measure the reaction kinetics of the reaction between mushroom tyrosinase and L-dopa.

Full text of DOI:10.1016/j.ijbiomac.2017.10.066

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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: www.elsevier.com/locate/ijbiomac
Determining mushroom tyrosinase inhibition by imidazolium ionic
liquids: A spectroscopic and molecular docking study
∗
Mark P. Heitz , Jason W. Rupp
Department of Chemistry and Biochemistry, The College at Brockport, SUNY, 228, Smith Hall 350 New Campus Drive, Brockport, NY, 14420, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 August 2017
Received in revised form
The inhibition effects of imidazolium ionic liquids (ILs) on the enzyme kinetics of mushroom tyrosi-
nase is reported. A simple UV-VIS spectrophotometric assay was used to measure the reaction kinetics
of the reaction between mushroom tyrosinase and L-dopa. Seven different imidazolium ILs, com-
3
0 September 2017
+
prised of 1-alkyl-3-methylimidazolium ([Imn1 ], n = 2, 4, 6) cations paired with several anions that
Accepted 11 October 2017
Available online xxx
−
−
−
−
included Cl , [NO3 ], methanesulfonate ([MeSO3 ]), trifluoromethanesulfonate (or triflate, [TFMS ]),
−
and bis(trifluoromethylsulfonyl)imide ([Tf2N ]). Lineweaver-Burk plots were generated from the recov-
ered kcat and Km parameters using four to six substrate concentrations per measurement. The results
Keywords:
show that mushroom tyrosinase activity was consistently inhibited by all of the ILs and that the type
Imidazolium ionic liquid
Tyrosinase inhibition
Molecular docking
+
−
of inhibition was non-competitive in nearly all cases. Only the data for [Im21 ][Tf2N ] suggested that
the inhibition mechanism was competitive with the substrate. Molecular docking simulations were per-
formed using AutoDock4.2 and AutoDock Vina and revealed that all cations docked in the L-dopa active
site. Anions showed varied results that included locations both within and outside of the active site.
©
2017 Elsevier B.V. All rights reserved.
1
. Introduction
interested in identifying tyrosinase inhibitors as a way to prevent
over riping of fruit [8,9]. The clear advantage of governing tyrosi-
nase chemistry in this application is increased product shelf life
with the potential to effectively increase agricultural yields while
simultaneously reducing the economic impact of waste.
The enzyme tyrosinase, found in melanocytes, plays an impor-
tant role in the biosynthesis of melanins and other polyphenolic
compounds [1]. Melanocytes are cells found in the basal layer of
the epidermis and are key in the production of melanin, the pig-
mentation molecule responsible for skin color and protection of
skin cells and DNA from UV damage [2]. Melanin is synthesized
by tyrosinase and its formation is rate-limited by the enzymatic
reaction. Even when melanocytes become cancerous the melanoma
can still produce melanin and it is of interest to note that the ele-
vated expression of the tyrosinase is found in melanoma cells [3]
and that the control of in vivo melanin production is regulated by
the inhibition of tyrosinase activity [4–7]. Previous research high-
lights that a strategy of targeting tyrosinase with inhibitors can be
used to treat melanogenic disorders [4,5,7]. Thus, understanding
enzyme-inhibitor interactions effects and how tyrosinase activity is
influenced can have a significant impact on the diagnosis and treat-
ment of melanin related diseases. Another important application of
tyrosinase chemistry is that it plays a key role in the browning of
fruit skin. As a result, the agricultural and food industries are con-
tinually seeking ways to reduce the impact of food waste and are
Tyrosinase is known to catalyze the hydroxylation of phenolic
substrates to catechol derivatives, which in turn are able to fur-
ther oxidize into orthoquinone products [6,7,10,11]. Biochemical
reactions that take place under physiological conditions often rely
on catalysts for efficient reactivity and thus enzymatic function
can have a significant impact on organisms at the cellular level.
Enzymes, like all other catalysts, are characterized by three funda-
mental properties: 1) increase reaction rates without altering the
position of chemical equilibrium; 2) are not consumed and are not
permanently altered by the reaction; and 3) acceleration of reaction
rates by altering substrate conformation so that the conforma-
tion becomes configured to form the transition state intermediate.
Catalytic activity involves enzyme/substrate binding to form an
enzyme-substrate complex (ES*) in a lock and key manner. Initially,
the substrate is bound through noncovalent interactions. It is often
the case that both enzyme and substrate are modified by an induced
fit process that causes the substrate stresses to further enhance the
conversion into the ES* transition state, which results from a weak-
ening of bonds. The ES* state is stabilized by tight enzyme binding
that lowers activation energy [10]. Substrates bind to the enzyme
active site with specificity and when bound convert into products
∗ Corresponding author.
E-mail address: mheitz@brockport.edu (M.P. Heitz).
https://doi.org/10.1016/j.ijbiomac.2017.10.066
0
141-8130/© 2017 Elsevier B.V. All rights reserved.
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2
that are subsequently released from the enzyme. Active sites can
be envisioned as grooves or clefts on the surface of the enzyme that
are composed of amino acids from different parts of the polypep-
tide chains. The specificity of active site interactions are unique to
each enzyme and are driven by the specific interactions between
the amino acids and substrate. Structurally, the tyrosinase active
site has been shown to consist of two Cu2+ ions coordinated to
two sets of three histidine residues [9]. The crystal structure has
been reported and the ribbon structure of tyrosinase with L-dopa
incorporated in the active site has been published and made avail-
able through the RCSB protein data bank (PDB), structure 4P6S [12].
According to this structure, there are two binding pockes for L-dopa
in each of the two chains reported. The binding pockets appear
on the enzyme’s outer surface and in both cases (for both chains)
involve (at least) HIS13, HIS60, HIS204, HIS208, ARG209 residues in
addition several other residues that are in positions of close contact
When incorporated into an enzyme, ILs have been reported to
have mixed effects on activity. In some cases ILs can enhance enzy-
matic activity and selectivity as highlighted in a recent mini-review
by Goldfeder and Fishman [33]. In contrast, the IL cations and
anion can disrupt the amino acid interactions and alter the enzyme
conformation through alteration of the hydrophobic effect (which
governs secondary and tertiary protein structure), hydrophobic
hydration, surface tension, and water–water interactions among
other factors, all of which induce a corresponding diminution of
enzymatic activity [34]. Typically, these structural changes cause
disruption to the enzyme’s active site and performance can be
compromised [35]. A large number of reports have demonstrated
that the activity effects in many IL systems can be predicted by
the Hofmeister series, where the relative kosmotropicity (order-
making or structure-making) or chaotropicity (disorder-making) of
the IL ions dictate the outcome of enzymatic activity, particularly
in bulk aqueous solutions where the IL ions tend to be solvent sep-
arated [36,37]. These general effects have been discussed at length
in several reviews [33–35,37–41].
(
∼4 Å).
Ionic liquids (ILs) are often touted as environmentally friendly
solvents that are capable of solubilizing a wide variety of poorly
water-soluble substrates. ILs, particularly imidazolium derived
ILs, have become broadly applied in many industrial applications
In this work, we report on the reaction kinetics between mush-
room tyrosinase and L-dopa measured in the presence of the seven
imidazolium-based ionic liquids, shown in Fig. 1. These imida-
zolium ILs were selected because of their favorable miscibility with
aqueous solutions. Moreover, given that imidazolium ILs are by far
the most widely studied and applied ILs, their extensive use sug-
gests that they would be the most likely candidates to appear in the
environment, whether through aqueous waste streams or direct
applications in agriculture [29,31]. We studied the effect of IL cation
hydrophobicity by increasing the alkyl chain substituent to deter-
mine its impact on enzymatic activity. To elucidate the effect of
[
13–19] and in agricultural applications such as pesticides and plant
growth regulation [20,21]. For example, ternary imidazolium IL
systems were studied for use as pretreatment media to reduce
biomass recalcitrance and to assess their ability to enhance the effi-
ciency of enzymatic saccharification for biofuel production [19].
Imidazolium ILs also have been used in the synthesis of new ILs
based on dicambra (a systemic herbicide) that were designed as
alternatives to using free dicambra [21]. Results from this work
showed that the ILs had improved properties of increased ther-
mal stability, reduced volatility, and improved efficacy compared
to free dicambra. The use of the imidazolium cation was selected
for its reported antimicrobial effects against gram-positive and
gram-negative bacteria, yeasts, molds, and fungi [21]. The antimi-
crobial effects of imidazolium ILs have also been studied using
human serum albumin (HSA) and demonstrated to have signifi-
cant antibacterial and antifungal activities [22]. In addition, ILs have
been cited as potentially useful for biocatalytic reactions [23–26].
The enzyme kinetics of yeast alcohol dehydrogenase was studied
fifteen IL cation/anion pairs, including eight imidazolium ILs and it
was reported that anion effects dominated the salt effects on this
enzyme [27]. These works, and many others, highlight one of the
attractive features of ILs and that is the physicochemical proper-
ties can be fine-tuned simply by selecting different combinations
of anions and cations. Moreover, attaching various substituents
to either the cation or anion makes ILs easy targets for use in
task-specific chemical applications. However, as is often the case,
chemical applications can signifcantly outpace a systematic, thor-
ough fundamental study of novel chemical systems and ILs are no
exception. The inherent problem becomes that broader impacts
are not well understood before process modification and imple-
mentation. For example, while ILs are used to design “greener”
chemical processes because of their low volatility, the actual envi-
ronmental impact is not well defined nor understood. However, this
area of focus has been receving increased attention [19–21,28–31].
Since many ILs exhibit significant water solubility, their presence
in aqueous waste streams and use in agriculture are clearly direct
pathways into the environment. Given the reported biodegrada-
tion variability of ILs and therefore the persistance of ILs in water
sources and soils, it is important to study the details of molecu-
lar interactions between biomolecules and ILs [28–31]. One recent
review article has discussed the topic of ILs as they relate to biolog-
ical activity and highlighted the wide range of effects of several IL
classes in the context of medicinal and pharmaceutical applications
+
anion, we selected the [Im21 ] cation to use with several anions
anticipating that [Im21+] would have simultaneously the highest
solubility and the weakest direct impact on the enzyme hydropho-
bic interactions. In the presence of [Im21+] the anion effect on the
resulting enzyme stability and activity should be most clearly delin-
eated. Since anions are reported to have the greater impact on
activity and stability [27,35], we were particularly interested to
determine the influence of specific ion effects on the tyrosinase
activity.
2. Materials and methods
2.1. Materials
Table 1 summarizes each of the ionic liquid abbreviations,
complete chemical names along with chemical abstract registry
numbers, and purity and sources of chemicals used in this work.
All enzyme and substrate solutions were prepared in 50 mM ionic
strength and pH = 6.8 potassium phosphate buffer. A concentration
of approximately 50 nM tyrosinase was constant across all exper-
iments reported. For convenience, we prepared a stock tyrosinase
solution in phosphate buffer such that we could easily micropipette
desired volumes for kinetic runs. In the same way, stock L-dopa
−
1
solutions were always prepared at 1.5 mg·mL . For a kinetic
series at a constant [IL], L-dopa concentrations were typically var-
ied between 0.5–3.8 mM by pipetting 200–1500 ␮L of stock the
L-dopa solution. Stock enzyme and substrate solutions were always
protected from light, both during experiments and when stored.
◦
Storage of all stock solutions was at 4 C when not in use.
2.2. Methods
2.2.1. Kinetic measurements
[
32].
To prepare for a series of experiments, solutions were freshly
made the afternoon prior to use and then equilibrated overnight at
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Fig. 1. Chemical structures of the cations and anions used in this work. Cations included 1-ethyl-3-methylimidazolium, [Im21+], 1-butyl-3-methylimidazolium,
Im41+], and 1-hexyl-3-methylimidazolium, [Im61+]. Anions included chloride, nitrate, methanesulfonate [MeSO3 ], trifluoromethanesulfonate [TFMS ], and
−
−
[
−
bis(trifluoromethylsulfonyl)imide [Tf2N ].
Table 1
Molecules and Ionic Liquids Studied.
Molecules/ILsa
L-dopa
CAS RNb
Chemical Name
Source (Purity) c
9
002−10-2
Tyrosinase
L-3,4-dihydroxylphenylalaine
Potassium phosphate, monobasic
Potassium phosphate, dibasic
1-ethyl-3-methylimidazolium nitrate
1-ethyl-3-methylimidazolium methanesulfonate
1-ethyl-3-methylimidazolium trifluoromethanesulfonate
1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide
1-butyl-3-methylimidazolium chloride
1-butyl-3-methylimidazolium methanesulfonate
1-hexyl-3-methylimidazolium chloride
Sigma Aldrich 50K units
Sigma Aldrich (98%)
Fisher (99%)
Acros (99%)
Fluka (97%)
EMD (98%)
EMD (98%)
EMD (98%)
EMD (98%)
59–92-7
7
1
778−77-0
6788−57-1
+
[
[
[
[
[
[
[
Im21 ][NO3-]
143314−14-1
516474−01-4
145022−44-2
174899−82-2
79917−90-1
342789−81-5
171058−17-6
+
-
Im21 ][MeSO3 ]
+
-
Im21 ][TFMS ]
+
-
Im21 ][Tf2N ]
+
-
Im41 ][Cl ]
+
-
Im41 ][MeSO3 ]
IoLiTec (95%)
Solvent Innovation (97%)
+
-
Im61 ][Cl ]
a
b
c
Chemical abbreviations.
CAS RN is the chemical abstract registry number.
Manufacturer purity.
◦
4
C, making only enough solution to insure that none lasted more
ing to avoid aerating the solution and creating an emulsion, and
then placing the cuvette immediately into the spectrometer and
starting the acquisition. This sequence was carefully performed
in an effort to be as systematic, consistent, and precise as possi-
ble. Absorption data was acquired as a function of reaction time at
475 nm, using 5 s intervals. The rate of dopaquinone formation was
derived by using the method of initial rate as defined by the slope
of the linear response window of [dopaquinone] versus time plots
at early reaction times. Dopaquinone concentrations were calcu-
lated directly from absorbance data using the absorptivity value of
than two or three days. In this way, we attempted to minimize
solution variability resulting from enzyme degradation. IL concen-
trations were made by micropipetting neat IL to produce solutions
that ranged from ∼5–250 mM.
The activity assay used for tyrosinase was based on spectropho-
tometric measurements using a Perkin-Elmer Lambda 800 UV/VIS
spectrometer set at 475 nm where we detected the dopaquinone
product formation. The reaction was performed in a standard 1 cm
path length quartz cuvette (23-Q-10, Starna, Atascadero, CA) with
a matched cuvette in the reference channel of the spectrometer
that contained a blank solution composed of buffer and ionic liq-
uid to correct the measured absorbance. Prior to a kinetic run,
−
1
−1
3600 M cm [42].
The tyrosinase/L-dopa system is well-described by Michaelis-
Menten kinetics. In this model, the IL inhibition behavior was
determined from Lineweaver-Burk (L-B, double reciprocal) plots,
◦
reagent solutions were equilibrated and maintained at 30 C using
a Neslab thermostated chiller. To prepare the reaction mixture, the
desired amounts of buffer, IL, and then enzyme solutions were first
micropipetted into a cuvette followed by thorough mixing for at
least 60 s to ensure solution homogenization. To initiate the reac-
tion, the desired amount of L-dopa was then micropipetted into the
cuvette followed by complete cuvette inversions for 2 s, not shak-
(1/V ) = (Km/Vmax)(1/[S]) + 1/Vmax where V0 is the initial reaction
0
velocity calculated from the absorbance versus time data, Km is
the Michaelis constant, Vmax is the maximum reaction velocity,
and [S] is the substrate concentration [43]. Fits to a linear model
allow extraction of the slope (=Km/Vmax), y-intercept (1/Vmax), and
x-intercept (= −1/Km). Assuming a two-step kinetic model, the
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product formation rate constant (k ) is defined as the limiting rate
2
Im21+][MeSO ]
-
[
constant of an enzyme-catalyzed reaction (kcat) where Vmax = k [E]
0.4
0.3
0.2
0.1
3
2
and [E] is the enzyme concentration [43]. From the L-B data, we
calculated the Km, kcat, and Vmax parameters and from these val-
kcat
3
.8 mM L-Dopa
ues the Km ratio. Finally, we used the neat buffer solution data as
a comparator to normalize the IL data and calculated the relative
ꢀ
ꢁ
ꢀ
ꢁ
kcat
Km
kcat
Km
activity,
IL/
Buffer.
2
.2.2. Computational methods
®
0
.5 mM L-dopa
90 120
We used Spartan’16 Parallel Suite [44] to compute all ionic
liquid parameters. Except for molecular mechanics and semi-
empirical models, the calculation methods used in Spartan have
been documented elsewhere [45]. In the present work, calcula-
tions were performed using the Hartree-Fock 3–21G level of theory
and for simulating ions in water the SM8 [46] water model was
used. The SM8 water model is an implicit continuum solvation
model that estimates the electrostatic polarization of a dielec-
tric medium using bulk solvent properties. For comparison, ion
properties were also calculated using DFT theory with the B3LYP
functional and 6–31G* basis set. The resulting hydrated ion ener-
gies were <1% different than the computed HF 3–21G method so a
more sophisticated level of theory was deemed not necessary for
the intended purposes in this work. Cation and anion structures
were first geometry-optimized to minimize the ion energy, after
which electrostatic potentials were determined. The final struc-
tures were exported into a Sybyl (.mol2) format in preparation for
docking studies.
Ligand docking simulations were performed using the open-
source programs for AutoDock4.2 [47] or AutoDock Vina [48] along
with AutoDockTools (ADT) [47], a graphical user interface compli-
ment to the AutoDock software suite. AutoDock4.2 uses an AMBER
based force field scoring function that estimates the binding free
energy of a ligand to its target. Vina was developed as a successor
to AutoDock 4.2 and boasts faster, more accurate results with less
direct investment required by the end user. Here, we used Vina to
perform blind docking simulations by searching the entire tyrosi-
nase structure to find the best docking sites for each of the IL ions.
To that end, we performed no less than five independent dock-
ing runs and averaged the results of each docking, in sequence, to
produce a representative result and the standard deviations associ-
ated with these replicate data. At times, the docked energies were
identical for the replicates and therefore yielded a standard devia-
tion of zero. In order to run the Vina docking program, both ligand
and receptor structures must be first prepared in a specific file for-
mat. We used ADT to create the necessary. pdbqt files that are read
by Vina. For the tyrosinase receptor, we used the crystallographic
structure 4P6S from the RCSB PDB website. To minimize the search
area on the receptor we removed chain B from the PDB file. We
then removed the two L-dopa molecules that were in chain A to
make the active site accessible to docking the IL ions. Ligand (IL
ions) structures were directly imported from the Spartan’16 file
as described above and the electrostatic charges were preserved.
In the simulation ligand positions were initialized with a random
starting position.
0
.0
0
30
60
Fig. 2. Absorbance traces for the tyrosinase/L-dopa reaction in 48.3 mM
−
◦
[
Im21+][MeSO3 ]; solution pH = 6.8 and T = 30 C. The arrow shows the absorbance
progression as a function of substrate concentrations at 0.5 mM (solid line), 1.0 mM
long dashes), 1.5 mM (medium dashes), 2.5 mM (short dashes) and 3.8 mM (dotted
(
line) L-dopa.
The initial reaction velocity was calculated from the absorbance
data using only data from 10 to 50 s reaction time where the
absorbance change was most linear. We examined various time
ranges and determined that using data over a longer window
introduced absorbance non-linearity. Fig. 3 shows the composite
results for the relative activity all systems reported here. Without
exception, all cation/anion IL combinations inhibited the tyrosinase
relative activity and activity decreased systematically as IL concen-
tration increased. The dotted line indicates 50% relative activity.
Table 2 collects the estimated IC50 values and these data quan-
tify the inhibition effectiveness for each of the ILs. There a large
variation in inhibition by [Im21+] ILs and the inhibition capacity
is strongly influenced by the anion present. The two [Im41+] ILs
display statistically the same level of inhibition. The longest chain
cation, [Im61+] along with [Im21+][Tf N ] both show substantial
−
2
capacity for tyrosinase inhibition. We have discussed these obser-
vations elsewhere and explained them in the context of anion
hydrophobicity and polarization coupled with the increased cation
hydrophobic effect [49].
3
.2. Inhibition types resulting from IL interactions with
mushroom tyrosinase
Having characterized the tyrosinase activity, we then focused
on determining the specific mode(s) of inhibition by the imida-
zolium ILs on tyrosinase. Each of the IL systems presented here
were processed as follows. A Lineweaver − Burk (L-B, double recip-
rocal) plot was generated for the data set, from which we extracted
the slope and intercept values that were used to calculate the Km
and Vmax parameters. This was repeated for each IL concentration
used in the system studied. The kinetic data was also used to quan-
tify the equilibrium constant for inhibitor binding. The left panel
of Fig. 4 shows an example for a complete set of L-B plots that
resulted from the reaction between tyrosinase and L-dopa in the
3
. Results and discussion
+
−
presence of [Im21 ][MeSO3 ]/phosphate buffer solution at pH = 6.8
◦
+
−
3.1. Tyrosinase activity in imidazolium ILs
and T = 30 C. The series of [Im21 ][MeSO3 ] concentrations shown
here are 4.8 (᭹), 24.1 (ꢀ), 38.6 (ꢀ), 48.3 (ꢀ), and 62.8 (᭿) mM. The
individual regression lines are fits to the linear model (see Meth-
ods section). Figs. S1–S5 in supporting information shows L-B plots
for the remaining ILs. Each of these data sets were reasonably well
described by a linear fit with r > 0.95, which was a typical fitting
result for all ILs studied in this work. The right panel shows two sec-
ondary plots for the L-B slope and L-B intercept that were used to
An absorbance data set was collected for kinetics determina-
tion that consisted of five L-dopa concentrations (0.5–3.8 mM) in
an IL/phosphate buffer solution at various IL concentrations. Fig. 2
presents a representative data set for the tyrosinase/L-dopa reac-
2
tion the presence of 48 mM [Im21+][MeSO3 ]. All data sets were
−
created in this way at each IL concentration for the ILs studied here.
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5
[
[
[
[
[
[
Im21 ][NO3
]
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
+
-
Im21 ][MeSO3
Im21+ ][TFMS- ]
+
-
Im21 ][Tf N ]
2
Im41+ ][Cl- ]
+
-
Im41 ][MeSO3
[Im61 ][Cl- ]
+
0
30
60
90
120
150
◦
Fig. 3. Tyrosinase relative activity in the presence of various ionic liquid concentrations at 30 C. Solid lines are included as a visual aid to distinguish the various IL data sets.
+
+
+
Symbols are organized by cation identity with circles = [Im21 ], squares = [Im41 ], and stars = [Im61 ]. The horizontal dotted line marks the 50% relative activity line.
Table 2
Inhibition Data for Mushroom Tyrosinase in Ionic Liquids.
Ionic Liquid
IC50 a / mM
Type
KEI b / mM
KEIS b / mM
Ratio
Im21 ][NO3-]
+
180 ± 20
54 ± 9
24 ± 4
–
20 ± 3
22 ± 2
9 ± 1
mixed
mixed
mixed
competitive
mixed
43 ± 18
22 ± 7
126 ± 29
62 ± 9
2.9
2.8
1.5
656
2.5
11.
2.3
[
[
[
[
[
[
[
+
-
Im21 ][MeSO3 ]
+
-
Im21 ][TFMS ]
24 ± 12
0.7 ± 0.1
43 ± 10
7 ± 4
37 ± 17
483 ± 93
110 ± 40
77 ± 17
8.9 ± 0.9
+
-
Im21 ][Tf2N ]
+
-
Im41 ][Cl ]
+
-
Im41 ][MeSO3 ]
mixed
mixed
+
-
Im61 ][Cl ]
3.8 ± 0.5
a
Estimates of these values were obtained by regressing the relative activity data and computing the corresponding 50% activity value from the regression equation.
Binding equilibrium constant values were calculated from Lineweaver-Burk slopes and intercepts. cRatio = KEIS/KEI.
b
Fig. 4. Left panel: Series of Lineweaver-Burk (L-B, double reciprocal) plots for mushroom tyrosinase/L-dopa reactions in solutions of [Im21+][MeSO3−]/phosphate buffer held
at pH = 6.8 and T = 30 C. [Im21+][MeSO3 ] concentrations are 4.8 (᭹), 24.1 (ꢀ), 38.6 (ꢀ), 48.3 (ꢀ), and 62.8 (᭿) mM. The regression lines are fits to a linear model, all r > 0.95.
Right panels: plots of slopes and intercepts recovered from the Lineweaver-Burk plot regression calculations. L-B slopes and intercepts were used to calculate KEI and KEIS
inhibition equilibrium constants, respectively.
◦
−
2
extract equilibrium constants (see Section 3.2.1 below for details)
8,50].
whereas a non-competitive or mixed inhibition model is revealed
by plots with lines that intersect in the second quadrant. The data
[
for [Im21+][MeSO3 ] shown in Fig. 4 is consistent with the lat-
ter model, mixed inhibition. In addition to the L-B plots for this
IL, further supporting evidence for mixed inhibition came from
the observation that the as the IL concentration increased, the Km
values increased while simultaneously the Vmax values decreased.
−
3
.2.1. Inhibition kinetics for tyrosinase activity in [Im21+] ILs
In the Michaelis-Menten kinetic scheme for enzyme inhibition,
the type of inhibition that governs the enzyme reaction is read-
ily recognizable by the pattern of resulting lines from a series of
L-B plots [43]. For plots that show a set of lines intersecting at
the y-intercept, this is indicative of a competitive inhibition model
From these data, we concluded that the [Im21+][MeSO3 ] inhibitor
−
(I) is able to bind not only to free tyrosinase (E) to make the
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6
enzyme-inhibitor (EI) complex, but also to the tyrosinase-l-dopa
ES*) complex to form the enzyme-substrate-inhibitor (ESI*) com-
hydrophobic character of the IL enhances the hydrophobic effect
on the tyrosinase [34,35,38]. Second, the L-B plots for these [Im41+
(
]
plex. We calculated the equilibrium binding constants for each of
these cases. For the (EI) complex, the slopes from each of the L-B
fits were used to make a secondary plot of L-B slope as a function of
IL concentration (see the upper graph, right panel of Fig. 4). These
data were regressed using a linear model to extract the x-intercept,
which directly yields the (EI) equilibrium binding constant (KEI)
ILs (not shown) both indicate mixed type inhibition since the family
of lines intersects in the second quadrant of the graph. Third, simi-
lar to the [Im21+] ILs, the KEI and KEIS values show that binding with
free enzyme is preferred over the ES* complex and KEIS/KEI ratios.
−
Fourth, the [MeSO3 ] binds more tightly to free tyrosinase com-
−
pared to Cl , by roughly a factor of six whereas in the ES* complex
[
8,50]. Similarly, to calculate the (ESI*) equilibrium binding con-
the preferential binding is less than a factor of two.
stant (KESI) the L-B intercepts were plotted as a function of [IL]
concentration (see the lower graph, right panel of Fig. 4). The val-
ues of KEI and KEIS for [Im21+][MeSO3 ] binding were 22 ± 7 and
3.2.3. Inhibition kinetic parameters for tyrosinase activity in
[Im61+] ILs
−
6
2 ± 9 mM, respectively, and are reported in Table 2. We observed
Although we only measured one [Im61+] IL, we can comment on
+
−
+
−
the cation effect by comparing to the [Im41+][Cl ] data. Increasing
the chain length to C6 has the expected effect of lowering the IC50
and decreasing the binding equilibrium constants. It appears that
increasing the imidazolium alkyl chain by only two carbons plays a
substantial role in the ensuing inhibition of tyrosinase, presumably
through increased screening of the hydrophobic effect [34]. While
−
similar behavior for [Im ][NO ] and [Im ][TFMS ] in that for
2
1
3
21
both of these cases, the ILs also displayed mixed inhibition charac-
teristics with KEI and K reported in Table 2. It is noteworthy that
EIS
[
Im21+][NO3 ] has binding equilibrium constants that are at least
−
twice that of the two methanesulfonate-based ILs. Larger values of
K are indicative of weaker inhibitor binding and this observation is
consistent with the relative activity behavior where [Im21+][NO3
−
]
the KEIS/KEI ratios for [Im41+][Cl ] and [Im ][Cl ] are statistically
−
+
−
61
has the least inhibitory effect on tyrosinase at any given IL concen-
tration [49]. Similarly, we can assess the binding affinity preference
of the ILs for either free or substrate-bound enzyme by examining
the KEIS/KEI ratios reported in Table 2. Since each of the KEIS/KEI
ratios are greater than 1 for these three ILs, this indicates that they
have a binding preference for free enzyme over the ES* complex.
But we also point out that the preference for free enzyme weakens
as anion hydrophobicity increases.
identical, the absolute magnitudes of KEI and KEIS are quite different,
by a factor of ∼11. This suggests that when bound, the effect of
+
+
[Im ] and [Im ] on tyrosinase and the ES* complex is likely very
41
61
similar for the two cations, however the difference in the binding
affinities is large with [Im61+] being more strongly bound to both
the free enzyme and the ES* complex.
3.3. Molecular docking simulations
Of particular interest in these experiments is the behavior of
tyrosinase with [Im21+][Tf N ], which is markedly different from
−
To aid experimental work, molecular docking simulations have
been used as a tool to augment the molecular level interpretation
of the data. A number of recent articles that discussed tyrosinase
inhibition [22,52–61] and protein-IL interactions [26,32,60,62–64]
have demonstrated the applicability of including docking simula-
tions in estimating potential sites for ligand binding.
2
all other ILs. Fig. 5 shows the L-B plots for the tyrosinase/L-dopa
kinetics that were measured using three [Im21 ][Tf N ] concentra-
tions. In consideration of the inhibition type, this data suggests that
the L-B regression lines intersect on the 1/V axis and thus is char-
+
−
2
+
acteristic of competitive inhibition. Compared to the other [Im21
]
ILs, here the apparent Km values increase while Vmax remains
effectively constant to within experimental uncertainty (data not
shown). As described above, we calculated the corresponding KI
value for the interactions between this IL and tyrosinase using
the L-B slopes and computed a value of 0.7 mM, see Table 2.
This KI value represents a significant increase in the IL affinity
for tyrosinase, an increase in excess of 30 times greater than
While the experimental kinetic results provides compelling evi-
dence to support mixed inhibition kinetics in all but [Im21+][Tf N ]
−
2
(competitive inhibition) and yield estimates of binding equilibrium
constants for IL binding to free tyrosinase and the ES* complex,
there is at least one other point of consideration that we now
address. Up to this point, we have not probed in any depth potential
sites within tyrosinase where IL ions might bind, either the active
site directly or associations with residues outside of the active site
that contribute to governing the tyrosinase activity. In an effort to
seek a molecular explanation for the observed inhibition behavior
of the ILs used here, we undertook a series of molecular dock-
ing studies using AutoDock4.2 and AutoDock Vina. The calculated
dockings were in general agreement between both programs but
Vina is reported to be more accurate [48] so we report the results
from that data. Docking calculations were performed using L-dopa
as a means of testing the agreement between Vina dockings and the
reported crystal structure for 4P6S [12]. The lowest energy dock-
ing configuration for L-dopa (-6.3 kcal/mol) is in good agreement
with experiment and places the L-dopa in proximity of HIS208,
VAL217, VAL218, ALA221, and the HIS60-bound metal atom. The
amino acids that compose the A-chain tyrosinase binding pocket
for L-dopa are shown in Fig. 6. The importance of free histidine
residues for ligand binding has been discussed and linked to tyrosi-
nase performance [55] and we note that this idea is consistent with
most of the docking sites that resulted from our studies here. Any
of the active site interactions between IL ions and tyrosinase in our
results included the metal-free HIS208 in most cases.
Im21+][MeSO ] and [Im ][TFMS ] and about 60 times greater
− −
+
[
3 21
+
−
+
than [Im21 ][NO3 ]. Comparing all of the [Im21 ] ILs, there is a
clear pattern of binding to tyrosinase that appears to follow the
anion hydrophobicity and polarizability. Of these four anions, rank-
ing by hydrophobicity results in the sequence of these ions as
−
−
−
−
[
NO3 ] < [MeSO3 ] < [TFMS ] < and [Tf N ], thus it was not unan-
2
−
ticipated that [Tf N ] preferred the relative hydrophobicity of the
2
enzyme compared to the phosphate buffer. Moreover, two recent
reviews have discussed the idea that anions are reported to have a
more active role in enzyme activity/stability through greater polar-
izability effects relative to cations [35,51], which is consistent with
our observations in [Im21+] ILs.
3
[
.2.2. Inhibition kinetics for tyrosinase activity in [Im41+] and
Im61+] ILs
The results from all of the kinetic measurements for the [Im41+]
and [Im61+] ILs are also summarized in Table 2. From these data,
we briefly highlight several features for the C and C6 alkyl chain
4
+
imidazolium derivatives. For the two [Im41 ] ILs, their behavior par-
allels that observed for the [Im21+] ILs. Several specific points are
apparent from these data. First, the IC50 data show that the pres-
ence of a longer alkyl chain on the imidazolium cation contributes
significantly to the inhibition of tyrosinase, by a factor of ∼2.5 on
Data for the top nine docking results for each ion used in this
work were thoroughly reviewed. We hasten to add a cautionary
note here that care must be taken to avoid over-interpreting dif-
ferences in docking energies. While it is no doubt customary to
+
−
+
−
going from [Im ][MeSO3 ] to [Im ][MeSO3 ]. The increased
2
1
41
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7
+
−
Fig. 5. Left panel: Series of Lineweaver-Burk (L-B, double reciprocal) plots for mushroom tyrosinase/L-dopa reactions in solutions of [Im21 ][Tf2N ]/phosphate buffer at
◦
+
−
2
pH = 6.8 and T = 30 C. [Im21 ][Tf2N ] concentrations are 38.8 (᭹), 50.4 (ꢀ), and 77.6 (ꢀ) mM. The regression lines are fits to a linear model, all r > 0.98. Right panel: slopes
recovered from the Lineweaver-Burk plot regression calculations and used to calculate the KEI inhibition equilibrium constants.
Fig. 6. Amino acids that compose the binding pocket (active site) for L-dopa (DAH305), image taken from PDB structure 4P6S [12].
Table 3
Molecular Docking Data Ionic Liquid Ions in Mushroom Tyrosinase.
Ionic Liquid
Energy
kcal mol-1
|deviation| a
/ kcal mol-1
Location b
Specific Interactions
/
L-dopa
−5.83 ± 0.37
−4.38 ± 0.22
−4.63 ± 0.26
−5.02 ± 0.20
−3.05 ± 0.07
−2.82 ± 0.09
−4.27 ± 0.07
−5.22 ± 0.14
A.S.
A.S.
A.S.
A.S.
HIS231
A.S.
A.S.
–
+
[
[
[
[
[
[
[
Im21
]
]
0.7
0.6
0.8
0.4
0.4
0.2
0.3
␲-␲ (HIS208)
␲-␲ (HIS208)
␲-␲ (HIS208)
H-bond (HIS231)
–
–
—
+
Im41
Im61+]
-
NO3 ]
-
MeSO3 ]
TFMS ]
Tf2N ]
-
-
A.S.
a
Absolute deviation between the maximum and minimum docked energy values that were associated with the top docking locations.
Most probable location of the primary docking answer. See text for additional docking location results. The abbreviation “A.S.” represents active site interactions.
b
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8
Fig. 7. Computational docking example results (from AutoDock Vina) for IL ion binding to mushroom tyrosinase. Panel A: lowest energy docking location for [Im61+], which
+
occurred in the tyrosinase active site (see Fig. 6). Also shown are the computed nearest neighbor residues for [Im61 ] that include (from top to bottom in the figure): PRO201,
−
HIS204, ASN205, HIS208, VAL218, and HIS42. Panel B: image of the lowest energy docking for [Tf2N ], which was in the tyrosinase active site. Nearest neighbor residues are
shown in the zoomed image and include (moving clockwise): ARG209, HIS208, ASN205, HIS204, PHE197, HIS60, VAL218, VAL217, GLY216.
report only the primary docking result, perhaps a better represen-
tation of the most probable ligand binding is likely represented by
an average of the top several docking sites. This is particularly true
for top dockings that differ by less than 0.5 kcal/mol, considering
that RT ≈ 0.5 kcal/mol and thermal randomization alone can influ-
ence the true docking site/orientation. As part of our evaluation of
docking results we also considered the energy range of top results
when selecting which dockings best represent chemical intuition.
So with these points in mind, the binding energies calculated by
averaging the three lowest energy dockings for the [Imx,1+] cations
and their associated standard deviations are reported in Table 3.
The [Im21+] results from the top three dockings across five repli-
cates showed an absolute energy difference of only 0.4 kcal/mol.
This small difference, given the 0.2 kcal/mol uncertainty, justified
averaging of these data. Moreover, for [Im21+] the top nine dock-
ing site energies ranged from −4.6 to −3.6 kcal/mol, only a 20%
from a close contact radius of 4 Å in Fig. 7, panel A. Dockings for
[Im21+] and [Im41+] are shown in supporting information, Figs.
S6 and S7, respectively. Residues common to all cation dockings
were HIS204, ASN205, HIS208, and VAL218. Regardless of the alkyl
chain on the imidazole ring all of the cations clearly showed ␲-
␲ interactions with HIS208. Some of the higher energy docking
orientations also suggested that there were ␲-␲ interactions with
HIS204. In all of the [Imx,1+] dockings, the differences in the docked
results stemmed only from orientation arrangements of the cation
within the active site. According to the energies, [Im61+] showed the
strongest interaction with tyrosinase by about 0.6 kcal/mol com-
pared to [Im21+]. We note that the collective cation data suggests a
very small but systematic decrease in energy on going from C2 to
C6, which is likely reflective of the longer alkyl chain having addi-
tional van der Waals interactions with the enzyme. Moreover, for
the longer alkyl chains conformational flexibility can easily be the
source of variation in different docking answers. We observed this
behavior in each of the cation results, but in particular with dock-
ings for [Im61+] because of the torsional flexibility of the C6 chain.
Since the top six docking locations for all cations are found in the
+
+
overall change. The [Im ] and [Im ] ions showed similar results
4
1
61
and were treated accordingly. We note that for all the imidazolium
cations, the primary docking location was always the tyrosinase
active site. As an example, we show the docked position of [Im61+
]
along with the identified nearest tyrosinase residues as determined
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9
−
active site, it appears that the enzyme-anion interactions play a
more significant role in the observed variation in tyrosinase relative
activity.
also note that [TFMS ] was a stronger inhibitor of the tyrosinase
+
relative activity (see comparable data for [Im21 ] in Fig. 3 and
Table 2). The docking energy for [TFMS ] was significantly less
than [MeSO3 ], by 1.4 kcal/mol, indicating stronger binding of this
fluorinated anion by tyrosinase.
−
−
Docking results for the anions used here are collected in Table 3
and arranged in order of the increased impact on tyrosinase rela-
−
tive activity. We were unable to perform dockings for Cl because
the pdbqt file preparation required a torsion tree root, which
apparently cannot be created for a monatomic ion. To provide a
−
3
.3.4. [Tf N ] dockings
2
−
All docking locations for [Tf N ] were in the L-dopa active site.
This result was consistent for all runs and for all nine results,
although for consistency the reported energy in Table 3 is only
for the average of the top three dockings from the replicate runs.
Of all anions studied here, [Tf N ] was by far the most consistent
with respect to location. Only the relative orientation of the anion
changed among all of the dockings. The larger variation in docking
energy is likely caused by the presence of highly electronegative
atoms (N, O, F, and S) that compose this anion and therefore dom-
inate the resulting set of interactions with the residues in the
binding pocket.
2
space-filling perspective we show a molecular surface image [65]
−
in Fig. 7, panel B, for the lowest energy [Tf N ] docking. Remaining
2
anion docking images are shown in Figs. S8–S10 of the supporting
information. For all docking in the active site, the ion fills the pocket
and effectively blocks access to L-dopa accounting for the compet-
−
2
−
itive portion of the mixed inhibition. Except for [NO3 ], the lowest
energy docking location was in the L-dopa active site for all of the
anions used here.
−
3
.3.1. [NO3 ] dockings
−
The Vina results suggested for [NO3 ] placed this ion at HIS231
with a 1.83 Å hydrogen-bond formed between the [NO3 ] oxy-
gen and the imidazole hydrogen. The bond energy was reported as
−
4. Summary and conclusions
−
7.6 kcal/mol. Active site dockings were also reported in replicate
runs with identical scores (ion binding energies) so evidently the
The myriad of ion combinations that can be used to form ILs
offers a nearly limitless set of choices for designing task-specific
systems. In this work we focused on the enzymatic activity of the
type 3[12] copper-containing oxidase mushroom tyrosinase and
the inhibition of activity alkylimidazolium ionic liquids. On the
whole, this work demonstrates the ability to use a small set of imi-
dazolium ILs to inhibit mushroom tyrosinase activity towards the
L-dopa substrate. Both the choice of ion identity and concentration
serve as an effective combination with which one can selectively
tailor tyrosinase inhibition.
−
most probable location for [NO3 ] is better thought of as a distri-
bution of equal energy locations. If [NO3 ] is equally likely to bind
in a non-active site location then this explains the observation that
−
−
[
NO3 ] has the least impact on tyrosinase relative activity, partic-
−
ularly at low [NO3 ] concentrations. Moreover, for the active site
dockings, no hydrogen bonding was observed. Other dockings for
−
[
NO3 ] also included ARG6 and ARG78, each of which displayed
two hydrogen bonds of 2.0 ± 0.1 Å.
−
3.3.2. [MeSO3 ] dockings
We have presented experimental data that characterized the
relative activity of this enzyme using the substrate L-dopa in
−
As mentioned above, the primary docking location for [MeSO3
]
◦
was the L-dopa active site and this was consistent over all repli-
cate docking runs. There were two other reproducible locations
identified by Vina that produced energies within 0.2 kcal/mol of
the lowest energy position. The second most consistent docking
set involved LYS19, SER110 AND GLN107. Interactions with these
residues included increased stabilization through hydrogen bond
formation with GLN107 and with SER110. The third preferred
docking was with ARG6 and ARG78, similar to [NO3 ] in both
location and the presence of hydrogen bonding interactions with
the [MeSO3 ] oxygen atoms. Distances here were of comparable
phosphate buffer solution at pH = 6.8 and T = 30 C using Michaelis-
Menten kinetic parameters to determine the enzymatic activity.
We calculated the relative activity as the ratio of (activity in the
presence of ILs)/(activity in buffer) and found that the relative
activity varied from about 15–95% over a small IL concentration
window of ∼10–100 mM. From the kinetic data, we determined
the inhibition type for the IL/tyrosinase system. In all solutions, the
presence of ILs resulted in mixed type inhibition with the exception
−
of [Im21+][Tf N ], which showed only competitive inhibition.
−
2
−
Our experimental results showed increased inhibition of
+
tyrosinase activity in the presence of the [Imx1+
] ILs in
lengths, at 2.0 ± 0.1 Å. In the relative activity context for the [Im
]
21
−
set of ILs, [MeSO ] ranked as the second least effective tyrosi-
proportion to cation alkyl chain length, and followed the
sequence [Im21 ] < [Im41+] < [Im61+], consistent with literature
reports [27,34,36,66,67]. The increased inhibition is connected to
the increased cation hydrophobicity. Molecular docking studies
with AutoDock Vina were used to reveal a set of most probable loca-
tions within tyrosinase that accommodated these cations. Without
exception, the cations all docked in the L-dopa active site of the
enzyme. The binding energies associated with the cation dock-
ings approached the L-dopa energy as alkyl chain length increased.
The [Im61+] energy was within 0.8 kcal/mol and slightly less than
the L-dopa energy. Moreover, the cation dockings all formed ␲-
interactions between the imidazole ring and the HIS208 residue.
From this data, we concluded that the presence of [Imx,1+] cations
created a competitive binding environment for the active site in
mushroom tyrosinase.
3
+
nase inhibition. Finally, for this anion, we also note that when the
cation dockings were also considered alongside this anion’s dock-
ings the more effective tyrosinase inhibition by [Im ][MeSO3
+
−
]
41
+
−
over [Im ][MeSO3 ] is clearly consistent with 1) the lower energy
2
1
[
Im41+] docking value and 2) the fact that even though [MeSO3
−
]
also docks outside of the L-dopa active site, the more strongly
bound [Im41+] docking in the active site had a greater influence
on tyrosinase inhibition.
−
3.3.3. [TFMS ] dockings
−
From a structural perspective, [TFMS ] is nearly identical to
−
[
MeSO3 ] with the exception of the perfluorinated methyl moi-
ety and therefore one might predict this ion should behave very
similarly to [MeSO3 ]. However, the data showed that all of the
−
top docking locations were localized in the L-dopa active site in
each of the replicate runs. In only one of the five runs performed
for this ion did the top three docking positions vary; that is, 1 in 15
runs produced a location variant. As one might anticipate, this addi-
tional docking site was at the LYS19, SER110 AND GLN107 residues,
Docking results showed that it was the IL anions that gov-
erned whether the observed IL inhibition was overall competitive.
The smaller, less polarizable anions were at least as likely to
bind to tyrosinase residues that were outside of the L-dopa active
site, which allowed for a cation/anion combination that resulted
in mixed inhibition behavior. As the anion size and polarization
−
similar to that observed for [MeSO3 ]. Of these two anions, we
Please cite this article in press as: M.P. Heitz, J.W. Rupp, Int. J. Biol. Macromol. (2017), https://doi.org/10.1016/j.ijbiomac.2017.10.066

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1
0
increased, we observed a concomitant increased in tyrosinase
inhibition. Additionally, the extent of halogenation as it governs
anion polarization changes also appeared to offer a convenient
characterization scheme to explain the observed experimental
anion inhibition effect. ILs results based on the [Im21+] cation
showed an increased inhibition trend that followed the sequence
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− − − −
NO3 ] < [MeSO3 ] < [TFMS ] < [Tf N ]. Docking results for these
2
[
anions showed that the latter two anions were docked primarily
−
−
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The authors gratefully acknowledge support from The College
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The authors declare no competing interests.
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