Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
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Tyrosinase-catalyzed hydroxylation of hydroquinone, a depigmenting agent, to hydroxyhydroquinone: A kinetic study María del Mar García-Molina a, Jose Luis Muñoz Muñoz a, Francisco Martinez-Ortiz b, José Rodriguez Martinez c, Pedro Antonio García-Ruiz d, José Neptuno Rodriguez-López a, Francisco García-Cánovas a,⇑ a GENZ: Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Campus de Excelencia Internacional ‘Mare Nostrum’, Universidad de Murcia, Espinardo, Murcia E-30100, Spain b Grupo de Investigación de Electroquímica Teórica y Aplicada, Departamento de Física-Química, Facultad de Química, Campus de Excelencia Internacional ‘Mare Nostrum’, Universidad de Murcia, Espinardo, Murcia E-30100, Spain c SUIC (Servicio de Instrumentación Científica), Universidad de Murcia, Spain d QCBA: Grupo de Química de Carbohidratos y Tecnología de Alimentos, Departamento de Química Orgánica, Facultad de Química, Campus de Excelencia Internacional ‘Mare Nostrum’, Universidad de Murcia, Espinardo, Murcia E-30100, Spain
a r t i c l e
i n f o
Article history: Received 28 March 2014 Accepted 24 April 2014 Available online xxxx Keywords: Tyrosinase Hydroquinone Kinetic 2-Hydroxy-p-benzoquinone Hydrogen peroxide Ascorbic acid
a b s t r a c t Hydroquinone (HQ) is used as a depigmenting agent. In this work we demonstrate that tyrosinase hydroxylates HQ to 2-hydroxyhydroquinone (HHQ). Oxy-tyrosinase hydroxylates HQ to HHQ forming the complex met-tyrosinase-HHQ, which can evolve in two different ways, forming deoxy-tyrosinase and p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone or on the other way generating met-tyrosinase and HHQ. In the latter case, HHQ is rapidly oxidized by oxygen to generate 2-hydroxy-p-benzoquinone, and therefore, it cannot close the enzyme catalytic cycle for the lack of reductant (HHQ). However, in the presence of hydrogen peroxide, met-tyrosinase (inactive on hydroquinone) is transformed into oxy-tyrosinase, which is active on HQ. Similarly, in the presence of ascorbic acid, HQ is transformed into 2-hydroxy-p-benzoquinone by the action of tyrosinase; however, in this case, ascorbic acid reduces met-tyrosinase to deoxy-tyrosinase, which after binding to oxygen, originates oxy-tyrosinase. This enzymatic form is now capable of reacting with HQ to generate p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone. The formation of HHQ during the action of tyrosinase on HQ is demonstrated by means of high performance liquid chromatography mass spectrometry (HPLC–MS) by using hydrogen peroxide and high ascorbic acid concentrations. We propose a kinetic mechanism for the tyrosinase oxidation of HQ which allows us the kinetic characterization of the process. A possible explanation of the cytotoxic effect of HQ is discussed. Ó 2014 Published by Elsevier Ltd.
1. Introduction Tyrosinase (E.C. 1.14.18.1) is a copper-containing enzyme widely distributed in nature. It catalyses two types of reactions:
Abbreviations: HQ, hydroquinone; HHQ, 2-hydroxyhydroquinone; PB, p-benzoquinone; HPB, 2-hydroxy-p-benzoquinone; D, o-diphenol; M, monophenol; AH2, ascorbic acid; Em , met-tyrosinase; Eox , oxy-tyrosinase; Ed , deoxy-tyrosinase; V HPB 0 , initial rate of HPB formation; V app max , apparent maximum velocity of tyrosinase acting app on HQ; K M , apparent Michaelis constant of tyrosinase acting on HQ; R ratio between [H2O2]0 and [HQ]0, V 0HPB;R , initial rate of HPB formation at a constant ratio [H2O2]0/[HQ]0; V app;R max , apparent maximum velocity of tyrosinase acting on HQ at a app;R constant ratio [H2O2]0/[HQ]0; K M , apparent Michaelis constant of tyrosinase acting on HQ at a constant ratio [H2O2]0/[HQ]0. ⇑ Corresponding author. Tel.: +34 868 884764; fax: +34 868 883963. E-mail address: [email protected] (F. García-Cánovas).
(a) the ortho-hydroxylation of monophenols to o-diphenols (monophenolase activity) and (b) the oxidation of o-diphenols to o-quinones (diphenolase activity). Both types of reaction require molecular oxygen as the second substrate of the enzyme.1–3 The first studies on the action of tyrosinase on hydroquinone (HQ) suggested that the enzyme does not act directly on this compound but that, in contrast, HQ was oxidized by the o-quinone generated by the enzyme acting on a o-diphenol substrate.4–6 More recent studies, using tyrosinase from the Harding-Passey melanoma, showed that HQ inhibits the melanin biosynthetic pathway from L-tyrosine and L-Dopa7–10 and, in subsequent studies, it was proposed that HQ inhibited the melanin biosynthesis pathway by acting as an alternative substrate to L-tyrosine and L-Dopa.11 As a consequence, it was suggested that the adverse physiological effects of HQ may be due to its oxidation by tyrosinase.12 These
http://dx.doi.org/10.1016/j.bmc.2014.04.048 0968-0896/Ó 2014 Published by Elsevier Ltd.
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oxidation is vicarious.22 However, our studies about the action of tyrosinase on monophenols and o-diphenols21,23–29 suggest that HQ is a substrate of tyrosinase but that the enzymatic activity is not evident because HQ is not capable of closing the enzymatic catalytic cycle by transforming the met-tyrosinase (Em ) form to oxy-tyrosinase (Eox ). This can be done by using an o-diphenol as a reductant.21,23–26 Em (inactive on monophenols) is transformed into Ed , which, by binding with oxygen, is transformed into Eox (active on monophenols). Alternatively, Em is converted into Eox by the addition of H2O2,28,30–32 as demonstrated by Mason (Schemes 1 and 2).33,34 Moreover, the addition of ascorbic acid, which acts as substrate of the enzyme,35 is capable of reducing Em to Ed , facilitating the transformation into the Eox which acts on HQ (Scheme 3). The action of the enzyme on HQ in the presence of H2O2 is depicted in Scheme 1. Note that in the proposed mechanism HHQ is not accumulated in the medium and so the experiments show no lag period. Hence HQ is the only monophenol that does not show a lag in the presence of H2O2.28 A kinetic analysis of
and subsequent studies suggested that HQ was hydroxylated to 2hydroxyhydroquinone (HHQ) through the action of tyrosinase, although the direct enzymatic formation de HHQ has not been demonstrated experimentally.13,14 Serious problems attributed to HQ are pigmentation of the eye and, in a small number of cases, permanent corneal damage.15 Due to this, dermatological depigmented treatments without HQ have been developed, which have been contrasted with others containing the compound.16–18 Attempts to inhibit HQ cytotoxicity have used resveratrol as an additional component.19 Actually, pharmaceutical products (creams and gels) contain up to 4% HQ are used, for example, despigmental topical gel (4%), melanase cream (2%), licoforte topical gel (4%), and pigmentase cream (4%).20 HQ, is an isomer of catechol, a known substrate of tyrosinase.21 However, it has recently been suggested that HQ is neither a substrate nor inhibitor of tyrosinase.22 The same authors suggest that these results agree with previous ones4–6 and conclude that HQ is oxidized in the presence of tyrosinase substrates so that the
OH
B2
B1H O N
2+ Cu
N
2+ Cu
N
N
O
N
O
O2.-
N H
EmHQ
O
OH
OH
HPB
k9
O2 HO
k-1
k1
HQ
OH
HHQ HO
OH
OH
B2
B1
B2H
B1H N 2+
2+ Cu
Cu N
k7
N
O N 2+
N
O
2+ Cu
Cu
N
N
O
N
H
N N
O N
N H
Em
H2O H2O2
k-2
k2
B2
B1H
O2
H2O
EmHHQ
k6
OH
N
+ Cu
+ Cu
N
N
N
N N
k5
k8
O
O
OH
B2
B1H
N
2+ Cu
N
O
2+ Cu
N
N N
O N
Ed
k-8
O
OH
HPB
HQ
O O
HO
k4
B2
B1H
OH
N
2+ O Cu
N
O N
2+ Cu
N N
N H
Eox
k-4
EoxHQ
Scheme 1. Structural mechanism proposed to explain the catalytic pathway of tyrosinase in its action on HQ in the presence of H2O2/Em met-tyrosinase, Em HQ mettyrosinase HQ complex axially bound to Cu atom with protonated base (B1H), Eox oxy-tyrosinase, Eox HQ oxy-tyrosinase HQ complex axially bound to Cu atom with protonated base (B1H), and the proton transferred to the peroxide, Em HHQ met-tyrosinase/2-hydroxyhydroquinone complex, Ed deoxy-tyrosinase, HQ ¼ hydroquinone, HHQ ¼ 2-hydroxyhydroquinone, HPB ¼ 2-hydroxy-p-benzoquinone.
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2.1.1. Effect of hydrogen peroxide The action of tyrosinase on HQ is shown in Figure 1A. In the absence of H2O2 the enzyme shows no activity (recording a), while in its presence the absorbance at 480 nm increases (recording b). As the enzyme concentration is increased the slope also increases, demonstrating the increase of absorbance with time (recordings b–f). The absorbance increase may be due to the hydroxylation of HQ to HHQ and its subsequent oxidation to 2-hydroxy-p-benzoquinone (HPB) (red) at pH 7. The formation of this red quinone is due to the presence of H2O2, which brings about the formation of Eox from Em (see Scheme 1).33,34 In this way the H2O2 helps identify HQ as a substrate of the enzyme.28,30–32 The action of the enzyme on HQ in the presence of H2O2 is shown in Scheme 1. Scheme 1 can be represented kinetically as Scheme 2 and its kinetic analysis is described in the Appendix. According to Eq. A1 inhibition should occurs as a result of the excess of HQ and this is confirmed in Figure 1B, which agrees with the mechanism proposed.
O2
k9
HPB
.
O2 HHQ
HQ
k1 EmHQ
k-1
k7
Em
EmHHQ
HPB
k6
H2O2
k-2
k2
Ed
H 2O
k-8
k5
k8
O2
HQ
k4
Eox
EoxHQ
k-4 Scheme 2. Proposed kinetic mechanism to explain tyrosinase action on HQ in the presence of H2O2,Em , Eox and Ed are met-, oxy- and deoxy-tyrosinase, respectively. HQ, HHQ and HPB represent hydroquinone, 2-hydroxyhydroquinone and 2hydroxy-p-benzoquinone, respectively. The enzymatic complex Em HQ, Em HHQ and Eox HQ are also showed.
the mechanism of Scheme 2 (derived from the structural mechanism shows in Scheme 1) permits the deduction of the analytical expression for the product accumulation rate. Furthermore, Scheme 3 depicts the action of the enzyme on HQ in the presence of ascorbic acid. The aim of this work was to demonstrate that HQ, a depigmenting agent used in pharmacology, is a substrate of tyrosinase. For this, we establish an experimental design that permits the kinetic characterisation of the enzyme’s action on HQ.1,21 A possible explanation of the cytotoxic effects of HQ are also discussed. 2. Results and discussion 2.1. HQ as a substrate of tyrosinase Experiments in the presence of hydrogen peroxide or ascorbic acid were carried out to demonstrate that HQ is a substrate of tyrosinase.
HQ k1
EmHQ
AH2 k2
Em
k-1
2.1.2. Effect of ascorbic acid Ascorbic acid (AH2) is a substrate of tyrosinase.35 Scheme 3 depicts the catalytic cycle of the enzyme in its action on this substrate. Ascorbic acid reduces the met-tyrosinase form to deoxytyrosinase, whose subsequent binding with oxygen generates oxy-tyrosinase, which acts on another molecule of AH2 regenerating met-tyrosinase and closing the catalytic cycle. The intermediate oxy-tyrosinase is capable of hydroxylate HQ (Scheme 3) to generate HHQ, which is subsequently oxidized to HPB. The concentrations of ascorbic used in the experiments depicted in Figure 2A do not reduce the HPB, so that Figure 2A shows that the absorbance at 480 nm (corresponding to HPB) increases with time. Increasing the enzyme concentration (Fig. 2A) augments the accumulation rate of the red quinone HPB. Figure 2B shows that the same effect is obtained when the concentration of AH2 is increased. Both results can be explained by the increased turnover of tyrosinase on AH2. Spectrophotometric identification of the product of the enzymatic reaction can be seen in Figure 2C (increased absorbance at 480 nm). In the absence of HQ or tyrosinase there is no increase in the absorbance at this wavelength (results not shown).
EmD
k-2
HHQ
k11 O2
k3
HPB -
O2
k10
A HPB Ed
EmHHQ k9 O2
k7 EoxAH2
AH2 k6 k-6
k-8
k5
k8 HQ Eox
k4 k-4
EoxHQ
Scheme 3. Action of tyrosinase on HQ in the presence of ascorbic acid, where: HQ ¼ Hydroquinone, HHQ ¼ 2-hydroxy-hydroquinone, HPB ¼ 2-hydroxi-p-benzoquinone, AH2 ¼ ascorbic acid, A ¼ dehydroascorbic acid, Em = met-tyrosinase, Ed = deoxy-tyrosinase and Eox = oxy-tyrosinase.
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0.15
120
B
A 100
0.10
(h)
(a)
0.05
60
V0
HPB
A480
(nM/s)
80
40
20
0.00
0
40
80
120
t (s)
0 0
5
10
15
20
25
[HQ]0(mM)
Figure 1. (A) Action of tyrosinase on HQ in the presence of hydrogen peroxide. Effect of varying the concentration of enzyme. Spectrophotometric recordings of the action of tyrosinase on HQ measured at a wavelength of 480 nm. The experimental conditions were: 30 mM phosphate buffer (pH 7.0), initial HQ concentration, [HQ]0 = 0.5 mM, initial H2O2 concentration, [H2O2]0 = 2.5 mM and initial enzyme concentrations, [E]0, were (nM): 0 (a), 10 (b), 35 (c), 55 (d), 75 (e), 90 (f), 115 (g) and 130 (h). B. Representation of values of the initial action rate of tyrosinase on HQ, maintaining [H2O2]0 and [E]0 constants. The conditions were: 30 mM phosphate buffer pH 7.0 [H2O2]0 = 40 mM, the HQ concentrations shown in the figure and [E]0 = 14 nM.
2.2. Identification of hydroxyhydroquinone as a product of the enzymatic reaction of tyrosinase on HQ To demonstrate that HPB is formed enzymatically through the oxidation of HHQ and not as a result of purely chemical reactions, such as the addition of water to p-benzoquinone (PB); several experiments were carried out at high ascorbic acid concentrations (to minimise the formation of quinone) and product formation was studied by HPLC–MS. The chromatograms displayed were recorded at 290 nm. Signals correspond to the following compounds identified by mass spectrometry and comparison with standards (Fig. 3A). Figure 3B shows the mass spectrum of the peak appearing in the chromatogram at 5.1 min. The kinetic of HHQ formation was then studied, taking aliquots at different times and revealing by HPLC–MS, following the evolution of the characteristic peak of HHQ. The results are shown in Figure 3C. Another method used to follow this reaction involved oxidizing aliquots of the reaction mixture with an excess of sodium periodate. The great difference in the molar absorbtivity coefficient between PB and HPB (Fig. 4A) enables the reaction in which HHQ is formed to be followed qualitatively (Fig. 4B). As the reaction time increases, the concentration of HHQ increases and, after oxidation with an excess of sodium periodate, the absorbance at 480 nm increases.36 It has been proposed that the generation of HPB in a reaction medium containing tyrosinase and HQ in the presence of L-Dopa occurs as a result of the water added by PB or through a disproportion reaction between HQ and PB and the formation of HPB in this reaction through the formation of a radical.22 However, as can be seen in Figure 5, the Cyclic Voltammetry experiments demonstrated the stability of PB at pH 7 (Fig. 5A) since at t = 0 and 30 min the recordings did not change. Figure 5B shows the cyclic voltagrams of a stoichiometric mixture of HQ and PB at the same times (t = 0 and 30 min). The recordings show that at this pH value and within this time period no HPB is formed. These data agree with those long described for the slow reaction of the addition of water to PB,37 with an apparent pseudo-first order constant = 3 107 s1. Although the reaction of hydroxylation of PB by photohydroxylation at 254 nm has been described, these results were obtained using a different experimental approach.38
Having demonstrated that the enzyme hydroxylate HQ to HHQ and then, according to Schemes 1–3, oxidizes this compound to HPB or releases HHQ to the medium, where it is rapidly oxidized by the oxygen in the solution to HPB, it is now possible to kinetically characterise the action of tyrosinase on HQ by spectrophotometrically following the formation of HPB at 480 nm. Based on the kinetic analysis developed in the Appendix, we propose the following steps for the kinetic characterization of the action of tyrosinase on HQ. 2.3. Kinetic chracterisation of HQ as tyrosinase substrate Step 1. Vary the concentration of H2O2 while maintaining the concentrations of HQ and tyrosinase constant. Follow the formation of HPB spectrophotometrically. The slopes of the lines, that is the initial velocity V HPB show hyperbolic dependence with 0 respect to [H2O2]0 according to Eq. A10. The hyperbolas obtained are depicted in Figure 6A (a–i). Step 2. Nonlinear regression analysis of the values of V HPB v ersus ½H2 O2 0 according to Eq. A10 to give the parameters 0 app V app max y K M , which are explained in Eqs. A11 and A12. app Step 3. Analysis of V app max and K M depending on [HQ]0 according to Eqs. A17 and A19, Figure 7A. This analysis provides the kinetic HQ information shown in Table 1. kcat ¼ k5 (Catalytic constant) and HQ K M ¼ k5 þ k4 =k4 (Michaelis constant). Figure 7B shows how (K app M ) depends on [HQ]0. Another experimental design involves using a constant ratio between [H2O2]0 and [HQ]0, that is [H2O2]0/[HQ]0 = R. From Eq. A1, it is possible to obtain Eq. A21. The values of V HPB;R show a 0 hyperbola when represented versus [HQ]0 (Fig. 8) and the nonlinear regression analysis of these data according to Eq. A21 provides app;R the values of V app;R . The representation of these values max and K M according to Eqs. A22 and A23 gives Figure 9A and 9A Inset, respectively. From Eqs. A19 and A22 K 1 , the dissociation constant of the complex Em ðHQ Þ,can be obtained (see Table 1). 2.4. Comparison with other monophenols The literature22 describes how the presence of the p-hydroxy group in HQ prevents binding to the tyrosinase active site,
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0.20
B
A 0.12
f
0.15
0.08
[E]0
0.10
[AH2]0
A480
A480
d
c 0.04
b
0.05
a 0.00
0.00 0
400
800
1200
1600
a 0
400
800
1200
1600
t (s)
t (s) 0.300
C
A
0.225
0.150
0.075
0.000 400
440
480
520
λ (nm) Figure 2. Action of tyrosinase on HQ in the presence of ascorbic acid. (A) Effect of varying the concentration of enzyme. Spectrophotometric recording of the action of tyrosinase on HQ measured at 480 nm in the presence of ascorbic acid, [AH2]0 = 2 mM. The experimental conditions were: 30 mM phosphate buffer (pH 7.0). [HQ]0 = 0.5 mM and [E]0 (lM) were: (a) 0.5, (b) 1.1, (c) 1.7 and (d) 2.2. B. Effect of varying the ascorbic acid concentration Spectrophotometric recording of the action of tyrosinase on HQ measured at 480 nm varying the concentration of ascorbic acid. The experimental conditions were: 30 mM phosphate buffer (pH 7.0). [HQ]0 = 0.5 mM, [E]0 = 1.7 lM and [AH2]0 (mM) were: (a) 1, (b) 3, (c) 2.5, (d) 2, (e) 1.5 and (f) 4. C. Spectrophotometric recordings of the action of tyrosinase on HQ in the presence of ascorbic acid. The experimental conditions were: 30 mM phosphate buffer (pH 7.0). [HQ]0 = 0.5 mM, [E]0 = 1.7 lM and [AH2]0 = 2 mM. Interval between recordings was 300 s.
although, as the same authors mention, other 4-substituents, such as alkyl, alkoxy and halo, are monoxygenase substrates.39,40 The action of tyrosinase on HHQ has recently been described, revealing that the position of a p-hydroxy does not hinder the action of tyrosinase.41 Moreover, the data obtained for the action of tyrosinase on HQ in the present study, compared with those previously described for other monophenols, are shown in Table 2.21 The chemical shift values indicate that HQ is a good tyrosinase subHQ strate. The values of kcat agree with those obtained for 4-methoxyphenol, 4-ethoxyphenol and phenol. Hence, the kinetic study of HQ using H2O2 provides data that agree with those obtained for the monophenols studied in the absence of H2O2. 3. Conclusions From these results, it can be concluded that HQ is a substrate of tyrosinase but the enzyme cannot express its activity in the absence of a reductant because of the characteristics of its catalytic cycle (Schemes 1–3).1,28 In the presence of H2O2 Em
(inactive on HQ) can pass to Eox , an intermediate that can act on HQ.28 In the presence of a reductor agent such as AH2, Em (inactive on HQ) generates Ed , which, in turn, passes to Eox , which acts on HQ. By means of these transformations of the intermediates in the catalytic cycle, tyrosinase can act on HQ, as a substrate. Moreover, the strong depigmenting effect of HQ, and hence its action on the melanogenesis pathway, is double: since it can act as reductant of the o-quinone of L-Dopa, o-dopaquinone, originating PB, and also, as shown in this work, by acting as a substrate of tyrosinase, it can redirect the melanin biosynthesis pathway towards the formation of HPB. Both quinone generation pathways involve the formation of two p-quinones, PB and HPB, which are stable, especially compared with o-quinones. This may involve the consumption of thiolic compounds in the medium and hence a change in the redox state of the cell, which would explain the cytotoxic effects described for the use of HQ.12,22,42 The confirmation that HQ is a substrate of tyrosinase may have important consequences for its use in pharmacology and cosmetics.
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A
B
C
Figure 3. (A) HPLC separation of a reaction mixture after reaction of 50 nM tyrosinase with [HQ]0 = 2 mM, [H2O2]0 = 1 mM and [AH2]0 = 30 mM in 30 mM sodium phosphate buffer, pH 7.0 at 25 °C. Analyses were performed 30 min after beginning the reaction. The signals correspond to: ascorbic acid (2.5 min), HHQ (5.1 min) and HQ (6.4 min), were registered at 290.4 nM B. Mass spectrum corresponding to 5.1 min reaction time (HHQ) of Figure 3A. C. Chromatograms corresponding to the fragment m/z 125 at different reaction times ( ) 500 s, (—) 1200 s and () 1600 s, showing the formation of the product HHQ. The experimental conditions were the same as Figure 3.A.
4. Material and methods 4.1. Reagents Mushroom tyrosinase or polyphenol oxidase (o-diphenol/O2 oxygen-oxidoreductase, EC1.14.18.1 was supplied by Sigma 4276 U/mg) (Madrid, Spain). The enzyme was purified as previously described.29 Protein concentration was determined by Bradford´s method using bovine serum albumin as standard.43 All the
substrates used in this work were purchased from Sigma (Madrid, Spain) and were prepared en 0.15 mM phosphoric acid to prevent autooxidation. Milli-Q system (MilliporeCorp.) ultrapure water was used throughout. 4.2. Spectrophotometric assays The enzymatic assays were carried out with a Perkin Elmer Lambda-35 spectrophotometer (Waltham, Massachusetts USA),
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2.0
A
B
400
0.2
1.0
0.0
0
450
900
1350
300
200
HPB
A
HPB
(nM/s)
0.4
V0
1.5
A480
0.6
1800
t (s)
0.5
100
PB
0.0 250
300
350
400
450
0 500
550
0
Figure 4. (A) Spectral characteristics of PB and HPB. The experimental conditions were 30 mM phosphate buffer, pH 7.0, la [PB]0 = 0.5 mM, HPB generated by stoichiometric oxidation with [NaIO4]0 = 0.5 mM and [HHQ]0=0.5 mM. B. Reaction of tyrosinase with HQ in the presence of a large excess of ascorbic acid. The experimental conditions were 30 mM phosphate buffer, pH 6.0 at 25 °C, [AH2]0 = 30 mM, [HQ]0 = 2 mM, [E]0 = 65 nM. At the times indicated in the figure, an aliquot was oxidized with excess sodium periodate ([NaIO4]0 = 35 mM) and the absorbance at 480 nM was measured.
16
I / nA
12 8 4 0
0.0
0.4
0.8
E/V 12
B
I / nA
8 4 0 -4 -8 -0.8
-0.4
0.0
60
80
100
120
0.4
Figure 6. Representation of initial formation rate of HPB (V HPB 0 ) in 30 mM phosphate buffer, pH 7.0, during the action of tyrosinase [E]0 = 28 nM on HQ, varying the concentration of H2O2 as indicated in the figure. The concentrations of HQ (mM) were: 0.07 (d), 0.1 (s) and 0.3 (.).0.4 (h), 0.5 (j), 0.75 (N) 1.2 (e) and 1.5().
with an excess of periodate and measuring the increase in absorbance at 480 nm, which corresponds mainly to HHQ formed. 4.3. HPLC–MS
A
-0.4
40
[H2O2] (mM)
λ
-4 -0.8
20
0.8
E/V Figure 5. (A) Cyclic voltammogram (cathodic scan, initial potential 0.6 V) of [PB]0 = 1 mM in 0.1 M phosphate buffer (pH = 7). (a) Fresh solution; (b) after 30 min. Scan rate: 0.05 V s1.B. Cyclic voltammogram (cathodic and anodic scan, initial potential 0.2 V) of [PB]0 = 0.5 mM and [HQ]0 = 0.5 mM in 0.1 M phosphate buffer (pH = 7). (a) Fresh solution; (b) after 30 min. Scan rate: 0.05 V s1.
on-line interfaced with a PC computer, where the kinetic data were recorded, stored and later analyzed. The conditions of the assay are specified in the corresponding figure legend.36,44 The reaction of tyrosinase on HQ in the presence of H2O2 and a large excess of AH2 was followed, oxidizing the reaction mixture
The reaction of tyrosinase with HQ was also followed by measuring the formation of HHQ in the presence of an excess of ascorbic acid by HPLC–MS. The enzyme was separated from the reaction intermediates by centrifugation using 10,000 MWL0 filters. The reaction products were analyzed in a high resolution liquid chromatograph. To separate and quantify the different compounds an AGILENT 1100 series HPLC (Germany) coupled to an AGILENT VL ion trap mass sepctrometer (Germany) was used. The separation programme used a gradient of (A) H2O with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The gradient began at time 0 with 3% B, raised to 50% B in 10 min. The initial value of 3% B was reached at 11 min, when the equipment was allowed to stabilize for 5 min. The flow rate was 0.8 ml/min. The signal at 290 nm was captured by the DAD, with an absorbance range of 190–350 nm at 2 nm steps. 10 lL of sample was injected onto a 5 lM SunFire™ C18 column, 4.6 mm 150 mm, thermostated at 25 °C. Mass spectrometry acquisition was carried out in scan mode, with a mass range of 50–500 m/z. The elecrospray source (ESI) was operated in negative mode. The drying temperature was 350 °C, nebulizer pressure was 60 psi and drying gas flow 9 l/m. The Extracted Ion Cromatogram (EIC) corresponding to each ion with its (MH), was used to monitor the different compounds 4.4. Electrochemistry Cyclic Voltammetry (CV) was performed using a computerdriven three electrode potentiostat constructed in the ‘Research Support Service of the University of Murcia’ (http://www.um.es/ sai). A three-electrode cell, with a 12.5 lm radius Pt working microdisk electrode (from CH Instruments) was used The reference electrode was a saturated calomel electrode (SCE), and the counter electrode was a platinum wire. Solutions were prepared with distilled deionized water (Milli-Q filtering system). Nitrogen gas was passed through solutions for de-aeration for 20 min prior to measurements (buffer and acidic substrate in separate parts) with
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750
80
A
B
600
(mM) app
450
KM
app
Vmax (nM/s)
60
40
300
20
150
0 0.0
0.5
1.0
1.5
0 0.0
2.0
0.5
1.0
1.5
2.0
[HQ]0 (mM)
[HQ]0 (mM)
app Figure 7. A. Representation of the values of V app max versus [HQ]0 B. Representation of the values of K M versus [HQ]0. The concentrations of HQ are indicated in the figure. Enzyme concentration, [E]0 = 28 nM.
Table 1 Kinetic constants that characterise the action of tyrosinase on hydroquinone. Chemical shift and pK values of hydroquinone.
Hydroquinone a
K HQ M (mM)
K 1 (mM)
k5 (s1)
pK1a
pK2a
d1
0.25 ± 0.04
4.56 ± 0.93
23.0 ± 1.1
8.2
10.1
153.30
(ppm)
From 47.
600
400
400 5 4 (mM)
300
3 2
app,R
app,R
200
V0
HPB,R
(nM/s)
300
KM
Vmax (nM/s)
500
200
1
100
100 0
0
5
10
15
20
25
R
0
0
0
2
4
6
5
10
15
20
25
R
[HQ]0 (mM) V HPB;R 0
0
V HPB;R 0
Figure 8. Representation of initial versus [HQ]0. The values of obtained at 480 nM for the reaction of tyrosinase on HQ at different [H2O2]0/[HQ]0 ratios (R) are shown in the figure. [E]0 = 28 nM. R values were: 2.5 (j), 5.0 (h), 6 (N), 7.5 (e), 10 (d), 15 (s) and 20 (.).
app;R Figure 9. Representation of V app;R max versus R. The values of V max obtained by analysis of the data in Figure 7 are represented versus the [H2O2]0/[HQ]0 ratio (R). Inset app;R Representation of K M versus [R].
Table 2 Kinetic constants for the monophenolase activity of mushroom tyrosinase
a nitrogen atmosphere maintained over the solution throughout the experiments. The platinum disk was polished prior to experiments using 1.0, 0.3 and 0.05 lm alumina-water slurry on soft lapping pads and after sonication for 10 min. 4.5. Kinetic analysis data For tyrosinase, the initial rate values (V0 ) were calculated from triplicate measurements at each reducing substrate concentration, and V0 versus [S]o data were adjusted to the Michaelis–Menten equation through the Sigma Plot 9.0 program for Windows,45 thus obtaining the maximum rate (Vmax ) and Michaelis constant (Km ).
*
Monophenol
K M (mM)
kcat (s1)
d3
4-Methoxyphenol* 4-Ethoxyphenol* Hydroquinone Phenol*
0.08 ± 0.003 0.17 ± 0.005 0.25 ± 0.04 0.70 ± 0.04
184.2 ± 6.1 132.0 ± 4.5 23.0 ± 1.1 12.7 ± 0.6
118.90 119.01 119.50 118.13
(ppm)
d4
(ppm)
152.29 152.39 153.30 158.15
Data taken from 21.
4.6. Determination of
13
C NMR chemical shifts
The carbon chemical shifts given in Tables 1 and 2 were obtained from the corresponding 13C NMR spectra, which were
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recorded at 298 K on a Bruker Avance 400 Hz instrument employing buffered solutions (at pH 7.0) of pure samples in D2O. Acknowledgements This paper was partially supported by Grants from Ministerio de Educación y Ciencia (Madrid, Spain) Project BIO2009-12956, Fundación Séneca (CARM, Murcia, Spain) Projects 08856/PI/08 and 08595/PI/08, Consejería de Educación (CARM, Murcia, Spain) BIO-BMC 06/01-0004.
V app max ¼
a1 ½HQ 0 ½E0 b5 þ b7 ½HQ 0
ðA13Þ
K app M ¼
b1 þ b3 ½HQ 0 þ b6 ½HQ 20 b5 þ b7 ½HQ 0
ðA14Þ
explained as: k5 ðk6 þk7 Þ ½HQ 0 ½E0 k5 þk6 þk7 6 þk7 Þðk4 þk5 Þ þ ½HQ 0 k4 ðK 5 þk6 þk7 Þ
V app max ¼ ðk
ðA15Þ
and
K app M
¼
K 1 k2 k8 ðk4 þk5 Þðk6 þk7 Þ K 1 k2 k4 k8 ðk5 þk6 þk7 Þ
k k k k ½HQ 2
4 þk5 ÞþK 1 k4 k5 k7 k8 ½HQ0 0 þ ½k2 k8 ðk6 þkK71 Þðk þ K 1 k24k45k87ðk85 þk6 þk k2 k4 k8 ðk5 þk6 þk7 Þ 7Þ
K 1 k2 k8 ðk6 þk7 Þðk4 þk5 Þ K 1 k2 k4 k8 ðk5 þk6 þk7 Þ
ðA16Þ
þ ½HQ 0
Appendix Applying the steady state approximation to the mechanism shown in Scheme 2 provides the following expression for the HPB formation rate:
V HPB ¼ 0
From Eq. A15, taking into consideration that the hydroxylation step controlled by k5 is the slowest (k5
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Tyrosinase-catalyzed hydroxylation of hydroquinone, a depigmenting agent, to hydroxyhydroquinone: A kinetic study María del Mar García-Molina a, Jose Luis Muñoz Muñoz a, Francisco Martinez-Ortiz b, José Rodriguez Martinez c, Pedro Antonio García-Ruiz d, José Neptuno Rodriguez-López a, Francisco García-Cánovas a,⇑ a GENZ: Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Campus de Excelencia Internacional ‘Mare Nostrum’, Universidad de Murcia, Espinardo, Murcia E-30100, Spain b Grupo de Investigación de Electroquímica Teórica y Aplicada, Departamento de Física-Química, Facultad de Química, Campus de Excelencia Internacional ‘Mare Nostrum’, Universidad de Murcia, Espinardo, Murcia E-30100, Spain c SUIC (Servicio de Instrumentación Científica), Universidad de Murcia, Spain d QCBA: Grupo de Química de Carbohidratos y Tecnología de Alimentos, Departamento de Química Orgánica, Facultad de Química, Campus de Excelencia Internacional ‘Mare Nostrum’, Universidad de Murcia, Espinardo, Murcia E-30100, Spain
a r t i c l e
i n f o
Article history: Received 28 March 2014 Accepted 24 April 2014 Available online xxxx Keywords: Tyrosinase Hydroquinone Kinetic 2-Hydroxy-p-benzoquinone Hydrogen peroxide Ascorbic acid
a b s t r a c t Hydroquinone (HQ) is used as a depigmenting agent. In this work we demonstrate that tyrosinase hydroxylates HQ to 2-hydroxyhydroquinone (HHQ). Oxy-tyrosinase hydroxylates HQ to HHQ forming the complex met-tyrosinase-HHQ, which can evolve in two different ways, forming deoxy-tyrosinase and p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone or on the other way generating met-tyrosinase and HHQ. In the latter case, HHQ is rapidly oxidized by oxygen to generate 2-hydroxy-p-benzoquinone, and therefore, it cannot close the enzyme catalytic cycle for the lack of reductant (HHQ). However, in the presence of hydrogen peroxide, met-tyrosinase (inactive on hydroquinone) is transformed into oxy-tyrosinase, which is active on HQ. Similarly, in the presence of ascorbic acid, HQ is transformed into 2-hydroxy-p-benzoquinone by the action of tyrosinase; however, in this case, ascorbic acid reduces met-tyrosinase to deoxy-tyrosinase, which after binding to oxygen, originates oxy-tyrosinase. This enzymatic form is now capable of reacting with HQ to generate p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone. The formation of HHQ during the action of tyrosinase on HQ is demonstrated by means of high performance liquid chromatography mass spectrometry (HPLC–MS) by using hydrogen peroxide and high ascorbic acid concentrations. We propose a kinetic mechanism for the tyrosinase oxidation of HQ which allows us the kinetic characterization of the process. A possible explanation of the cytotoxic effect of HQ is discussed. Ó 2014 Published by Elsevier Ltd.
1. Introduction Tyrosinase (E.C. 1.14.18.1) is a copper-containing enzyme widely distributed in nature. It catalyses two types of reactions:
Abbreviations: HQ, hydroquinone; HHQ, 2-hydroxyhydroquinone; PB, p-benzoquinone; HPB, 2-hydroxy-p-benzoquinone; D, o-diphenol; M, monophenol; AH2, ascorbic acid; Em , met-tyrosinase; Eox , oxy-tyrosinase; Ed , deoxy-tyrosinase; V HPB 0 , initial rate of HPB formation; V app max , apparent maximum velocity of tyrosinase acting app on HQ; K M , apparent Michaelis constant of tyrosinase acting on HQ; R ratio between [H2O2]0 and [HQ]0, V 0HPB;R , initial rate of HPB formation at a constant ratio [H2O2]0/[HQ]0; V app;R max , apparent maximum velocity of tyrosinase acting on HQ at a app;R constant ratio [H2O2]0/[HQ]0; K M , apparent Michaelis constant of tyrosinase acting on HQ at a constant ratio [H2O2]0/[HQ]0. ⇑ Corresponding author. Tel.: +34 868 884764; fax: +34 868 883963. E-mail address: [email protected] (F. García-Cánovas).
(a) the ortho-hydroxylation of monophenols to o-diphenols (monophenolase activity) and (b) the oxidation of o-diphenols to o-quinones (diphenolase activity). Both types of reaction require molecular oxygen as the second substrate of the enzyme.1–3 The first studies on the action of tyrosinase on hydroquinone (HQ) suggested that the enzyme does not act directly on this compound but that, in contrast, HQ was oxidized by the o-quinone generated by the enzyme acting on a o-diphenol substrate.4–6 More recent studies, using tyrosinase from the Harding-Passey melanoma, showed that HQ inhibits the melanin biosynthetic pathway from L-tyrosine and L-Dopa7–10 and, in subsequent studies, it was proposed that HQ inhibited the melanin biosynthesis pathway by acting as an alternative substrate to L-tyrosine and L-Dopa.11 As a consequence, it was suggested that the adverse physiological effects of HQ may be due to its oxidation by tyrosinase.12 These
http://dx.doi.org/10.1016/j.bmc.2014.04.048 0968-0896/Ó 2014 Published by Elsevier Ltd.
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oxidation is vicarious.22 However, our studies about the action of tyrosinase on monophenols and o-diphenols21,23–29 suggest that HQ is a substrate of tyrosinase but that the enzymatic activity is not evident because HQ is not capable of closing the enzymatic catalytic cycle by transforming the met-tyrosinase (Em ) form to oxy-tyrosinase (Eox ). This can be done by using an o-diphenol as a reductant.21,23–26 Em (inactive on monophenols) is transformed into Ed , which, by binding with oxygen, is transformed into Eox (active on monophenols). Alternatively, Em is converted into Eox by the addition of H2O2,28,30–32 as demonstrated by Mason (Schemes 1 and 2).33,34 Moreover, the addition of ascorbic acid, which acts as substrate of the enzyme,35 is capable of reducing Em to Ed , facilitating the transformation into the Eox which acts on HQ (Scheme 3). The action of the enzyme on HQ in the presence of H2O2 is depicted in Scheme 1. Note that in the proposed mechanism HHQ is not accumulated in the medium and so the experiments show no lag period. Hence HQ is the only monophenol that does not show a lag in the presence of H2O2.28 A kinetic analysis of
and subsequent studies suggested that HQ was hydroxylated to 2hydroxyhydroquinone (HHQ) through the action of tyrosinase, although the direct enzymatic formation de HHQ has not been demonstrated experimentally.13,14 Serious problems attributed to HQ are pigmentation of the eye and, in a small number of cases, permanent corneal damage.15 Due to this, dermatological depigmented treatments without HQ have been developed, which have been contrasted with others containing the compound.16–18 Attempts to inhibit HQ cytotoxicity have used resveratrol as an additional component.19 Actually, pharmaceutical products (creams and gels) contain up to 4% HQ are used, for example, despigmental topical gel (4%), melanase cream (2%), licoforte topical gel (4%), and pigmentase cream (4%).20 HQ, is an isomer of catechol, a known substrate of tyrosinase.21 However, it has recently been suggested that HQ is neither a substrate nor inhibitor of tyrosinase.22 The same authors suggest that these results agree with previous ones4–6 and conclude that HQ is oxidized in the presence of tyrosinase substrates so that the
OH
B2
B1H O N
2+ Cu
N
2+ Cu
N
N
O
N
O
O2.-
N H
EmHQ
O
OH
OH
HPB
k9
O2 HO
k-1
k1
HQ
OH
HHQ HO
OH
OH
B2
B1
B2H
B1H N 2+
2+ Cu
Cu N
k7
N
O N 2+
N
O
2+ Cu
Cu
N
N
O
N
H
N N
O N
N H
Em
H2O H2O2
k-2
k2
B2
B1H
O2
H2O
EmHHQ
k6
OH
N
+ Cu
+ Cu
N
N
N
N N
k5
k8
O
O
OH
B2
B1H
N
2+ Cu
N
O
2+ Cu
N
N N
O N
Ed
k-8
O
OH
HPB
HQ
O O
HO
k4
B2
B1H
OH
N
2+ O Cu
N
O N
2+ Cu
N N
N H
Eox
k-4
EoxHQ
Scheme 1. Structural mechanism proposed to explain the catalytic pathway of tyrosinase in its action on HQ in the presence of H2O2/Em met-tyrosinase, Em HQ mettyrosinase HQ complex axially bound to Cu atom with protonated base (B1H), Eox oxy-tyrosinase, Eox HQ oxy-tyrosinase HQ complex axially bound to Cu atom with protonated base (B1H), and the proton transferred to the peroxide, Em HHQ met-tyrosinase/2-hydroxyhydroquinone complex, Ed deoxy-tyrosinase, HQ ¼ hydroquinone, HHQ ¼ 2-hydroxyhydroquinone, HPB ¼ 2-hydroxy-p-benzoquinone.
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2.1.1. Effect of hydrogen peroxide The action of tyrosinase on HQ is shown in Figure 1A. In the absence of H2O2 the enzyme shows no activity (recording a), while in its presence the absorbance at 480 nm increases (recording b). As the enzyme concentration is increased the slope also increases, demonstrating the increase of absorbance with time (recordings b–f). The absorbance increase may be due to the hydroxylation of HQ to HHQ and its subsequent oxidation to 2-hydroxy-p-benzoquinone (HPB) (red) at pH 7. The formation of this red quinone is due to the presence of H2O2, which brings about the formation of Eox from Em (see Scheme 1).33,34 In this way the H2O2 helps identify HQ as a substrate of the enzyme.28,30–32 The action of the enzyme on HQ in the presence of H2O2 is shown in Scheme 1. Scheme 1 can be represented kinetically as Scheme 2 and its kinetic analysis is described in the Appendix. According to Eq. A1 inhibition should occurs as a result of the excess of HQ and this is confirmed in Figure 1B, which agrees with the mechanism proposed.
O2
k9
HPB
.
O2 HHQ
HQ
k1 EmHQ
k-1
k7
Em
EmHHQ
HPB
k6
H2O2
k-2
k2
Ed
H 2O
k-8
k5
k8
O2
HQ
k4
Eox
EoxHQ
k-4 Scheme 2. Proposed kinetic mechanism to explain tyrosinase action on HQ in the presence of H2O2,Em , Eox and Ed are met-, oxy- and deoxy-tyrosinase, respectively. HQ, HHQ and HPB represent hydroquinone, 2-hydroxyhydroquinone and 2hydroxy-p-benzoquinone, respectively. The enzymatic complex Em HQ, Em HHQ and Eox HQ are also showed.
the mechanism of Scheme 2 (derived from the structural mechanism shows in Scheme 1) permits the deduction of the analytical expression for the product accumulation rate. Furthermore, Scheme 3 depicts the action of the enzyme on HQ in the presence of ascorbic acid. The aim of this work was to demonstrate that HQ, a depigmenting agent used in pharmacology, is a substrate of tyrosinase. For this, we establish an experimental design that permits the kinetic characterisation of the enzyme’s action on HQ.1,21 A possible explanation of the cytotoxic effects of HQ are also discussed. 2. Results and discussion 2.1. HQ as a substrate of tyrosinase Experiments in the presence of hydrogen peroxide or ascorbic acid were carried out to demonstrate that HQ is a substrate of tyrosinase.
HQ k1
EmHQ
AH2 k2
Em
k-1
2.1.2. Effect of ascorbic acid Ascorbic acid (AH2) is a substrate of tyrosinase.35 Scheme 3 depicts the catalytic cycle of the enzyme in its action on this substrate. Ascorbic acid reduces the met-tyrosinase form to deoxytyrosinase, whose subsequent binding with oxygen generates oxy-tyrosinase, which acts on another molecule of AH2 regenerating met-tyrosinase and closing the catalytic cycle. The intermediate oxy-tyrosinase is capable of hydroxylate HQ (Scheme 3) to generate HHQ, which is subsequently oxidized to HPB. The concentrations of ascorbic used in the experiments depicted in Figure 2A do not reduce the HPB, so that Figure 2A shows that the absorbance at 480 nm (corresponding to HPB) increases with time. Increasing the enzyme concentration (Fig. 2A) augments the accumulation rate of the red quinone HPB. Figure 2B shows that the same effect is obtained when the concentration of AH2 is increased. Both results can be explained by the increased turnover of tyrosinase on AH2. Spectrophotometric identification of the product of the enzymatic reaction can be seen in Figure 2C (increased absorbance at 480 nm). In the absence of HQ or tyrosinase there is no increase in the absorbance at this wavelength (results not shown).
EmD
k-2
HHQ
k11 O2
k3
HPB -
O2
k10
A HPB Ed
EmHHQ k9 O2
k7 EoxAH2
AH2 k6 k-6
k-8
k5
k8 HQ Eox
k4 k-4
EoxHQ
Scheme 3. Action of tyrosinase on HQ in the presence of ascorbic acid, where: HQ ¼ Hydroquinone, HHQ ¼ 2-hydroxy-hydroquinone, HPB ¼ 2-hydroxi-p-benzoquinone, AH2 ¼ ascorbic acid, A ¼ dehydroascorbic acid, Em = met-tyrosinase, Ed = deoxy-tyrosinase and Eox = oxy-tyrosinase.
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0.15
120
B
A 100
0.10
(h)
(a)
0.05
60
V0
HPB
A480
(nM/s)
80
40
20
0.00
0
40
80
120
t (s)
0 0
5
10
15
20
25
[HQ]0(mM)
Figure 1. (A) Action of tyrosinase on HQ in the presence of hydrogen peroxide. Effect of varying the concentration of enzyme. Spectrophotometric recordings of the action of tyrosinase on HQ measured at a wavelength of 480 nm. The experimental conditions were: 30 mM phosphate buffer (pH 7.0), initial HQ concentration, [HQ]0 = 0.5 mM, initial H2O2 concentration, [H2O2]0 = 2.5 mM and initial enzyme concentrations, [E]0, were (nM): 0 (a), 10 (b), 35 (c), 55 (d), 75 (e), 90 (f), 115 (g) and 130 (h). B. Representation of values of the initial action rate of tyrosinase on HQ, maintaining [H2O2]0 and [E]0 constants. The conditions were: 30 mM phosphate buffer pH 7.0 [H2O2]0 = 40 mM, the HQ concentrations shown in the figure and [E]0 = 14 nM.
2.2. Identification of hydroxyhydroquinone as a product of the enzymatic reaction of tyrosinase on HQ To demonstrate that HPB is formed enzymatically through the oxidation of HHQ and not as a result of purely chemical reactions, such as the addition of water to p-benzoquinone (PB); several experiments were carried out at high ascorbic acid concentrations (to minimise the formation of quinone) and product formation was studied by HPLC–MS. The chromatograms displayed were recorded at 290 nm. Signals correspond to the following compounds identified by mass spectrometry and comparison with standards (Fig. 3A). Figure 3B shows the mass spectrum of the peak appearing in the chromatogram at 5.1 min. The kinetic of HHQ formation was then studied, taking aliquots at different times and revealing by HPLC–MS, following the evolution of the characteristic peak of HHQ. The results are shown in Figure 3C. Another method used to follow this reaction involved oxidizing aliquots of the reaction mixture with an excess of sodium periodate. The great difference in the molar absorbtivity coefficient between PB and HPB (Fig. 4A) enables the reaction in which HHQ is formed to be followed qualitatively (Fig. 4B). As the reaction time increases, the concentration of HHQ increases and, after oxidation with an excess of sodium periodate, the absorbance at 480 nm increases.36 It has been proposed that the generation of HPB in a reaction medium containing tyrosinase and HQ in the presence of L-Dopa occurs as a result of the water added by PB or through a disproportion reaction between HQ and PB and the formation of HPB in this reaction through the formation of a radical.22 However, as can be seen in Figure 5, the Cyclic Voltammetry experiments demonstrated the stability of PB at pH 7 (Fig. 5A) since at t = 0 and 30 min the recordings did not change. Figure 5B shows the cyclic voltagrams of a stoichiometric mixture of HQ and PB at the same times (t = 0 and 30 min). The recordings show that at this pH value and within this time period no HPB is formed. These data agree with those long described for the slow reaction of the addition of water to PB,37 with an apparent pseudo-first order constant = 3 107 s1. Although the reaction of hydroxylation of PB by photohydroxylation at 254 nm has been described, these results were obtained using a different experimental approach.38
Having demonstrated that the enzyme hydroxylate HQ to HHQ and then, according to Schemes 1–3, oxidizes this compound to HPB or releases HHQ to the medium, where it is rapidly oxidized by the oxygen in the solution to HPB, it is now possible to kinetically characterise the action of tyrosinase on HQ by spectrophotometrically following the formation of HPB at 480 nm. Based on the kinetic analysis developed in the Appendix, we propose the following steps for the kinetic characterization of the action of tyrosinase on HQ. 2.3. Kinetic chracterisation of HQ as tyrosinase substrate Step 1. Vary the concentration of H2O2 while maintaining the concentrations of HQ and tyrosinase constant. Follow the formation of HPB spectrophotometrically. The slopes of the lines, that is the initial velocity V HPB show hyperbolic dependence with 0 respect to [H2O2]0 according to Eq. A10. The hyperbolas obtained are depicted in Figure 6A (a–i). Step 2. Nonlinear regression analysis of the values of V HPB v ersus ½H2 O2 0 according to Eq. A10 to give the parameters 0 app V app max y K M , which are explained in Eqs. A11 and A12. app Step 3. Analysis of V app max and K M depending on [HQ]0 according to Eqs. A17 and A19, Figure 7A. This analysis provides the kinetic HQ information shown in Table 1. kcat ¼ k5 (Catalytic constant) and HQ K M ¼ k5 þ k4 =k4 (Michaelis constant). Figure 7B shows how (K app M ) depends on [HQ]0. Another experimental design involves using a constant ratio between [H2O2]0 and [HQ]0, that is [H2O2]0/[HQ]0 = R. From Eq. A1, it is possible to obtain Eq. A21. The values of V HPB;R show a 0 hyperbola when represented versus [HQ]0 (Fig. 8) and the nonlinear regression analysis of these data according to Eq. A21 provides app;R the values of V app;R . The representation of these values max and K M according to Eqs. A22 and A23 gives Figure 9A and 9A Inset, respectively. From Eqs. A19 and A22 K 1 , the dissociation constant of the complex Em ðHQ Þ,can be obtained (see Table 1). 2.4. Comparison with other monophenols The literature22 describes how the presence of the p-hydroxy group in HQ prevents binding to the tyrosinase active site,
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0.20
B
A 0.12
f
0.15
0.08
[E]0
0.10
[AH2]0
A480
A480
d
c 0.04
b
0.05
a 0.00
0.00 0
400
800
1200
1600
a 0
400
800
1200
1600
t (s)
t (s) 0.300
C
A
0.225
0.150
0.075
0.000 400
440
480
520
λ (nm) Figure 2. Action of tyrosinase on HQ in the presence of ascorbic acid. (A) Effect of varying the concentration of enzyme. Spectrophotometric recording of the action of tyrosinase on HQ measured at 480 nm in the presence of ascorbic acid, [AH2]0 = 2 mM. The experimental conditions were: 30 mM phosphate buffer (pH 7.0). [HQ]0 = 0.5 mM and [E]0 (lM) were: (a) 0.5, (b) 1.1, (c) 1.7 and (d) 2.2. B. Effect of varying the ascorbic acid concentration Spectrophotometric recording of the action of tyrosinase on HQ measured at 480 nm varying the concentration of ascorbic acid. The experimental conditions were: 30 mM phosphate buffer (pH 7.0). [HQ]0 = 0.5 mM, [E]0 = 1.7 lM and [AH2]0 (mM) were: (a) 1, (b) 3, (c) 2.5, (d) 2, (e) 1.5 and (f) 4. C. Spectrophotometric recordings of the action of tyrosinase on HQ in the presence of ascorbic acid. The experimental conditions were: 30 mM phosphate buffer (pH 7.0). [HQ]0 = 0.5 mM, [E]0 = 1.7 lM and [AH2]0 = 2 mM. Interval between recordings was 300 s.
although, as the same authors mention, other 4-substituents, such as alkyl, alkoxy and halo, are monoxygenase substrates.39,40 The action of tyrosinase on HHQ has recently been described, revealing that the position of a p-hydroxy does not hinder the action of tyrosinase.41 Moreover, the data obtained for the action of tyrosinase on HQ in the present study, compared with those previously described for other monophenols, are shown in Table 2.21 The chemical shift values indicate that HQ is a good tyrosinase subHQ strate. The values of kcat agree with those obtained for 4-methoxyphenol, 4-ethoxyphenol and phenol. Hence, the kinetic study of HQ using H2O2 provides data that agree with those obtained for the monophenols studied in the absence of H2O2. 3. Conclusions From these results, it can be concluded that HQ is a substrate of tyrosinase but the enzyme cannot express its activity in the absence of a reductant because of the characteristics of its catalytic cycle (Schemes 1–3).1,28 In the presence of H2O2 Em
(inactive on HQ) can pass to Eox , an intermediate that can act on HQ.28 In the presence of a reductor agent such as AH2, Em (inactive on HQ) generates Ed , which, in turn, passes to Eox , which acts on HQ. By means of these transformations of the intermediates in the catalytic cycle, tyrosinase can act on HQ, as a substrate. Moreover, the strong depigmenting effect of HQ, and hence its action on the melanogenesis pathway, is double: since it can act as reductant of the o-quinone of L-Dopa, o-dopaquinone, originating PB, and also, as shown in this work, by acting as a substrate of tyrosinase, it can redirect the melanin biosynthesis pathway towards the formation of HPB. Both quinone generation pathways involve the formation of two p-quinones, PB and HPB, which are stable, especially compared with o-quinones. This may involve the consumption of thiolic compounds in the medium and hence a change in the redox state of the cell, which would explain the cytotoxic effects described for the use of HQ.12,22,42 The confirmation that HQ is a substrate of tyrosinase may have important consequences for its use in pharmacology and cosmetics.
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A
B
C
Figure 3. (A) HPLC separation of a reaction mixture after reaction of 50 nM tyrosinase with [HQ]0 = 2 mM, [H2O2]0 = 1 mM and [AH2]0 = 30 mM in 30 mM sodium phosphate buffer, pH 7.0 at 25 °C. Analyses were performed 30 min after beginning the reaction. The signals correspond to: ascorbic acid (2.5 min), HHQ (5.1 min) and HQ (6.4 min), were registered at 290.4 nM B. Mass spectrum corresponding to 5.1 min reaction time (HHQ) of Figure 3A. C. Chromatograms corresponding to the fragment m/z 125 at different reaction times ( ) 500 s, (—) 1200 s and () 1600 s, showing the formation of the product HHQ. The experimental conditions were the same as Figure 3.A.
4. Material and methods 4.1. Reagents Mushroom tyrosinase or polyphenol oxidase (o-diphenol/O2 oxygen-oxidoreductase, EC1.14.18.1 was supplied by Sigma 4276 U/mg) (Madrid, Spain). The enzyme was purified as previously described.29 Protein concentration was determined by Bradford´s method using bovine serum albumin as standard.43 All the
substrates used in this work were purchased from Sigma (Madrid, Spain) and were prepared en 0.15 mM phosphoric acid to prevent autooxidation. Milli-Q system (MilliporeCorp.) ultrapure water was used throughout. 4.2. Spectrophotometric assays The enzymatic assays were carried out with a Perkin Elmer Lambda-35 spectrophotometer (Waltham, Massachusetts USA),
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2.0
A
B
400
0.2
1.0
0.0
0
450
900
1350
300
200
HPB
A
HPB
(nM/s)
0.4
V0
1.5
A480
0.6
1800
t (s)
0.5
100
PB
0.0 250
300
350
400
450
0 500
550
0
Figure 4. (A) Spectral characteristics of PB and HPB. The experimental conditions were 30 mM phosphate buffer, pH 7.0, la [PB]0 = 0.5 mM, HPB generated by stoichiometric oxidation with [NaIO4]0 = 0.5 mM and [HHQ]0=0.5 mM. B. Reaction of tyrosinase with HQ in the presence of a large excess of ascorbic acid. The experimental conditions were 30 mM phosphate buffer, pH 6.0 at 25 °C, [AH2]0 = 30 mM, [HQ]0 = 2 mM, [E]0 = 65 nM. At the times indicated in the figure, an aliquot was oxidized with excess sodium periodate ([NaIO4]0 = 35 mM) and the absorbance at 480 nM was measured.
16
I / nA
12 8 4 0
0.0
0.4
0.8
E/V 12
B
I / nA
8 4 0 -4 -8 -0.8
-0.4
0.0
60
80
100
120
0.4
Figure 6. Representation of initial formation rate of HPB (V HPB 0 ) in 30 mM phosphate buffer, pH 7.0, during the action of tyrosinase [E]0 = 28 nM on HQ, varying the concentration of H2O2 as indicated in the figure. The concentrations of HQ (mM) were: 0.07 (d), 0.1 (s) and 0.3 (.).0.4 (h), 0.5 (j), 0.75 (N) 1.2 (e) and 1.5().
with an excess of periodate and measuring the increase in absorbance at 480 nm, which corresponds mainly to HHQ formed. 4.3. HPLC–MS
A
-0.4
40
[H2O2] (mM)
λ
-4 -0.8
20
0.8
E/V Figure 5. (A) Cyclic voltammogram (cathodic scan, initial potential 0.6 V) of [PB]0 = 1 mM in 0.1 M phosphate buffer (pH = 7). (a) Fresh solution; (b) after 30 min. Scan rate: 0.05 V s1.B. Cyclic voltammogram (cathodic and anodic scan, initial potential 0.2 V) of [PB]0 = 0.5 mM and [HQ]0 = 0.5 mM in 0.1 M phosphate buffer (pH = 7). (a) Fresh solution; (b) after 30 min. Scan rate: 0.05 V s1.
on-line interfaced with a PC computer, where the kinetic data were recorded, stored and later analyzed. The conditions of the assay are specified in the corresponding figure legend.36,44 The reaction of tyrosinase on HQ in the presence of H2O2 and a large excess of AH2 was followed, oxidizing the reaction mixture
The reaction of tyrosinase with HQ was also followed by measuring the formation of HHQ in the presence of an excess of ascorbic acid by HPLC–MS. The enzyme was separated from the reaction intermediates by centrifugation using 10,000 MWL0 filters. The reaction products were analyzed in a high resolution liquid chromatograph. To separate and quantify the different compounds an AGILENT 1100 series HPLC (Germany) coupled to an AGILENT VL ion trap mass sepctrometer (Germany) was used. The separation programme used a gradient of (A) H2O with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The gradient began at time 0 with 3% B, raised to 50% B in 10 min. The initial value of 3% B was reached at 11 min, when the equipment was allowed to stabilize for 5 min. The flow rate was 0.8 ml/min. The signal at 290 nm was captured by the DAD, with an absorbance range of 190–350 nm at 2 nm steps. 10 lL of sample was injected onto a 5 lM SunFire™ C18 column, 4.6 mm 150 mm, thermostated at 25 °C. Mass spectrometry acquisition was carried out in scan mode, with a mass range of 50–500 m/z. The elecrospray source (ESI) was operated in negative mode. The drying temperature was 350 °C, nebulizer pressure was 60 psi and drying gas flow 9 l/m. The Extracted Ion Cromatogram (EIC) corresponding to each ion with its (MH), was used to monitor the different compounds 4.4. Electrochemistry Cyclic Voltammetry (CV) was performed using a computerdriven three electrode potentiostat constructed in the ‘Research Support Service of the University of Murcia’ (http://www.um.es/ sai). A three-electrode cell, with a 12.5 lm radius Pt working microdisk electrode (from CH Instruments) was used The reference electrode was a saturated calomel electrode (SCE), and the counter electrode was a platinum wire. Solutions were prepared with distilled deionized water (Milli-Q filtering system). Nitrogen gas was passed through solutions for de-aeration for 20 min prior to measurements (buffer and acidic substrate in separate parts) with
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750
80
A
B
600
(mM) app
450
KM
app
Vmax (nM/s)
60
40
300
20
150
0 0.0
0.5
1.0
1.5
0 0.0
2.0
0.5
1.0
1.5
2.0
[HQ]0 (mM)
[HQ]0 (mM)
app Figure 7. A. Representation of the values of V app max versus [HQ]0 B. Representation of the values of K M versus [HQ]0. The concentrations of HQ are indicated in the figure. Enzyme concentration, [E]0 = 28 nM.
Table 1 Kinetic constants that characterise the action of tyrosinase on hydroquinone. Chemical shift and pK values of hydroquinone.
Hydroquinone a
K HQ M (mM)
K 1 (mM)
k5 (s1)
pK1a
pK2a
d1
0.25 ± 0.04
4.56 ± 0.93
23.0 ± 1.1
8.2
10.1
153.30
(ppm)
From 47.
600
400
400 5 4 (mM)
300
3 2
app,R
app,R
200
V0
HPB,R
(nM/s)
300
KM
Vmax (nM/s)
500
200
1
100
100 0
0
5
10
15
20
25
R
0
0
0
2
4
6
5
10
15
20
25
R
[HQ]0 (mM) V HPB;R 0
0
V HPB;R 0
Figure 8. Representation of initial versus [HQ]0. The values of obtained at 480 nM for the reaction of tyrosinase on HQ at different [H2O2]0/[HQ]0 ratios (R) are shown in the figure. [E]0 = 28 nM. R values were: 2.5 (j), 5.0 (h), 6 (N), 7.5 (e), 10 (d), 15 (s) and 20 (.).
app;R Figure 9. Representation of V app;R max versus R. The values of V max obtained by analysis of the data in Figure 7 are represented versus the [H2O2]0/[HQ]0 ratio (R). Inset app;R Representation of K M versus [R].
Table 2 Kinetic constants for the monophenolase activity of mushroom tyrosinase
a nitrogen atmosphere maintained over the solution throughout the experiments. The platinum disk was polished prior to experiments using 1.0, 0.3 and 0.05 lm alumina-water slurry on soft lapping pads and after sonication for 10 min. 4.5. Kinetic analysis data For tyrosinase, the initial rate values (V0 ) were calculated from triplicate measurements at each reducing substrate concentration, and V0 versus [S]o data were adjusted to the Michaelis–Menten equation through the Sigma Plot 9.0 program for Windows,45 thus obtaining the maximum rate (Vmax ) and Michaelis constant (Km ).
*
Monophenol
K M (mM)
kcat (s1)
d3
4-Methoxyphenol* 4-Ethoxyphenol* Hydroquinone Phenol*
0.08 ± 0.003 0.17 ± 0.005 0.25 ± 0.04 0.70 ± 0.04
184.2 ± 6.1 132.0 ± 4.5 23.0 ± 1.1 12.7 ± 0.6
118.90 119.01 119.50 118.13
(ppm)
d4
(ppm)
152.29 152.39 153.30 158.15
Data taken from 21.
4.6. Determination of
13
C NMR chemical shifts
The carbon chemical shifts given in Tables 1 and 2 were obtained from the corresponding 13C NMR spectra, which were
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recorded at 298 K on a Bruker Avance 400 Hz instrument employing buffered solutions (at pH 7.0) of pure samples in D2O. Acknowledgements This paper was partially supported by Grants from Ministerio de Educación y Ciencia (Madrid, Spain) Project BIO2009-12956, Fundación Séneca (CARM, Murcia, Spain) Projects 08856/PI/08 and 08595/PI/08, Consejería de Educación (CARM, Murcia, Spain) BIO-BMC 06/01-0004.
V app max ¼
a1 ½HQ 0 ½E0 b5 þ b7 ½HQ 0
ðA13Þ
K app M ¼
b1 þ b3 ½HQ 0 þ b6 ½HQ 20 b5 þ b7 ½HQ 0
ðA14Þ
explained as: k5 ðk6 þk7 Þ ½HQ 0 ½E0 k5 þk6 þk7 6 þk7 Þðk4 þk5 Þ þ ½HQ 0 k4 ðK 5 þk6 þk7 Þ
V app max ¼ ðk
ðA15Þ
and
K app M
¼
K 1 k2 k8 ðk4 þk5 Þðk6 þk7 Þ K 1 k2 k4 k8 ðk5 þk6 þk7 Þ
k k k k ½HQ 2
4 þk5 ÞþK 1 k4 k5 k7 k8 ½HQ0 0 þ ½k2 k8 ðk6 þkK71 Þðk þ K 1 k24k45k87ðk85 þk6 þk k2 k4 k8 ðk5 þk6 þk7 Þ 7Þ
K 1 k2 k8 ðk6 þk7 Þðk4 þk5 Þ K 1 k2 k4 k8 ðk5 þk6 þk7 Þ
ðA16Þ
þ ½HQ 0
Appendix Applying the steady state approximation to the mechanism shown in Scheme 2 provides the following expression for the HPB formation rate:
V HPB ¼ 0
From Eq. A15, taking into consideration that the hydroxylation step controlled by k5 is the slowest (k5
