Study of tyrosine and dopa enantiomers as tyrosinase substrates initiating l‐ and d‐melanogenesis pathways

Tyrosinase starts melanogenesis and determines its course, catalyzing the oxidation by molecular oxygen of tyrosine to dopa, and that of dopa to dopaquinone. Then, nonenzymatic coupling reactions lead to dopachrome, which evolves toward melanin. Recently, it has been reported that d‐tyrosine acts as tyrosinase inhibitor and depigmenting agent. The action of tyrosinase on the enantiomers of tyrosine (l‐tyrosine and d‐tyrosine) and dopa (l‐dopa and d‐dopa) was studied for the first time focusing on quantitative transient phase kinetics. Post‐steady‐state transient phase studies revealed that l‐dopachrome is formed more rapidly than d‐dopachrome. This is due to the lower values of Michaelis constants for l‐enantiomers than for d‐enantiomers, although the maximum rates are equal for both enantiomers. A deeper analysis of the inter‐steady‐state transient phase of monophenols demonstrated that the enantiomer d‐tyrosine causes a longer lag period and a lower steady‐state rate, than l‐tyrosine at the same concentration. Therefore, d‐melanogenesis from d‐tyrosine occurs more slowly than does l‐melanogenesis from l‐tyrosine, which suggests the apparent inhibition of melanin biosynthesis by d‐tyrosine. As conclusion, d‐tyrosine acts as a real substrate of tyrosinase, with low catalytic efficiency and, therefore, delays the formation of d‐melanin.


Introduction
Tyrosinase (monophenol, o-diphenol: oxygen oxidoreductase, EC 1.14.18.1.) is a copper enzyme that is widely distributed among microorganisms, plants, and animals, in which it shows great similarity between different species [1,2]. It mainly catalyzes two types of reactions: the hydroxylation of monophenols to o-diphenols and the oxidation of these o-diphenols to their corresponding o-quinones. Both reactions use molecular oxygen as oxidant substrate [3,4]. The monophenolase activity of tyrosinase is the rate-limiting step in the melanin biosynthesis pathway (Fig. S1A).
Several works have studied the stereoselectivity of different tyrosinases [5][6][7][8], but no complete kinetic study has been made of the steady-state and transient phase of this process. Steadystate studies of tyrosinase carried out several years ago indicated that the active site of the enzyme had some degree of stereoselectivity [5]. Research with the enzyme obtained from hamster melanoma indicated that the V max values were the same for the stereoisomers l-and d-tyrosine and l-and d-dopa. However, the K M values were higher for the d-isomers [6]. Tyrosinase purified from human melanoma cells showed a higher K M for the d-dopa enantiomer (3 mM) than for the l-dopa enantiomer (0.5 mM) [7]. Although the participation of d-tyrosine in melanogenesis has been studied, there have been few reports on the subject [8]. Applied Biochemistry As melanoma tyrosinase VH 421 cells showed less affinity for d-dopa than for l-dopa, it was proposed that d-dopa should be added as a cofactor in l-tyrosine assays [9]. Stereoselectivity has also been described in a tyrosinase resistant to organic solvents of Streptomyces sp. [10]. Moreover, mutations in the tyrosinase gene of Ralstonia solanacearum led to an increase in the k cat for d-tyrosine [11]. Mushroom tyrosinase also showed stereoselectivity when acting on betaxanthins [12]. Using the enzyme from avocado, the same value for V max was described for l-tyrosine and d-tyrosine [13], and the same pattern was obtained for tyrosinase from Vibrio tyrosinaticus [14], but not for tyrosinase from Pseudomonas sp. [15]. Studies with tyrosinase from Bacillus megaterium acting on dopa reveal that (K D M > K L M ) in aqueous and organic solvents, for the wild-type [16] and the R209H [17] and V218F [18] mutants.
In addition, it was demonstrated that the inactivation of tyrosinase in its action on l-dopa and d-dopa is also stereoselective, the enzyme showing stereoselectivity in the substrate binding (K D M > K L M ), but not in the suicide inactivation constant [19]. A similar event occurred in the inactivation of tyrosinase acting on l-and d-ascorbic acid as the enzyme showed greater affinity for l-ascorbic, although the maximum inactivation rate was the same [20]. Recently, d-tyrosine has been described as a regulator of melanin biosynthesis through the competitive inhibition of tyrosinase activity and the amino acid has been proposed as a possible new skin whitening agent [21].
Enantioselectivity studies of tyrosinase from the creosote bush (Larrea tridentata) led to the suggestion that polyphenol oxidase hydroxylates only to the (+) larreatricin form [22]. However, it has recently been shown that this enzyme acts on the two isomers: (+) larreatricin and (−) larreatricin [23]. As regards the kinetics, the values obtained for K M and k cat do not show the same relationship with the values from NMR experiments, as occurs in the action of mushroom tyrosinase on l-tyrosine and d-tyrosine and l-dopa and d-dopa [24]. As discussed previously [24], the enzyme has greater affinity (lower K M ) for the L forms. However, the V max are the same for both stereoisomers because this parameter depends on the nucleophilic potency of the oxygen of the phenolic hydroxyl, and the NMR of the stereoisomers is the same for the carbon that supports the hydroxyl group. The NMR for larreatricin is described in the Supplementary Material of Ref. [23] and the chemical shift values, δ, are the same for positions 4 and 4' (157.01 ppm), which are the carbons bearing the hydroxyl and which are similar to carbon C-4 of l-tyrosine and d-tyrosine (158.88 and 158.89 ppm, respectively) [24]. The authors of Ref. [23] tested four enzymes: larreatricin hydroxylase from L. tridentata (LtPPO), apple tyrosinase (MdPPO1), the fungal tyrosinase from Agaricus bisporus (AbPPO4), and the proteolytically activated form of the same (AbPPO4-act). The data shown in Table 1 of Ref. [23] do not correspond to those described in Ref. [24] perhaps because the insolubility of the substrate (larreatricin), the presence of SDS, and the presence of methanol make the measurements difficult to compare. On Highlights r The action of tyrosinase on the enantiomers of tyrosine (ltyrosine and d-tyrosine) and dopa (l-dopa and d-dopa) was studied for the first time focusing on quantitative transient phase kinetics.
r Post-steady-state transient phase studies revealed that ldopachrome is formed more rapidly than d-dopachrome, due to the lower values of Michaelis constants for lenantiomers than for d-enantiomers, although the maximum rates are equal for both enantiomers.
r Enantiomer d-tyrosine causes a longer lag period, and a lower steady-state rate, than l-tyrosine at the same concentration. Therefore, d-melanogenesis from d-tyrosine occurs more slowly than does l-melanogenesis from ltyrosine.
the other hand, it has been reported that human tyrosinase do not oxidize d-tyrosine, that mushroom tyrosinase is not stereoselective on R-and S-rhododendrol, and that human tyrosinase is more efficient on R-rhododendrol [25].
The availability of a highly sensitive method for measuring monophenolase and diphenolase activities [26,27] enabled the stereoselectivity of mushroom, pear, and strawberry tyrosinase to be studied in the steady state [24,28,29]. However, in the present paper, we study, for the first time, l-melanogenesis and d-melanogenesis in the formation of l-dopachrome and d-dopachrome, respectively, from the approaches of the inter-steady-state transient phase for monophenols and poststeady-state transient phase for monophenols and diphenols. The kinetic study should help clarify the recent controversy regarding the role of d-tyrosine in melanogenesis and the methodology for kinetic characterization of the stereoselectivity of tyrosinases.

Materials
Mushroom tyrosinase (2,687 U/mg) was purchased from Sigma (Madrid, Spain) and purified as described previously [30]. Bradford's method was used to determine the protein content using bovine serum albumin as standard [31].
l-Tyrosine, l-dopa, d-tyrosine, and d-dopa (Fig. S1B) were obtained from Sigma. Stock solutions of these compounds were prepared in 0.15 mM phosphoric acid to prevent autooxidation. Milli-Q system (Millipore Corp, Billerica, MA) ultrapure water was used throughout.

Methods
Enzyme activity assay. The enzymatic activity was determined by spectrophotometric techniques using Perkin-Lambda-35 spectrophotometer interconnected online to a computer, in which the kinetic data were recorded, stored, and subsequently

Substrate
Steady-state constants 13  Steady-state assays: diphenolase activity on l-dopa and d-dopa. The steady-state rates of tyrosinase in its action on l-and d-dopa were obtained by measuring the dopachrome formation over time at 475 nm [35]. The experimental conditions are detailed in the figure caption.
Post-steady-state assays: diphenolase activity on l-dopa and d-dopa. The absorbance variation over time was followed at 475 nm, with high enzyme (95 nM) and low substrate concentrations . These conditions provide a first-order kinetic behavior with respect to the substrate. The enzymatic system evolved in the post-steadystate transient phase until the substrate was fully consumed. This type of transient phase is similar to that seen during active enzyme depletion, in the irreversible inhibition [36] or suicide inactivation of enzymes [37].
Steady-state assays: monophenolase activity of tyrosinase on l-tyrosine and d-tyrosine with o-diphenol (l-dopa or d-dopa) added at t → 0. Steady-state rates for the action of tyrosinase acting on l-and d-tyrosine were obtained by measuring the dopachrome formation over the time at 475 nm. A lag period was avoided by adding the necessary quantity of o-diphenol to reach the steady-state, [D] ss , before adding the enzyme to begin the enzymatic reaction [35].
[D] ss = R [M] 0 , where R = 0.042 in the conditions used in the reaction [35]. In this way, the steady-state phase was reached at the beginning of the reaction, t → 0.
Post-steady-state transient phase: monophenolase activity of tyrosinase on l-tyrosine and d-tyrosine. The variation of absorbance over time was measured at 475 nm. Having a high enzyme concentration (305 nM) and low substrate concentration (20 nM , first-order kinetic behavior with respect to the substrate was induced [36,37], in the same way as the diphenolase activity (section Post-Steady-State Assays: Diphenolase Activity on l-Dopa and d-Dopa).
Inter-steady-state transient phase: monophenolase activity of tyrosinase on l-tyrosine and d-tyrosine with no addition of o-diphenol (l-dopa or d-dopa) at t → 0. Enzyme activity was measured at 475 nm, and the formation of dopachrome, without adding o-diphenol before the enzymatic reaction begins, was followed. The concentration of enzyme was 20 nM. The recording pointed to a lag period (τ ) between the initial (t → 0) and final steady-states, during which o-diphenol was accumulated in the medium until it reached [D] ss = R [M] 0 in the final steady state [38]. Such an inter-steady-state phase is also characteristic during the slow inhibition of enzymes in the presence of substrate [39].
Kinetic data analysis.  [40], which provided the maximum rate (V max ) and the apparent Michaelis constant (Kapp). The REFERASS computer program was used to obtain the rate equations of these mechanisms [41], as detailed in the Appendix of the Supplementary Material (Eqs. A1-A14). Some results of the corresponding experimental procedure can be seen in the figures of the Supplementary Material (Figs. S1 and S2).
NMR assays. The 13C-NMR spectra of the l-tyrosine, dtyrosine, l-dopa, and d-dopa considered here were obtained in a Varian Unity spectrometer of 300 MHz [24,42]. The electronic density of a carbon atom is correlated with the value of its chemical displacement (δ) in 13 C-NMR [43,44]. Moreover, the nucleophilic power or electron-donating capacity of the oxygen atom of one phenolic hydroxyl has been seen to be inversely correlated with the δ value obtained in 13 C-NMR in the case of the carbon atom that binds to the hydroxyl group [45].

Results and Discussion
In previous studies of the steady-state kinetics of tyrosinase, it was shown that tyrosinase has greater affinity for the l-isomers than for the d-isomers, that is, the Michaelis constant (K M ) was lower for l-enantiomers. However, the maximum rates (V max ) were the same for both isomers. These data agree with the values from NMR experiments, since the electron density of the carbons that support the phenolic hydroxyls is identical for the l-and d-isomers (δ 3 and δ 4 are equal), and Applied Biochemistry thus the nucleophilicity of the oxygens is the same and the nucleophilic attack determines the rate [24,28,29]. However, the time dependence of melanogenesis differs between the l-and d-isomers of tyrosine and is not well explained in the literature where the conclusions are occasionally erroneous [24], as it is shown in Fig. 1 of Ref. [24]. The spectrophotometric recordings for the product generation with time in this figure did not achieve the real steady state in short period times due to the enzyme concentration added to the reaction was low. Because of that, apparently, the lag period for d-tyrosine was shorter than l-tyrosine. This situation can be avoided adding higher enzyme concentrations to the assays reaching the steady state in shorter periods as is presented in this paper (see below) [24].

Steady-state studies
In these steady-state tests, the accumulation of product versus time was measured in such a way that the amount of substrate remained practically constant, since the measurements were made at short times.
Experimental results of diphenolase activity. The action of tyrosinase on l-dopa and d-dopa showed hyperbolic behavior (Figs. S2A and inset.). Analysis of the data by means of nonlinear regression according to the Michaelis equation (Eq. A2) provided the corresponding values of . These data agree with the values from NMR experiments (Table 1) and the previously published results [24].
To explain the results shown in Table 1, we propose a kinetic mechanism (Fig. 1), in which there are no M or M-enzyme intermediates.
Experimental results of monophenolase activity. It is known that tyrosinase can act on both stereoisomers of monophenols (Fig. S1B), such as l-tyrosine and d-tyrosine [24,28,29]. In the monophenolase activity of tyrosinase, the spectrophotometric recording of the absorbance with respect to time at 475 nm shows a lag period (Fig. 2) [46]. The lag period is the time taken by the enzyme, acting on l-tyrosine or d-tyrosine, to accumulate a certain amount of o-diphenol in the steady-state ([D] ss [D] ss ) [47]. In our study, the amount of [D] ss [D] ss differed according to whether the l-or d-isomer was being studied, and always must fulfil Eq. (1):  [47,48]. For l-isomer: For d-isomer: The values of R L and R D were determined as indicated in Fig. 2. In the steady state, the excess of accumulated o-diphenol is oxidized by periodate, and is converted into dopachrome, and the value of [D L ] ss or [D D ] ss is calculated from the increase in absorbance at 475 nm taking into account the respective molar absorptivities (Fig. 2) [35,48]. These data facilitate the study of monophenolase activity because it is possible to add to each monophenol concentration the quantity of o-diphenol  [24]. Table 1 shows the results obtained. Note that the difference in K app M values for the monophenolic stereoisomers is greater than in the case of o-diphenolics.
To explain the results shown in Table 1 for monophenols, the kinetic mechanism depicted in Fig. 1 is proposed.

Studies of post-steady-state transient phase
In these experiments, the enzyme rapidly reaches the steady state at t → 0 and the substrate concentration varies so that the enzyme is in a post-steady-state transient phase, until the substrate is depleted.
The most straightforward kinetic would be: where S indicates monophenol (M) or o-diphenol (D) and Cr indicates the product of the reaction, dopachrome. The substrate concentration through the action of tyrosinase would evolve as follows: and the velocity of dopachrome formation would be: Integrating Eq. (5): gives Eq. (8): Integrating Eq. (6), taking into account Eq. (8): gives Eq. (10): where λ is the catalytic power. Experimental results of diphenolase activity.
which agrees with the experimental data (Tables 1 and 2).

Effect of substrate stereoisomerism on the lag period of monophenolase activity of tyrosinase. Study of inter-steady-state transient phase
In its action on monophenols, tyrosinase shows a lag period before it reaches the final steady state. This lag period "τ " is the time necessary to accumulate in the medium a certain concentration of o-diphenol in the medium as a result of the chemical evolution of o-dopaquinone [46][47][48]. This concentration of o-diphenol is described by Eq. (1). The analytical expression of the lag period is obtained by applying the balance of matter in the steady-state and gives [46]: (14) In For each of the isomers, the expression of the lag period takes the form of: (15) and

Ratio between Michaelis constants and catalytic powers of the enantiomers of tyrosine and dopa
Steady state Post-steady-state transient phase  (Table 1), for the same monophenol concentration: Furthermore, the equation for the lag phase, [Eqs. (15) and (16)], gives: Consequently, according to Eqs. (15) and (16), the lag period is greater for isomer d-than for l-, which means that in the qualitative study published by our group previously, the enzymatic reactions had not reached the steady-state. [24] Inter-steady-state transient phase of the monophenolase activity without addition of o-diphenol at t → 0: experimental results. The assays of monophenolase activity of tyrosinase acting on l-or d-tyrosine show a lag period that lasts until the system reaches the final steady state (Eqs. 15 and 16). This lag period increases when the substrate concentration increases, as shown in Fig. 7 for the l-isomer. Similar behavior was observed for the d-isomer but, in this case, the lag periods were longer and the steady-state rate was lower (Fig. 8). A comparison of the experiments involving the l-and d-tyrosine forms using the low and same concentration of enzyme, as well as the same concentration of substrate, shows that the amount of dopachrome that leads to a given amount of melanin in a given time is much lower in the case of the d-isomers [21,25]. This would explain (Eqs. 15, 16, and 20) why the d-tyrosine form is sometimes not considered as a tyrosinase substrate [21,25].
In this work, dopachrome is considered to be the final product of the proximal phase of melanogenesis. However, dopachrome is not completely stable, but evolves into melanin, and the same occurs with dopaminochrome [48][49][50], during the distal phase of melanogenesis. As can be seen in Fig. 9, the chemical evolution of l-dopachrome and d-dopachrome shows the same kinetics. Therefore, the only difference lies in the proximal phase of melanogenesis because the formation of d-dopachrome from d-tyrosine is slower than l-dopachrome production from l-tyrosine.
Recently, the study of tyrosinase stereoselectivity has grown in importance due to: (i) its reactivity toward d and l-tyrosine, its physiological substrate [21]; (ii) the action of human tyrosinase on rhododendrol, which induces leukoderma [25]; and (iii) the origin of antiviral and anticancer properties of creosote bush lignans in vivo, which has also been studied, considering the action of polyphenol oxidase on (+) and (−) larreatricin. [22,23] It has been proposed that d-tyrosine negatively regulates melanin biosynthesis by competitively inhibiting tyrosinase activity in human MNT1 melanoma cells and primary human melanocytes [21]. However, it must be taken into account that these experiments were carried out with the same concentration of substrate (l-tyrosine or d-tyrosine), the same concentration of enzyme and always the same reaction time. According to the data presented in Ref. [21], the effect of dtyrosine increases the lag period and slows down dopachrome formation. Thus, melanin accumulation is lower from the d-enantiomer than from l-tyrosine when they are compared under the same conditions. In Fig. 3 of Ref. [21], the accumulation of melanin increases as the concentration of d-tyrosine increases (Figs. 3a and 3d). These experiments indicate that d-tyrosine competes with l-tyrosine for the active site of the enzyme, but acts as a competitive substrate that generates the same product (o-dopaquinone → dopachrome) [48,49]. This means that d-tyrosine cannot be proposed as a new skin-whitening agent.

Conclusions
The action of tyrosinase on the stereoisomers of tyrosine and dopa was studied following three experimental kinetic approaches. Our studies of the steady state showed that V D max and K D m are higher than V L max and K L m for both tyrosine and dopa. The study based on the post-steady-state transient phase, provided data which agreed with those obtained for the steadystate approach. However, the results obtained for the intersteady-state transient phase demonstrate that the lag period is longer and the steady-state rate is slower for d-tyrosine than for l-tyrosine. Thus, d-tyrosine delays d-melanogenesis compared with l-melanogenesis from l-tyrosine. In the case of d-dopa, the rate of d-dopachrome formation is slower than that of ldopachrome from l-dopa, but the limiting process is d-tyrosine hydroxylation. Consequently, the above experimental results demonstrate that d-tyrosine is not a true tyrosinase inhibitor and is capable of preventing or significantly decreasing lmelanogenesis. d-Tyrosine is an alternative substrate of tyrosinase that originates d-melanogens and d-melanin, although more slowly than the corresponding biosynthesis of l-melanogens and l-melanin from l-tyrosine.