Structural and kinetic considerations on the catalysis of deoxyarbutin by tyrosinase

Deoxyarbutin, a potent inhibitor of tyrosinase, could act as substrate of the enzyme. Oxytyrosinase is able to hydroxylate deoxyarbutin and finishes the catalytic cycle by oxidizing the formed o-diphenol to quinone, while the enzyme becomes deoxytyrosinase, which evolves to oxytyrosinase in the presence of oxygen. This compound is the only one described that does not release o-diphenol after the hydroxylation step. Oxytyrosinase hydroxylates the deoxyarbutin in ortho position of the phenolic hydroxyl group by means of an aromatic electrophilic substitution. As the oxygen orbitals and the copper atoms are not coplanar, but in axial/equatorial position, the concerted oxidation/reduction cannot occur and the release of a copper atom to bind again in coplanar position, enabling the oxidation/reduction or release of the o-diphenol from the active site to the medium. In the case of deoxyarbutin, the o-diphenol formed is repulsed by the water due to its hydrophobicity, and so can bind correctly and be oxidized to a quinone before being released. Deoxyarbutin has been characterized with: kcatD-Arb = 1.95 ± 0.06 s-1 and KMD-Arb = 33 ± 4 μM. Computational simulations of the interaction of β-arbutin, deoxyarbutin and their o-diphenol products with tyrosinase show how these ligands bind at the copper centre of tyrosinase. The presence of an energy barrier in the release of the o-diphenol product of deoxyarbutin, which is not present in the case of β-arbutin, together with the differences in polarity and, consequently differences in their interaction with water help understand the differences in the kinetic behaviour of both compounds. Therefore, it is proposed that the release of the o-diphenol product of deoxyarbutin from the active site might be slower than in the case of β-arbutin, contributing to its oxidation to a quinone before being released from the protein into the water phase.

Introduction Tyrosinase (EC 1.14.18.1) uses molecular oxygen as cosubstrate to catalyse the ortho-hydroxylation of monophenols to o-diphenols (monophenolase activity), and the oxidation of o-diphenols to o-quinones (diphenolase activity). The catalytic centre of tyrosinase has two copper atoms each coordinated with three histidine residues, similar to of hemocyanin and catechol oxidase [1,2]. These copper atoms have different oxidation and coordination modes, depending on the enzymatic form: Cu 2+ Cu 2+ in E m (metatyrosinase); Cu 1+ Cu 1+ in E d (deoxytyrosinase); Cu 2+ Cu 2+ O 2À 2 in E ox (oxytyrosinase) [2]. This enzyme is responsible for the browning of fruits, vegetables, fungi and crustaceans and is essential in the melanogenesis process of human skin pigmentation for protection from UVinduced damage. Nevertheless, its excessive accumulation can produce hyperpigmentation disorders such as freckles, solar lentigines, ephelide, and melasma [3][4][5], which have led to the development of mechanisms for inhibition of the enzyme [6][7][8][9][10]. Among these compounds are oxyresveratrol [11], 4-hexylresorcinol [12], 4-n-butylresorcinol [13] and ellagic acid [14], which are used in the cosmetics and pharmaceutical industries, although they have recently been characterized as substrates of the enzyme [15][16][17][18][19]. Tyrosinase acts on them when it becomes E ox , which can be attained in the presence of: a) a reductant, such as ascorbic acid (AH 2 ), to convert E m to E d , which evolves to E ox in the presence of oxygen; b) hydrogen peroxide (H 2 O 2 ), to transform E m into E ox directly; c) o-diphenol (only necessary in catalytic quantities if there is ascorbic acid to keep the quantity of o-diphenol constant in the reaction medium) to produce the conversion of E m to E d , which, with oxygen, becomes E ox [20].
In order to develop a potent inhibitor of the enzyme, an HQ-derivate called deoxyarbutin (D-Arb) (4-(oxan-2-yloxy)phenol) was synthesized [27], for which a K i 10 times lower than that for HQ and 350 times lower than that for arbutin was obtained. Initial studies on the safety and efficacy of the tyrosinase inhibitor D-Arb compared with HQ, demonstrate that D-Arb is 23 times more potent [28]. Moreover, a study comparing HQ, arbutins and D-Arb demonstrated that D-Arb diminishes the expression of tyrosinase [29].
For its part, second generation inhibitors of tyrosinase have been synthesized from D-Arb: deoxyfuran (dF), thiodeoxyarbutin (tdA) and flurodeoxyarbutin (fdA), all of which have been demonstrated to be more potent inhibitors than HQ [30] (Fig 1). Regarding the safety of D-Arb compared with arbutins, the Scientific Committee on Consumer Safety (SCCS) has stated that, although the use of D-Arb up to 3% in face creams has been considered safe, the possible formation of HQ make this concentration dangerous [31]. Moreover, the SCCS has stated that the limit in cosmetics should be 2% for face creams and 0.5% in body lotions in the case of α-arbutin [32], and 7% for face creams in the case of β-arbutin (β-Arb) [33].
The toxicity of D-Arb and HQ towards melanosomes, has been studied in vivo and in vitro, observing that the external membrane of melanosomes was broken in the presence of 5% HQ for 10 days, but not in the presence of D-Arb. Moreover, HQ induced a slightly higher amount of hydroxyl free radicals whereas D-Arb showed moderate hydroxyl radical-scavenging activity, meaning that this latter compound could be used as skin lightening agent and antioxidant with lower cytotoxicity [34].
Derivates of HQ and arbutins, such as D-Arb, could release HQ, which can be toxic for bone marrow due to its catalysis to benzene metabolites [35]. Despite the fact that D-Arb is a more potent inhibitor than HQ and arbutins, its stability under certain conditions remains a problem for use in cosmetics and medicines. It has been demonstrated that D-Arb is a thermolabile and photolabile compound in aqueous solutions [36,37]. Thus, new formulations using anhydrous emulsion systems such as polyol-in-silicone have been developed to stabilize D-Arb [38]. Moreover, nanostructured lipid carriers have been tested to improve the application of D-Arb and hence, its depigmenting ability [39].
The fact that tyrosinase shows activity on D-Arb without the addition of H 2 O 2 , AH 2 or catalytic amounts of o-diphenol encouraged us to see whether it really is a potent inhibitor, as previous works indicated, or an alternative substrate, and to characterize it kinetically.

Materials and methods Materials
Mushroom tyrosinase (3130 U/mg) was supplied by Sigma (Madrid, Spain) and purified as previously described [40]. The protein concentration was determined by Bradford's method [41], using bovine serum albumin as the standard. The substrates and solvents used were hydrogen peroxide (H 2 O 2 ), tert-butylcatechol (TBC), L-tyrosine, L-dopa, D-Arb and dimethylformamide (DMF), which were obtained from Sigma (Madrid, Spain). Stock solutions of L-dopa, L-tyrosine, and TBC were prepared in 0.15 mM phosphoric acid to prevent auto-oxidation. Milli-Q system (Millipore Corp, Billerica, MA.) ultrapure water was used throughout. D-Arb was purified to prevent possible contamination by o-diphenol by passing it through a column (1 cm diameter) containing 2g of aluminium oxide suspended in 0.5 M ammonium acetate pH 6.1 [42].This solution was further purified by Chelex-100 chromatography (100-200 mesh, Na + form, Biorad) to remove traces of metal ions. Moreover, D-Arb was solubilised in DMF (8%). The chemical structures of β-Arb and D-Arb are shown in Fig 1.

Spectrophotometric assays
The enzymatic assays were carried out with a PerkinElmer Lambda-35 spectrophotometer, online interfaced with a compatible PC 486DX microcomputer controlled by UV-Winlab software, where the kinetic data were recorded, stored, and analyzed. All of the assays were carried out at 25˚C, using 30 mM phosphate buffer at pH 7.25 and the rest of the experimental conditions are specified in the corresponding figure legend [43,44]. Three repetitions of each experiment were made.

Determination of kinetic parameters
The experimental assays were always carried out in saturating conditions of O 2 [45][46][47]. Initial rate values (V DÀ Arb 0 ), calculated and represented with different concentration of substrate, were fitted to the Michaelis−Menten equation using the Sigma Plot 9.0 program for Windows [48], which provided the maximum rate (V DÀ Arb max ) and the Michaelis constant (K DÀ Arb M ) in the presence or the absence of inhibitor.
Action of tyrosinase on D-Arb after adding hydrogen peroxide or ascorbic acid The action of tyrosinase on D-Arb may occur through the oxy form of the enzyme. As the H 2 O 2 transforms E m into E ox and the ascorbic acid (AH 2 ) reduces E m to E d , which becomes E ox in the presence of oxygen, tyrosinase could act on D-Arb in the presence of any of these compounds.
Determination of 13 C NMR chemical shifts 13 C NMR spectra of D-Arb and β-Arb were obtained on a Bruker Avance 300 MHz instrument using DMSO as solvent (S1 Fig). The δ values were measured relative to those for tetramethylsilane using the carbon signals of the deuterated solvent. The maximum line width accepted in the NMR spectra was 0.06 Hz, so that the maximum accepted error for each peak was ± 0.03 ppm.

Simulation assays
Simulations revealed the kinetic behaviour of the different concentrations of the ligand and enzymatic species involved in the reaction mechanisms proposed for tyrosinase. The respective systems of differential equations were solved numerically for particular sets of values of the rate constants and initial concentrations. Numerical integration is based on the Runge-Kutta-Fehlberg algorithm [49], implemented on a PC-compatible computer program (WES) [50]. Simulations intend to reproduce the qualitative dependences of the integrated mechanism in relation with the experimental data. The kinetic rate constants used in the simulations with Ltyrosine and L-dopa (S18 Fig) agree with previous studies carried out by our group [40,44,51]. In the case of D-Arb, we use k DÀ Arb cat ¼ k 10 = 1.5 s -1 , K DÀ Arb M ¼ ðk 10 þ k À 9 Þ=k 9 = 0.033 mM, which were obtained along this work, and the rest of constants are estimates to reproduce the qualitative experimental dependences.

Molecular dynamics (MD)
The molecular structure of tyrosinase was taken from the Protein Databank (PDB ID:2Y9W, Chain A) [52], corresponding to the deoxy form of tyrosinase from Agaricus bisporus. Information on the chemical structures for β-Arb and D-Arb is available in the PubChem Substance and Compound database [53] through the unique chemical structure identifier CID: 346 for β-Arb and CID: 1745519 for D-Arb. MD simulations were conducted using GROMACS, version 5.0.7 [54]. The protein was oriented with the z-axis running from the copper ions to the outside of the protein structure along the ligand entry/exit path. To parameterize the protein and ions the 53A6 GROMOS force field [55] included in GROMACS package was used. Ligands were parameterized with the same force field using the Automated Topology Builder server [56] version 2.2 using Quantum Mechanical optimization in water at the B3LYP/6-31G Ã level. The hydration free energy in SPC water calculation was validated by the ATB server for the GROMOS force field by using a set of 459 molecules of known experimental values [56]. To further refine the ligand topologies a more extensive validation would be required.
The initial configuration was constructed by placing the ligands in the active site of the protein and the system was solvated with water from a simple point charge (SPC) water model [57] at a density of 33.5 water molecules/nm 3 in a box of 7.2x7.2x7.2 nm 3 size. After a steepestdescent minimization step the system was equilibrated in an NpT ensemble for 2 ns with a time step of 2 fs, using a V-rescale [58] and Berendsen barostat [59] to regulate temperature and pressure respectively. Position restraints were applied on the protein including the copper ions. Production run were performed in the same conditions with a Parrinello-Rahman barostat for 1 ns.

Potential of mean force
The ortho-hydroxy derivatives of β-Arb and D-Arb were constructed with PyMOL 1.8.2.1 [60]. MD simulations were carried out as described above for arbutin and D-Arb to obtain the equilibrated structures of the hydroxy-ligands in the active site. To calculate potentials of mean force (PMF) the final configuration of the production run was used as the initial structure for the "pulling" simulation. The centre of mass distances (COM) between the carbon atom at para position of the aromatic ring of each ligand and the centre of mass of the copper ions were calculated, and then pulled away from the copper site along the z-axis in a time of 500 picoseconds (ps) using a spring constant of 3000 kJ -1 nm -2 and a pulling rate of 0.0034 nm ps -1 . This pulling was carried out to create a set of starting configurations. The values of the spring constant and pulling rate were chosen after testing many combinations to find an optimal pair of numbers that did not cause instabilities in the system. A total of 40 windows were used distributed within a 1.7 nm COM separation. In each window, a short equilibration of 1 ns was followed by a 5 ns production run for umbrella sampling. A harmonic force with a force constant of 3000 kJ mol -1 nm -2 was applied for each umbrella-sampling window. Additional windows were generated to improve sampling. Thus, a total of 160 and 254 windows were finally used for β-ArbOH and D-ArbOH, respectively. To generate the potentials of mean force, the weighed histogram analysis method (WHAM) was used [61].

D-Arb apparently inhibits the monophenolase and diphenolase activities of tyrosinase
When these activities of the enzyme on L-tyrosine and L-dopa are studied in the presence of D-Arb, apparent inhibition is observed (Fig 2A and 2B). When the type of inhibition was studied (Fig 2A Inset and 2B Inset), the results showed an apparent competitive inhibition. The K app i values are shown in Table 1.

Total oxygen consumption test
In previous works [18,26], we developed a test to elucidate whether a compound is a substrate or inhibitor of tyrosinase. The method consists of a spectrophotometric measurement at 410 nm of the formation of the o-tert-butylquinone generated by the action of tyrosinase on TBC in the absence (recording "a") and presence of increasing concentrations of D-Arb (recordings "b-d") (Fig 3). The absorbance at 410 nm and the reaction time increase as the concentration of D-Arb increases, which indicates that a product with a higher absorbance than that of otert-butylquinone comes from D-Arb. Moreover, the formation of a new product is detected in the spectrophotometric recordings of the action of tyrosinase in a medium with TBC and D-Arb (Fig 3 Inset).
Action of tyrosinase on D-Arb after adding hydrogen peroxide or ascorbic acid E m becomes E ox in the presence of H 2 O 2 [62], and it was expected that this form of tyrosinase would be able to hydroxylate D-Arb. The result of the action of tyrosinase in the presence of H 2 O 2 is shown in S2 Fig and the

Action of tyrosinase on D-Arb without the addition of hydrogen peroxide or ascorbic acid
The experiments represented in Fig 4A show that tyrosinase acts on D-Arb without any reductant or H 2 O 2 , behaviour which has not been observed previously in similar compounds https://doi.org/10.1371/journal.pone.0187845.g002 Table 1. Type and kinetic constants for the apparent inhibition of tyrosinase by D-Arb and β-Arb.   (monophenols). E ox is the only form of tyrosinase which can hydroxylate D-Arb, but the cycle of E ox has to be completed to maintain the activity (Fig 5). According to the experimental and simulated results shown in Fig 4B, the stoichiometry of product (P) formation with respect to the oxygen consumed would be 1:1.

Compound
The initial formation rate of P is given by the equation: As the enzyme is saturated by oxygen ([O 2 ] 0 ! 1) [45][46][47], we obtain: k 11 k À 15 ðk À 9 þk 10 Þ k 9 k 15 ðk 10 þk 11 Þ þ k 11 k À 15 ðk À 9 þk 10 Þ k 9 k 12 k 15 ðk 10 þk 11 Þ ½DÀ Arb 0 þ k 11 ðk À 9 þk 10 Þ as k 11 >> k 10 , so: so being As K DÀ Arb M ¼ ðk À 9 þk 10 Þ=k 9 and K 12 = k −12 /k 12 , if we consider that K 9 = k −9 /k 9 could have a similar value to K 12 (since they refer to the bond to the same substrate of two different forms of the enzyme, oxy and metatyrosinase, but the same active site), K DÀ Arb M would be similar to K 12 , since k 10 has a low value. If this constant has a high value, K DÀ Arb M would be higher than K 12 , as it happens in the case of true substrates of tyrosinase [63,64]. where The following characteristics of the action of the enzyme on D-Arb should be noted: There is no lag period because the product (P) is a stable quinone (Fig 4A). Moreover, D-Arb was purified by passing it through a column containing aluminium oxide in order to eliminate possible contamination with o-diphenol. S4 Fig shows the spectra before (a) and after (b) purification in the column, the oxidation by sodium periodate (the absorbance would change in the presence of o-diphenol) (S4 Fig Inset A) and the activity of tyrosinase on the eluted D-Arb with no lag (S4 Fig Inset B). The absence of variation in the absorbance after the oxidation with sodium periodate and the absence of the lag period in the activity of tyrosinase on the eluted D-Arb demonstrate that there was no contamination by o-diphenol.

Kinetic characterization of D-Arb as substrate of tyrosinase
The experiments mentioned above demonstrate that E ox is able to act on D-Arb and, so, the proposed mechanism in

Formation and properties of the product of the enzymatic reaction (P)
The enzyme hydroxylates D-Arb to D-ArbOH in a step controlled by k 10 , and subsequently oxidizes this compound to a quinone (P), in which the p-quinoid canonical form contributes significantly to the hybrid resonance, in a stage controlled by k 11 . The substrate is consumed by the enzymatic action (S5 Fig Inset), which allows the molar absorptivity value (ε) to be determined (S5  (Table 2). Note the low value of k DÀ Arb cat , which agrees with the chemical shift values of the carbon with the phenolic hydroxyl group ( Table 2). The low value of K DÀ Arb M ( Table 2) also agrees with the low value of k DÀ Arb cat and the high value of k 9 , which reflects the hydrophobicity of D-Arb. The differences with β-Arb (Table 2) is that this compound have many hydroxyl groups, which interact with residues of the active site of the enzyme.
Generation and disappearance of the oxy form of tyrosinase The system changes when a micromolar range of H 2 O 2 is added (Fig 5). The reason for this is that H 2 O 2 increases the concentration of E ox and so the activity is higher (Fig 6)

Computational simulations of substrates binding
The chemical structures of β-Arb and D-Arb (Fig 1) suggest that the compounds have a very different behaviour with respect to environmental polarity. β-Arb contains four hydroxyl groups, D-Arb lacks, so that the partition coefficient (log P) is -1.35 and +2.40 for β-Arb and D-Arb, respectively, as calculated by the XLogP3 program [66]. These values indicate that β-Arb is preferably partitioned in water while D-Arb is partitioned in octanol. Therefore, polar interactions, like hydrogen bonds with the proteins and the water molecule of the solvent, are very important for defining the ligand binding interactions that take place in the substrate binding pocket. The oxy form of tyrosinase was solvated with water in a rectangular box. MD  in a radius of 4 Å from the ligand molecule are depicted to exclude water molecules that are outside the interaction distance. Of note is the difference in the amount of water around both ligands. β-Arb is surrounded by many more water molecules than D-Arb, as might be expected from their polarity differences.
Another view of Fig 7 is shown in S12 Fig to highlight the position of the oxane ring of D-Arb, which is more restricted to the cavity of the active site, while the pyranose ring of β-Arb is more exposed to the outermost part of the active site. It is therefore plausible that the amount of water in the active site might be an important factor to take into consideration to modulate the affinity of ligands. Usually, water molecules are removed from the protein in docking studies. However, some structurally conserved water molecules participating in substrate binding are found in the active site of the crystallized tyrosinase, and have been proposed as being involved in substrate binding and catalysis [67][68][69]. All these results reveal the importance of water molecules in the process of binding to the active site.

Computational simulations of o-diphenol binding
This study has demonstrated that the o-diphenol product of D-Arb is not released from the enzyme but is oxidized to the corresponding quinone, while other substrates, like β-Arb, are hydroxylated by oxytyrosinase, releasing o-diphenol. This result suggests that the o-diphenol product of D-Arb is somehow trapped in the active site long enough to be oxidized before being released from the enzyme. In a search for possible reasons to explain this behaviour the o-diphenol products of D-Arb (D-ArbOH) and β-Arb (β-ArbOH) were studied by MD in the met form of tyrosinase.
The distribution of water molecules along the z-axis from copper atoms to the outside of the cavity of the substrate binding pocket of metatyrosinase was determined in the presence of bound o-diphenols. For this purpose, the quantity of water molecules in a cylinder of 0.6 nm radius centred at the o-diphenol molecule was determined. S13 Fig shows the histograms of the distribution of the water molecules. β-ArbOH, bound to the copper atoms goes towards the water layer, the pyranose ring being surrounded by water molecules (S13A Fig). In contrast, D-ArbOH interacts with water almost exclusively trough ether oxygen atoms. The oxane ring excludes water molecules due to its high hydrophobicity and the oxane ring is rotated with respect to the phenyl ring increasing the distance from the water layer and avoiding the interaction with water in the outer region of the binding pocket (S13B Fig). It is feasible to think that D-ArbOH has an energetic barrier formed by the water layer, which would slow down its release from the cavity, contributing to keeping D-ArbOH longer in the active centre and facilitating its oxidation to quinone.
Potential of mean force (PMF) profiles for the dissociation process of o-diphenols from metatyrosinase along the z-axis are presented in Fig 8. The greatest z-distance values correspond to the o-diphenols in the bulk solution. One of the differences between the two o-diphenols that deserves to be highlighted is that the PMF profile of D-ArbOH shows an energy barrier of about 3 kcal/mol at a distance of 0.8 nm, which is not observed in the β-ArbOH PMF profile. Attempts to calculate the free energy of the o-diphenol dissociation provide very high values. However, it has to be taken into account that the potential of mean force includes the contribution of all the components of the system. Since the protein is computed in MD under position restraint, the main uncertainty arises from the water molecules which have freedom of movement and change their electrostatic interactions.
To throw light on the events occurring in the energy barrier of the PMF profile of D-ArbOH (0.7-1 nm of Fig 8B) the most relevant hydrogen bond interactions were determined for both o-diphenols using GROMACS tools (S14 Fig). One important thing to mention is that the amount of water bound to β-ArbOH is much higher all along the trajectory compared with D-ArbOH (S14 Fig). β-ArbOH forms a total of 9 hydrogen bonds at the active site (z = 0.42 nm), decreases to 8 and finally reaches 12 in the bulk solution (z > 1.1 nm). However, D-ArbOH forms about 3 hydrogen bonds at the active site, decreases to 1-2 above 0.6-0.75 nm zdistance and increases to 5 in the bulk solution. This means that the pyranose ring of β-ArbOH is always bound to water, as was described for β-Arb binding to oxytyrosinase (Fig 7A). The hydrogen bonds between all the oxygen atoms of D-ArbOH and different aminoacid residues and water were measured. Among then, only significant differences were found in the 0-7-1 nm z-distance between the phenolic groups of D-ArbOH and water molecules (S14B Fig) and N260  (S14C Fig). It can be seen that the hydroxyl group of C2 (C 2 -OH) forms 2 hydrogen bonds at the active site (0.41 nm) while that of hydroxyl group at C1 (C 1 -OH) does not (S14B Fig). Between 0.6 and 0.72 nm, the loss of hydrogen bonds appears to be concomitant with the formation of one hydrogen bond with N260 and vice versa (S14C Fig). Afterwards, C 1 -OH displaces C 2 -OH from the N260 interaction (0.76 nm) and at 0.86 nm the C 1 -OH releases N260 and binds to water (S14C Fig). Above 1 nm, D-ArbHO is too far from N260 and only interacts with water. All these events occur to overcome the energy barrier of the PMF (Fig 8B).
It has been shown that the pyranose ring of β-ArbOH is always linked to water molecules of the bulk solution, even at the binding site (S12A, S13A and S15A Figs). S15B-S15F

Discussion
D-Arb is an apparent inhibitor of tyrosinase, which is why it is used as depigmenting agent in cosmetics and medicines. Its effectiveness as inhibitor of the activity of the enzyme on L-tyrosine and L-dopa [70] is higher than that of its precursors α and β-Arb, which were studied by our group in a previous work [26]. The aim of synthesising D-Arb was to obtain a more effective and less cytotoxic inhibitor than HQ and arbutins [28], although the SCCS decided in 2016 that D-Arb could only be used at up to 3% [31]. The kinetic study carried out by us supports the view that this compound is indeed effective at lower concentrations.
When the type of inhibition of tyrosinase by D-Arb was studied, an apparent competitive inhibition in both activities was observed (Fig 2A and 2B Inset). A mechanism to explain the action of tyrosinase on D-Arb in the presence of L-tyrosine is proposed in S16 Fig, while a possible mechanism for the action of tyrosinase on D-Arb and L-dopa is shown in S17 Fig Simula-tion of the mechanisms provided similar results (S18A and S18B Fig) to those obtained in Fig  2A and 2B, which are described in Table 1. The inhibition was seen to be competitive and the constant were K app I = 0.070 ± 0.004 and K app I = 0.062 ± 0.004 for the monophenolase and diphenolase activities, respectively.
In addition, the total oxygen consumption test (Fig 3A) demonstrated that D-Arb is a substrate of tyrosinase, since the absorbance and the reaction time of increase with increasing concentrations of this compound. Furthermore, a new maximum was obtained in the spectrophotometric recording when the enzyme acts on a mixture of TBC and D-Arb (Fig 3B).
D-Arb is a derived from a monophenol, so the presence of E ox is necessary to run the catalytic cycle. This form of the enzyme can be generated by H 2 O 2 , AH 2 and TBC (o-diphenol) and its catalytic activity is shown in Figs S2, S3, 3A and 3B respectively. Hence, various mechanisms are proposed to explain the action of tyrosinase in the presence of H 2 O 2 , AH 2 and TBC in S19, S20 and S21 Figs, respectively. However, the most interesting observation is that tyrosinase can act on D-Arb without the addition of these compounds (Fig 4A).
Therefore, the mechanism proposed in Fig 5 shows that tyrosinase can act directly on D-Arb through its E ox form, hydroxylating D-Arb to its o-diphenol and, subsequently, it does not release the o-diphenol, but transforms it to its quinone, so, the catalytic cycle is completed.
Note that this case differs from the action of tyrosinase on β-Arb [26]. In this previous work with β-Arb, we did not add o-diphenol, H 2 O 2 or AH 2 to the medium, but 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH). This potent nucleophile attacks the o-quinone and, after various reactions, leads to the accumulation of o-diphenol in the medium, which, in turn, activates the system, transforming E m to E d , which becomes E ox in the presence of O 2 [71,72].
In the mechanism described above (Fig 5): a. There cannot be lag period in the accumulation of the product (P), such as that shown in Fig 4A. b. Activity should be increased in the presence of micromolar quantities of H 2 O 2 , since E m becomes E ox , as demonstrated in Fig 6A. Note that the system already contains around 20% E ox at the beggining, according to the results. This percentage agrees with the values (3-30%) described in the bibliography [73]. c. The system responds to the addition of catalase. This enzyme breaks down H 2 O 2 , and so the E ox becomes E m and the activity disappears as is shown in Fig 6B. d. V P 0 seems to have a linear dependence on the enzyme concentration and a hyperbolic dependence on the substrate concentration (Fig 4A).
The different tests mentioned above ("a-d") can be carried out by numerical integration (S22, S23, S6 and S24 Figs, respectively) of the mechanism described in Fig 5. Thus, the obtained data confirm us the proposed mechanism, and by fitting the initial rate values by non-linear regression to Eq 7, we obtain the k DÀ Arb cat and K DÀ Arb M values shown in Table 2. The k DÀ Arb cat agrees with the chemical shift values of the carbon with the phenolic hydroxyl group and is very similar to those of α and β-Arb ( Table 2). The K DÀ Arb M value is very low compared to that of β-Arb (Table 2), which can be explained by the hydrophobicity of the pyranose ring, which, in turn, increases the affinity of tyrosinase towards the substrate.
The characteristics shown by D-Arb acting as substrate of tyrosinase support our proposed mechanism. Regarding the monophenolase activity, we propose that the monophenol makes a nucleophilic attack on E ox through the oxygen of OH group, transferring a proton to the peroxide group and giving rise to a hydroperoxide group, which, in turn, electrophilically attacks the ortho position of the benzene ring. The originated o-diphenol is in an axial/equatorial position and cannot be oxidized by the enzyme, since the oxygen orbitals are not coplanar with the copper atoms [42,51]. Subsequently, one of the copper bonds is broken and, in this way, when the o-diphenol is only bound to a copper atom, it can be released and the complex E m D-ArbOH can evolve to E m + D-ArbOH or bind in diaxial position to be oxidized, giving rise to E d + P. We emphasise the importance of this stage for the enzyme to act on monophenols.
D-Arb as a substrate differs from those described to date [18,26] since it involves the step from E m D-ArbOH to E d + P as the only way the product can be generated. The above experiments and the previous works confirm the proposed mechanism (Fig 5) [74]. Therefore, in summary: a) E ox is the only form able to act on D-Arb; b) H 2 O 2 (μM) increases the concentration of E ox and hence, the activity of the enzyme; c) pre-incubation with catalase decreases the activity of the enzyme. When D-Arb is compared with other alternative substrates of tyrosinase such as HQ or β-Arb, it can be observed that this compound does not need any compound (H 2 O 2 , AH 2 or o-diphenol) to show activity.
The computational simulations showed that the N260 residue actively participates in the ligand binding process. Many authors have proposed N260 as a key residue in substrate binding in mushroom tyrosinase [75][76][77][78][79][80][81][82]. Besides, it has been demonstrated that an asparagine is essential to properly orientate the conserved water for proton transfer from a monophenol [69]. The presence of structural water in the copper centre of tyrosinase has been revealed in different crystallized tyrosinases and catechol oxidases, for example in sweet potato COx [83], Streptomyces tyrosinase [84], Agaricus bisporus tyrosinase [52], Manduca sexta tyrosinase [85] and tyrosinase from Bacillus megaterium [67].
The present study has demonstrated the importance of water molecules not only for their direct participation in substrate binding through aminoacid residues (i.e. N260), but also for acting as solvents facilitating or hindering substrate binding and/or dissociation. The simulations results point to the different behaviour of D-Arb and β-Arb in the active centre of tyrosinase as a result of their differences in polarity. Consequently, their interactions with the solvent might be a critical issue for defining the binding properties. It has been shown that D-Arb faces a barrier to scape of the binding pocket, partly composed of the solvent, which is mostly excluded from the binding cavity due to the presence of a hydrophobic environment created by D-Arb. This barrier is absent in the case of β-Arb, which is always in close contact with water molecules through hydrogen bond interactions. Taking into account all of the above it is feasible to propose that the high hydrophobicity of the oxane ring of D-Arb might contribute to keeping the o-diphenol product in the copper centre long enough to be further oxidized to the corresponding quinone. On the other hand, the o-diphenol product of β-Arb, with a polar pyranose ring, might be readily released from the active site to the water phase, avoiding oxidation to quinone.
In conclusion, this work demonstrates that D-Arb only apparently inhibits tyrosinase, since it is really an alternative substrate of the enzyme. This compound is the only substrate described to date that is hydroxylated by the enzyme without the generated o-diphenol being released to the medium. On the contrary, it is oxidized by the enzyme, which becomes E d and releases the quinone (P). This imply that D-Arb should be used with caution as depigmenting agent, since the generated quinone could react with potent nucleophiles, such as cysteine and glutathione, disturbing the redox state of the cell.  ] were used to simulate the activity of the enzyme on D-Arb (0.2 mM). The rate constants were: k 8 = 2.3 x 10 8 M -1 s -1 , k -8 = 1.07 x 10 3 s -1 , k 9 = 1.6 x 10 5 M -1 s -1 , k -9 = 3.8 s -1 , k 10 = 1.5 s -1 , k 11 = 400 s -1 , k 12 = 1.6 x 10 5 M -1 s -1 , k -12 = 3.8 s -1 , k 15