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Action of tyrosinase on caffeic acid and its n-nonyl ester. Catalysis and suicide inactivation
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Action of tyrosinase on caffeic acid and its n-nonyl ester. Catalysis and suicide inactivation Antonio Garcia-Jimenez a , Jose Antonio Teruel-Puche b , Pedro Antonio Garcia-Ruiz c , Adrian Saura-Sanmartin d , Jose Berna d , Jose Neptuno Rodríguez-López a , Francisco Garcia-Canovas a,∗ a GENZ-Group of research on Enzymology, Department of Biochemistry and Molecular Biology-A, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, E-30100, Espinardo, Murcia, Spain1 b Group of Molecular Interactions in Membranes, Department of Biochemistry and Molecular Biology-A, University of Murcia, E-30100, Espinardo, Murcia, Spain c University of Murcia, Faculty of Veterinary, Group of Chemistry of Carbohydrates, Industrial Polymers and Additives, Department of Organic Chemistry, E-30100 Murcia, Spain d Group of Synthetic Organic Chemistry, Department of Organic Chemistry, Faculty of Chemistry, University of Murcia, E-30100 Espinardo, Murcia, Spain
a r t i c l e
i n f o
Article history: Received 28 July 2017 Received in revised form 24 October 2017 Accepted 24 October 2017 Available online xxx Keywords: Tyrosinase Caffeic acid n-Nonyl caffeate
a b s t r a c t Different mechanisms for inhibiting tyrosinase can be designed to avoid postharvest quality losses of fruits and vegetables. The action of tyrosinase on caffeic acid and its n-nonyl ester (n-nonyl caffeate) was characterized kinetically in this work. The results lead us to propose that both compounds are suicide substrates of tyrosinase, for which we establish the catalytic and inactivation efficiencies. The ester is more potent as inactivator than the caffeic acid and the number of turnovers made by one molecule of the enzyme before its inactivation (r) is lower for the ester. We proposed that the anti-browning and antibacterial properties may be due to suicide inactivation processes. © 2017 Published by Elsevier B.V.
1. Introduction The process of food browning due to the oxidation of polyphenols is notorius since it leads to a loss of nutritional, aesthetic and organoleptic properties, and, so, of the food’s commercial value [1–4]. Tyrosinase (EC 1.14.18.1), a copper-containing enzyme, is one of the main agents responsible for this process. Its catalytic centre has two copper atoms, each coordinated with three histidine residues [5,6]. This enzyme catalyzes the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the subsequent oxidation of o-diphenols to o-quinones (diphenolase activity) [7]. Recently, a study was published on the protective effect of n-nonyl caffeate on the Chinese olive (Canarium album) [8], the quality of which during postharvest is affected by browning and bacterial putrefaction [9]. Tyrosinase inhibitors reduce these effects
and, so, have important applications in this respect [10–13]. In the case of Chinese olive, it was demonstrated that n-nonyl caffeate can prevent postharvest putrefaction because of its anti-browning and anti-bacterial effect [8]. Indeed, it was seen to attack Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Salmonella and Klebsiella pneumoniae, but not Pseudomonas aeruginosa or Agrobacterium tumefaciens. A kinetic analysis of this compound led the same authors to propose that n-nonyl caffeate is a reversible inhibitor of tyrosinase at low concentrations and an irreversible inhibitor at high concentrations [8]. It was this atypical behaviour of n-nonyl caffeate that led us to study the action of the enzyme on this compound based in the knowledge gained in our previous studies about the action mechanism of tyrosinase [7]. Therefore, n-nonyl caffeate and caffeic acid (CAFA), from which it is derived, were studied to understand the mechanism involved, while considering the possibility that such compounds, being substrates of tyrosinase, may give rise to unstable o-quinones.
∗ Corresponding author at: Departamento Bioquímica y Biología Molecular A, Facultad de Veterinaria, Campus de Espinardo, E-30100, Spain. E-mail address: [email protected] (F. Garcia-Canovas). 1 www.um.es/genz. https://doi.org/10.1016/j.ijbiomac.2017.10.151 0141-8130/© 2017 Published by Elsevier B.V.
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Fig. 1. Chemical structures of (a) CAFA, (b) n-nonyl caffeate, (c) DHPAA, (d) PCA and (e) DHPPA.
2. Materials and methods
ratio [21–24]. The corresponding analytic expression and data analysis provided r.
2.1. Materials
2.2. Methods 2.2.1. Synthesis method of n-nonyl caffeate Catalytic amounts of sulfuric acid were added to a solution of CAFA (100 mg, 0.6 mM) in nonanol (10 mL) and the reaction mixture was stirred at 100 ◦ C for 6 h. After this time, nonanol was removed by vacuum distillation, and the resulting solid was crystallized from ether, yielding the product of interest as a white solid. 2.2.2. Tyrosinase activity Spectrophotometric assays were carried out with a PerkinElmer Lambda-35 spectrophotometer (using water in the reference cuvette), online interfaced with a compatible PC 486DX microcomputer controlled by UV-Winlab software, where the kinetic data were recorded, stored, and analyzed. The diphenolase activity on Ldopa and the monophenolase activity on L-tyrosine were followed by measuring the increase of absorbance at = 475 nm led by the formation of dopachrome. For its part, the diphenolase activity on TBC (4-tert-butylcatechol) was measured at = 410 nm [16]. The suicide inactivation of tyrosinase acting on CAFA was followed by measuring the disappearance of NADH [17–20], originated by its oxidation by the o-quinone, at 380 nm. Finally, the suicide inactivation of tyrosinase acting on n-nonyl caffeate was followed by reacting the enzyme with this compound in the presence of AH2 and measuring the on-going residual activity of tyrosinase in a high concentration of TBC [17–20]. All the experiments were made in 30 mM phosphate buffer pH 7.0. In order to calculate the parameter r (the partition ratio or the turnover numbers made by one mol of the enzyme before its inactivation) when tyrosinase acts on n-nonyl caffeate, the enzyme was incubated with this compound while bubbling with air. After that, aliquots were taken at t → ∞ and the activity was assayed with TBC, providing the values of residual activity vs the substrate/enzyme
13 C
NMR assays NMR spectra of n-nonyl caffeate, CAFA and DHPPA were obtained on a Bruker Avance 300 MHz instrument. In the case of DHPAA and PCA, the instrument used was a Bruker Avance 400 MHz. In all the cases DMSO was used as solvent. The ␦ 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. 2.2.3.
Mushroom tyrosinase (3130 U/mg), reduced nicotinamide adenine dinucleotide (NADH), ascorbic acid (AH2 ), 3-(3,4-dihydroxyphenyl)propionic acid (DHPPA), 3,4dihydroxyphenylacetic acid (DHPAA), protocatechuic acid (PCA) and CAFA were purchased from Sigma (Madrid, Spain). The enzyme was purified as previously described [14] and the protein concentration was determined by Bradford’s method [15], using bovine serum albumin as the standard. Stock solutions of CAFA and n-nonyl caffeate were prepared in 0.15 mM phosphoric acid (aqueous solution) to prevent auto-oxidation and, this latter compound was also solubilised in DMSO (4% (v/v)) as cosolvent. Milli-Q system ultrapure was used throughout.
13 C
2.2.4. Computational docking The chemical structure of n-nonyl caffeate was constructed with PyMOL 1.8.4.0 [25] and its geometry was optimized with MOPAC2012 software [26] and PM7 semiempirical Hamiltonian. Rotatable bonds in the ligand were assigned by AutoDockTools4 program [27,28]. Oxytyrosinase was prepared as previously described [29]. Ligand was docked into the catalytic site of mushroom tyrosinase from Agaricus bisporus (PDB code: 2Y9W) using AutoDock Vina [30]. AutoDock Vina parameters were as follows: receptor file; ligand file; xyz centre coordinate of the pocket residue centred in the two copper ions; search space in each dimension, 11.3 Å; exhaustiveness, 24; and generation number of binding modes, 10. 2.2.5. Data analysis The experimental disappearance of NADH with time follows the equation:
[NADH] = [NADH]0 − [Q] = [NADH]0 − c1 1 − e
−c t
2
+ c3 + c4t
(1) where [Q ] is the concentration of the product of the enzymatic reaction, t is time and in which ci (i = 1–4) can be obtained by nonlinear regression [31]. The parameter c1 is equal to [Q ]∞ = [NADH]0 − [NADH]f (the concentration of product obtained at the end of the reaction) and c2 is equal to the corresponding . The coefficient c3 represents the uncertainty at zero time absorbance caused by the addition of the enzyme at the start of the reaction, while c4 corresponds to the slow spontaneous oxidation of o-diphenol and NADH. The effects of both experimental artefacts should be computer subtracted in further NADH against time. 3. Results and discussion CAFA and n-nonyl caffeate are ortho-diphenolic compounds (Fig. 1), and so can be chemically (by sodium periodate) [32] and enzymatically (by tyrosinase) oxidized [7].
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Fig. 2. A. Scan spectra of the evolution of CAFQ obtained by the oxidation of CAFA by NaIO4 in default. The experimental conditions were [CAFA]0 = 150 M and [NaIO4]0 = 50 M. Recordings were made every 2 min. Inset. Scan spectra (recordings “a–i” and spectrophotometric recording (“g”) at 550 nm of the evolution of the quinone of n-nonyl caffeate obtained by the oxidation of this compound by NaIO4 in default. The experimental conditions were [n-nonyl caffeate]0 = 200 M and [NaIO4 ]0 = 60 M. Recordings were made every 2 min. B. Scan spectra of the evolution of CAFQ obtained by the oxidation of CAFA by NaIO4 in excess. The experimental conditions were [CAFA]0 = 50 M and [NaIO4 ]0 = 400 M. Recordings were made every 2 min. Inset. Scan spectra of the evolution of the quinone of n-nonyl caffeate obtained by the oxidation of this compound by NaIO4 in excess. The experimental conditions were [n-nonyl caffeate]0 = 200 M and [NaIO4 ]0 = 600 M. Recordings were made every 2 min.
3.1. Characteristics of the o-quinones of the CAFA and n-nonyl caffeate Sodium periodate (NaIO4 ) is known to oxidize o-diphenols to oquinones [32]. Fig. 2A shows the spectrophotometric recordings of the oxidation of CAFA by NaIO4 in default. Note the presence of two imperfect isosbestic points due to the evolution of the o-quinone in the presence of o-diphenol and the accumulation of intermediate compounds as is represented in Fig. 1SM, which shows the possible set of reactions until the formation of a practically stable product [33]. The same experiment in the case of n-nonyl caffeate is shown in Fig. 2A Inset, where an increase of absorbance after an initial decay can be seen in the form of scans (recordings “a–i”) and as a spectrophotometric recording at 550 nm (recording “j”). When CAFA is oxidized by NaIO4 in excess, the isosbestic points are more clearly defined (Fig. 2B), since no intermediate products are accumulated in the medium (Fig. 2SM). The oxidation of n-nonyl caffeate by NaIO4 in excess generates an o-quinone, which evolves in the same way as that of CAFA (Fig. 2B Inset). 3.2. Action of tyrosinase on CAFA and n-nonyl caffeate The action of tyrosinase on CAFA and n-nonyl caffeate are represented in Fig. 3 Inset, respectively, the spectra being similar to those obtained with NaIO4 in default (Fig. 2A Inset). Thus, CAFA and n-nonyl caffeate are oxidized by tyrosinase, generating o-quinones. 3.3.
13 C
Fig. 3. A. Scan spectra of the action of tyrosinase on CAFA. The experimental conditions were [E]0 = 30 nM and [CAFA]0 = 50 M. Recordings were made every 2 min. B. Scan spectra of the action of tyrosinase on n-nonyl caffeate. The experimental conditions were [E]0 = 30 nM and [n-nonyl caffeate]0 = 50 M. Recordings were made every 2 min.
feate, DHPPA, DHPAA and PCA (the last three compounds shown for comparison). The ␦3 and ␦4 values are shown in Table 1.
NMR
The NMR spectra of CAFA and n-nonyl caffeate showed the chemical shifts of the carbon with the hydroxyl groups (C-3 and C-4), which indicated the potency of the nucleophilic attack on the copper atom of the active site by the oxygen of the hydroxyl group [34]. Hence, Fig. 3SM shows the NMR spectra of CAFA, n-nonyl caf-
3.4. Effect of CAFA and n-nonyl caffeate on the monophenolase and diphenolase activities of tyrosinase on L-tyrosine and L-dopa When the action of tyrosinase on L-tyrosine is assayed (Fig. 4A), the formation of dopachrome (recording “a”) shows a lag period, which corresponds to the time needed for the system to accumulate
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Fig. 4. A. Spectrophotometric recordings at 475 nm of the monophenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of CAFA (M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 100 nM and [L-tyrosine]0 = 0.5 mM. Inset. Spectrophotometric recordings at 475 nm of the monophenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of n-nonyl caffeate (M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 100 nM and [L-tyrosine]0 = 0.5 mM. B. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of CAFA (M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 40 nM and [L-dopa]0 = 0.5 mM. Inset. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of n-nonyl caffeate (M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 40 nM and [L-dopa]0 = 0.5 mM. Table 1 Values of the chemical shifts of the different suicide substrates obtained by 13 C NMR for the C-3 and C-4. Compound
␦4
␦3
DHPAA DHPPA CAFA n-nonyl caffeate PCA
145.7 145.9 149.0 149.2 150.8
144.8 144.3 146.5 146.4 145.6
ever, this set of data can also be explained by a suicide inactivation process, as is detailed in the following mechanism: The kinetic mechanism proposed to explain the suicide inactivation of tyrosinase is described in Fig. 7. The inactivation constant of tyrosinase ki72 is the lowest, so the enzymatic forms of the mechanism of Fig. 7 are in the steady state (ES ), which evolves through a transition phase towards the inactivation of the enzyme [17]. The variation of [ES ] and [Q] with time is given by: d [ES ] = −ki7 f(E ox − S)1 [E S ] 2 dt
a certain amount of o-diphenol in the steady state [35]. The addition of CAFA (Fig. 4A) or n-nonyl caffeate (Fig. 4A Inset) reduces the lag period and apparently activates the enzyme, since the rate is increased (recordings “b-f”). In the case of the diphenolase assay, the addition of CAFA (Fig. 4B) or n-nonyl caffeate (Fig. 4B Inset) apparently activates the enzyme when the formation of dopachrome is measured (recordings “b–f”). This happens because these compounds act as electron donors, converting Em to Ed , and the product formed in each case absorbs in the visible spectrum, the catalytic constant for these compounds being higher than for L-dopa (107.4 ± 3.1 s−1 ) and Ltyrosine (7.9 ± 0.3 s−1 ) [34].
and d[Q ] = 2k7 f(E ox − S)1 [E S ] 2 dt
(3)
where f (E ox − S)1 represents the fraction of the species (Eox − S)1 in the steady state (Es ). The integration of Eq. (2) and Eq. (3), considering [ES ] = [E]0 and [Q] = 0 at t = 0, yields: [Q] = [Q]∞ (1 − e−t )
(4)
with [Q]∞ =
3.5. Inactivation of tyrosinase by CAFA and n-nonyl caffeate Fig. 5A and B show the spectrophotometric recordings of the action of the enzyme on L-dopa in the presence of CAFA and n-nonyl caffeate, but starting the reaction with the addition of L-dopa. In this case, the previous contact of the enzyme with CAFA or n-nonyl caffeate inactivates it, which is particularly evident when the concentration of n-nonyl caffeate is increased (Fig. 6). For this reason, it was proposed that n-nonyl caffeate is a reversible inhibitor at low concentrations and irreversible at high concentrations [8]. How-
(2)
2k7 2kcat [E]0 = i 2 [E]0 = 2r[E]0 max k7
(5)
2
where the partition ratio (r) is r=
kcat max
(6)
the apparent inactivation constant () =
max [S]0 S + KM [S]0
(7)
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Fig. 5. A. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the absence of (a) and the presence of different concentrations of CAFA: b) 40, c) 120, d) 250 and e) 400; pre-incubating the assay with this compound and starting the reaction with L-dopa. The rest of the experimental conditions were [E]0 = 10 nM and [L-dopa]0 = 0.5 mM. B. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the absence of (a) and the presence of different concentrations of n-nonyl caffeate: b) 40, c) 120, d) 250 and e) 400; pre-incubating the enzyme with this compound and starting the reaction with L-dopa. The rest of the experimental conditions were [E]0 = 10 nM and [L-dopa]0 = 0.5 mM.
and the initial rate in the steady state gives V0 =
2kcat [S]0 [E]0
(8)
S + KM [S]0
with max =
k7i k3 k7 k7 2
1
3
k7 k7 k7 + k3 k7 k7 + k3 k7 k7 + k3 k7 k7 1
2
3
2
3
1
3
1
(9)
2
and kcat =
k7 k3 k7 k7 2
1
3
k7 k7 k7 + k3 k7 k7 + k3 k7 k7 + k3 k7 k7 1
2
3
2
3
1
3
1
(10)
2
The stoichiometry of Q when it is reduced by NADH is: [NADH] = [NADH]0 − Q
(11)
giving [NADH] = [NADH]f + [NADH]∞ e−t
(12)
with [NADH]f ([NADH]f value at t → ∞, [NADH]∞ = [NADH]0 − [NADH]f = [Q]∞ ), being (the apparent constant of suicide inactivation of tyrosinase in the presence of CAFA). The suicide inactivation can also be studied by measuring the residual activity of the enzyme, maintaining the concentration of substrate constant by means of a reductant, such as AH2 , which transforms the o-quinone into o-diphenol, and measuring the residual activity with a high concentration of TBC. Therefore, the variation of the active enzyme (Es ) corresponds to Eq. (2) and its expression with time is: −ki f (Eox −S)1 t
[ES ] = [E0 ]e
72
(13)
and the matter balance: [E 0 ] = [Es ] + [Ei ]
(14)
so [E s ] = [E0 ] − [Ei ] = [E0 ] e
−ki f (Eox −S)1 t 72
(15)
and Ar = A0 e−t
(16)
Fig. 6. Effect of the concentration of tyrosinase in its action on L-dopa (0.5 mM) in the absence (䊉) and the presence of different concentrations of n-nonyl caffeate (M): () 20 and (䊏) 60; pre-incubating the enzyme with this compound and starting the reaction with L-dopa.
where Ar is the residual activity corresponding to [E 0 ] − [Ei ] at time t and A0 is the initial activity. The apparent inactivation constant () depends on the concentration of substrate according to Eq. (7). 3.5.1. Suicide inactivation of tyrosinase acting on caffeic acid When tyrosinase acts on CAFA, an unstable o-caffeoquinone (CAFQ) is originated (Fig. 3). The spectrophotometric recordings made at short times allows the characterization of CAFA as substrate of the enzyme, as previously described [36]. In this way, the kinetic constants of the initial steady state are obtained: Michaelis CAFA constant (K CAFA M ), the catalytic constant (kcat ) and the catalytic efficiency (kcat /KM ) (Table 2). At long times, the initial steady state evolves through a transition phase towards the inactivation of the enzyme, in a process that allows new parameters to be determined: maximum apparent inactivation constant (max ), the inactivation efficiency (max /KM )
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Fig. 7. Kinetic mechanism proposed to explain the suicide inactivation of tyrosinase acting on o-diphenols. Em , metatyrosinase; Em S complex between Em and S; Ed , deoxytyrosinase; Eox , oxytyrosinase; Eox S, complex between Eox and S; (Eox -S)1 , axial binding complex of protonated S and Eox ; (Eox -S)2 , diaxial binding complex of deprotonated S and Eox ; (Eox -S)3 , axial binding complex of deprotonated S and Eox ; Ei , inactive enzyme.
Table 2 Kinetic constants which characterize the suicide inactivaction of tyrosinase by DHPPA, DHPAA, CAFA, n-nonyl caffeate and PCA. kcat
KM −1
(s DHPPA DHPAA CAFA n-nonyl caffeate PCA
)
(mM)
607.2 ± 25.1 433.1 ± 25.9 403 ± 8.30
0.70 ± 0.09 1.30 ± 0.10 0.70 ± 0.05
8.1 ± 0.3
0.07 ± 0.01
max x 103
kcat /KM (mM
−1 −1
s
)
867 ± 117 333 ± 34 576 ± 43 870 ± 110 116 ± 17
and the partition ratio r. In this way, the study of CAFA as substrate of tyrosinase is complete. 3.5.1.1. Effect of the concentration of substrate. The inactivation of tyrosinase at different concentrations of substrate (CAFA) was measured by following the disappearance of NADH (Fig. 8A) at 380 nm. The data analysis provides the apparent inactivation constant () if the spontaneous oxidation of CAFA and NADH are taken into account (see data analysis) and the curves are fitted to Eq. (12). The value of [NADH]∞ does not vary as expected in a suicide inactivation, while max and KM are obtained by means of an analysis of vs [S]0 (Fig. (8)A Inset) according to Eq. (7). This value of KM agrees with that obtained with the data for the steady state [36]. The parameter r can be derived from max and kcat , according to Eq. (6). 3.5.1.2. Effect of the concentration of enzyme. The concentration of tyrosinase was varied with a fixed concentration of substrate and adding AH2 to maintain the concentration of the substrate constant during each assay, since this compound reduces the o-quinone. The residual activity was measured with high concentrations of TBC (Fig. 8B). For its part, , which is obtained by analyzing these data according to Eq. (16) does not vary when the concentration of enzyme changes, as Eq. (7) predicted (Fig. 8B Inset). Therefore, the characterization of CAFA as suicide substrate is complete and all the parameters can be gathered, as in Table 2: kcat , KM , kcat /KM (catalytic efficiency), max , max /KM (inactivation efficiency) and r. 3.5.2. Suicide inactivation of tyrosinase acting on n-nonyl caffeate The low solubility and the broad spectra make a kinetic study of the suicide inactivation of tyrosinase in its action on n-nonyl caffeate difficult. To make the characterization, the residual activity of the enzyme was measured with high concentrations of TBC, maintaining the concentration of substrate constant in each experiment by the presence of AH2 , which reduces de o-quinone to n-nonyl caffeate. 3.5.2.1. Effect of the concentration of substrate. The effect of the concentration of substrate is shown in Fig. 9A and the data are fitted
−1
(s
)
6.93 ± 0.27 6.89 ± 0.37 12.7 ± 0.8 0.85 ± 0.03
max /KM x 103
r
Ref.
87646 ± 1315 62838 ± 1187 34300 ± 645 27200 ± 536 10186 ± 224
[17] [17] This paper This paper [17]
(mM−1 s−1 ) 9.9 ± 1.3 5.3 ± 0.5 18.1 ± 1.7 32.0 ± 4.0 12.1 ± 1.8
to Eq. (16), providing the apparent inactivation constant. Moreover Fig. 9A Inset show the dependence of vs [S]0 , whose slope corresponds to max /KM (inactivation efficiency), according to Eq. (7).
3.5.2.2. Effect of the concentration of enzyme. The effect of the concentration of tyrosinase is shown in Fig. 9B, while the data analysis according to Eq. (16) allows us to obtain the value of the apparent inactivation constant, whose independence with respect to the concentration of the enzyme is depicted in Fig. 9B Inset, which agrees with Eq. (7).
3.5.2.3. Deduction and calculation of parameter r. The determination of r for CAFA was made applying Eq. (6). In the case of n-nonyl caffeate, it was obtained measuring the residual activity at a constant concentration of enzyme after consumption of the different concentrations of substrate: Step 1: the enzyme is pre-incubated with increasing concentrations of substrate and bubbling with air in the medium to maintain constant the concentration of oxygen. Step 2: the residual activity (Ar ) at t → ∞ is determined with a high concentration of TBC. Step 3: the Ar /A0 is represented vs [S]0 /[E]0 , which give rises to a straight line (Fig. 10). The points at which the straight line cuts the axis, provide r. Step 4: deduction of the analytic expression of r and calculation of its value. Fig. 7 indicates that the action of tyrosinase on o-diphenols causes the suicide inactivation of the enzyme, but releases the product o-quinone (Q), both in the catalytic and the suicide pathways. In this way, matter balance: S0 = S + Q
(17)
where S0 is the initial concentration of o-diphenol (n-nonyl caffeate in this case), S is the concentration of substrate after the inactivation and Q is the product.
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Fig. 8. A. Effect of the concentration of substrate on the suicide inactivation during the action of tyrosinase on caffeic acid using NADH as reductant. The disappearance of NADH was measured at 380 nm. The experimental conditions were [E]0 = 20 nM, [NADH]0 = 2 mM and [CAFA]0 (mM): a) 0.1, b) 0.2, c) 0.25, d) 0.35, e) 0.4, f) 0.6, g) 0.8 and h) 1. Inset. Variation of the apparent inactivation constant () with the concentration of CAFA. The experimental conditions were the same as in the main figure. B. Effect of the concentration of the enzyme on the suicide inactivation caused by the action of tyrosinase on CAFA using AH2 as reductant. Different concentrations of tyrosinase (nM): a) 10, b) 20, c) 30 and d) 50; were pre-incubated in the presence of AH2 (0.7 mM) and CAFA (0.5 mM). Subsequently, various aliquots (10% of the total on the medium assay) were taken to test the activity of tyrosinase on TBC (3 mM) after different times of pre-incubation. Inset. Variation of the apparent inactivation constant () with the concentration of enzyme. The experimental conditions were the same as in the main figure.
Fig. 9. A. Effect of the concentration of substrate on the suicide inactivation caused by the action of tyrosinase on n-nonyl caffeate using AH2 as reductant. Tyrosinase (30 nM) was pre-incubated in the presence of ascorbic acid (0.7 mM) and different concentrations of n-nonyl caffeate (M): a) 10, b) 20, c) 30 and d) 50. Subsequently, various aliquots (10% of the total on the medium assay) were taken to test the activity of tyrosinase on TBC (3 mM) after different times of pre-incubation. Inset. Variation of the apparent inactivation constant () with the concentration of n-nonyl caffeate. The experimental conditions were the same as in the main figure. B. Effect of the concentration of the enzyme on the suicide inactivation caused by the action of tyrosinase on n-nonyl caffeate using AH2 as reductant. Different concentrations of tyrosinase (nM): a) 10, b) 20, c) 30 and d) 50; were pre-incubated in the presence of ascorbic acid (0.7 mM) and n-nonyl caffeate (30 M). Subsequently, various aliquots (10% of the medium assay) were taken to test the activity of tyrosinase on TBC (3 mM) after different times of pre-incubation. Inset. Variation of the apparent inactivation constant () with the concentration of enzyme. The experimental conditions were the same as in the main figure.
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turnovers of the enzyme, giving rise to o-quinones. Hence Eq. (24) becomes: 2 [S0 ] Ar [ES ] =1− = A0 2(r + 1) [E 0 ] [E0 ]
(25)
and the cut point on the X axis (Ar /A0 = 0) is: [S0 ] =r+1 [E0 ]
(26)
or [S0 ] −1 [E0 ]
r=
(27)
In the case of n-nonyl caffeate, the catalytic efficiency (kcat /KM ) (Table 2) can be obtained from r (Fig. 10) and the inactivation efficiency (max /KM ) (Fig. 9A), resulting: r=
kcat /KM kcat = max max /KM
(28)
Hence, the catalytic efficiency is: Fig. 10. Determination of parameter r by means of the sensivity of tyrosinase to inactivation at different molar ratios of n-nonyl caffeate/enzyme. Plot of residual activity against the [n-nonyl caffeate]/[tyrosinase] ratio. Residual activity was measured with TBC as substrate after incubations. During the assay, air was bubbled through to maintain the concentration of oxygen saturating. The line was fitted and the value of the intercept at the X-axis was used to calculate r.
The number of turnovers realized by the enzyme before its inactivation (r) is: [Q] − 2 [E i ] /2 r= [Ei ]
(18)
since two molecules of Q are released in each turnover, even in the inactivation pathway. The expression of Q can be obtained from Eq. (18): [Q] = 2(r + 1) [E i ]
(19)
The expression of [Ei ] can be obtained according to: [S0 ] = [S] + 2(r + 1) [E i ]
(20)
[S0 ] − [S] [E i ] = 2(r + 1)
(21)
The matter balance of the enzyme is: [E S ] = [E0 ] − [Ei ]
(22)
where [ES ] is the active enzyme. [ES ] [S0 ] − [S] =1− 2(r + 1) [E 0 ] [E0 ]
(23)
If the substrate is consumed ([S] = 0), Eq. (23) becomes: Ar [ES ] [S0 ] = =1− A0 2(r + 1) [E 0 ] [E0 ]
(24)
Taking into account the evolution of the quinone from n-nonyl caffeate (see Fig. 1BSM), the oxidation of the compound by the enzyme is analogous to the oxidation by NaIO4 in default. Assuming that the reaction starts with two molecules of n-nonyl caffeate, one of them is oxidized to o-quinone, which reacts with the other molecule of substrate, giving rise to a dimmer, which, in turn, changes to the leuco form. The leuco form is oxidized by the enzyme, giving rise to another quinone, which also becomes leuco. Finally, this latter compound is oxidized by tyrosinase, giving rise to an o-quinone, which becomes a diquinone by means of another enzymatic oxidation. Therefore, one initial molecule is oxidized three times and the other is oxidized only once, which means two
kcat = r max KM KM
(29)
as is shown in Table 2. In this way, the kinetic characterizations of CAFA and n-nonyl caffeate as suicide substrates of tyrosinase is complete. 3.6. Molecular docking The docking of n-nonyl caffeate at the binuclear copper active site of oxytyrosinase is shown in Fig. 11 with a dissociation constant of 0.5 mM, which is lower than that reported for CAFA (1.18 mM) [36]. The hydrogen atom of the C1-OH phenolic group is 2.7 Å from the oxygen atom of the amide bond of N260 and 3.9 Å from the oxygen atom of the carboxamide group of N260. The hydrogen atom of the C2-OH phenolic group is 2.9 Å from an oxygen atom of the peroxide ion and 3.8 Å from the oxygen atom of the carboxamide group of N260. Therefore, hydrogen bonds interactions between the phenolic groups of the ligand and the protein are possible. Besides, the aliphatic chain of the nonyl group is 3.6 Å from the aliphatic chain of V283, contributing to the stabilization of the docking configuration by hydrophobic interactions (Fig. 11). The disposition of the phenyl ring and the phenolic groups of n-nonyl caffeate is similar to that reported for CAFA previously [36]. The presence of the long alkyl chain of the nonyl group makes the phenyl ring rotates in its plane and only one hydroxyl group is bonded to the peroxide atom instead of two, as in the case of CAFA [36]. However, this configuration would allow electrostatic interactions with N260, hydrophobic interactions with V283 and, therefore, a lower Kd value than that of CAFA. The docking of n-nonyl caffeate to deoxytyrosinase has already been reported [8]. This docking proposed interactions with N260 and V283, nevertheless, the oxygen atoms of the phenolic groups interact with copper ions. In our study, the phenolic groups are located further from the copper ions because of the presence of the peroxide ion in the oxytyrosinase. Thus, the phenolic group would interact with the peroxide ion of oxytyrosinase and with copper ions of deoxytyrosinase. However, the docking to oxytyrosinase is the most significant, since the oxygen saturates the enzyme (deoxytyrosinase) [37] and, so, the substrate does not bind when the studied concentrations are used, as indicated in Fig. 11. In summary, the results obtained from the docking studies show that the dissociation constant for n-nonyl caffeate binding to oxytyrosinase (0.5 mM) is lower than that for CAFA (1.18 mM). These data could explain the fact that the catalytic efficiency and the inactivation efficiency are higher for n-nonyl caffeate, which may be
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Fig. 11. Computational docking of n-nonyl caffeate. Lowest energy AutoDock-Vina of n-nonyl caffeate at the tyrosinase active site is shown as green sticks. Distances (Å) are shown by dotted yellow lines. The atom colors are as follows: red = oxygen, blue = nitrogen, brown = copper, carbon = green and white = hydrogen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
associated with a lower KM , since the ␦4 values are similar (Table 1) and, so, the kcat values should be similar too. It is proposed that the hydrophobic chain of n-nonyl caffeate could lead to a high affinity for the enzyme, since the hydrophobic repulsion with the medium pushes the compound into the active site. This indicates that the n-nonyl caffeate is a more potent suicide substrate for tyrosinase than CAFA.
4. Conclusion When the inhibition of tyrosinase by CAFA and n-nonyl caffeate was studied in this work, both compounds were seen to act as suicide substrates of the enzyme due to their o-diphenolic structure. The inhibitory mechanism is the same at low and high concentrations, but the inactivation rate varies. The irreversible inactivation (suicide kinetic) is slow at low concentrations, and fast at high concentrations. n-Nonyl caffeate has a higher catalytic efficiency (870 ± 110 mM−1 s−1 ) and inactivation efficiency (32.0 ± 4.0 mM−1 s−1 ) than the other studied o-diphenols with a similar structure (DHPPA, DHPAA, CAFA and PCA). Since the ␦4 values for CAFA and n-nonyl caffeate are similar, the catalytic constants might be expected similar too, so the differences in the catalytic efficiency (1.5 times) and the inactivation efficiency (1.8 times) could be caused by the lower KM value for n-nonyl caffeate, which, in turn, is due to the presence of a hydrophobic chain. This behaviour as suicide substrate could explain its antibrowning effect and antibacterial activity against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Salmonella and Klebsiella pneumoniae.
Conflict of interest The authors declare no conflict of interest and funds.
Acknowledgements This work was supported by the Fundación Seneca (CARM, Murcia, Spain) under Projects 19545/PI/14, 19304/PI/14 and 19240/PI/14; MINECO under Projects SAF2016-77241-R and CTQ2014-56887-P (Co-financing with Fondos FEDER); and University of Murcia, Murcia under Projects UMU15452 and UMU17766. A. Garcia-Jimenez has a FPU fellowship from the University of Murcia.
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Action of tyrosinase on caffeic acid and its n-nonyl ester. Catalysis and suicide inactivation Antonio Garcia-Jimenez a , Jose Antonio Teruel-Puche b , Pedro Antonio Garcia-Ruiz c , Adrian Saura-Sanmartin d , Jose Berna d , Jose Neptuno Rodríguez-López a , Francisco Garcia-Canovas a,∗ a GENZ-Group of research on Enzymology, Department of Biochemistry and Molecular Biology-A, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, E-30100, Espinardo, Murcia, Spain1 b Group of Molecular Interactions in Membranes, Department of Biochemistry and Molecular Biology-A, University of Murcia, E-30100, Espinardo, Murcia, Spain c University of Murcia, Faculty of Veterinary, Group of Chemistry of Carbohydrates, Industrial Polymers and Additives, Department of Organic Chemistry, E-30100 Murcia, Spain d Group of Synthetic Organic Chemistry, Department of Organic Chemistry, Faculty of Chemistry, University of Murcia, E-30100 Espinardo, Murcia, Spain
a r t i c l e
i n f o
Article history: Received 28 July 2017 Received in revised form 24 October 2017 Accepted 24 October 2017 Available online xxx Keywords: Tyrosinase Caffeic acid n-Nonyl caffeate
a b s t r a c t Different mechanisms for inhibiting tyrosinase can be designed to avoid postharvest quality losses of fruits and vegetables. The action of tyrosinase on caffeic acid and its n-nonyl ester (n-nonyl caffeate) was characterized kinetically in this work. The results lead us to propose that both compounds are suicide substrates of tyrosinase, for which we establish the catalytic and inactivation efficiencies. The ester is more potent as inactivator than the caffeic acid and the number of turnovers made by one molecule of the enzyme before its inactivation (r) is lower for the ester. We proposed that the anti-browning and antibacterial properties may be due to suicide inactivation processes. © 2017 Published by Elsevier B.V.
1. Introduction The process of food browning due to the oxidation of polyphenols is notorius since it leads to a loss of nutritional, aesthetic and organoleptic properties, and, so, of the food’s commercial value [1–4]. Tyrosinase (EC 1.14.18.1), a copper-containing enzyme, is one of the main agents responsible for this process. Its catalytic centre has two copper atoms, each coordinated with three histidine residues [5,6]. This enzyme catalyzes the hydroxylation of monophenols to o-diphenols (monophenolase activity) and the subsequent oxidation of o-diphenols to o-quinones (diphenolase activity) [7]. Recently, a study was published on the protective effect of n-nonyl caffeate on the Chinese olive (Canarium album) [8], the quality of which during postharvest is affected by browning and bacterial putrefaction [9]. Tyrosinase inhibitors reduce these effects
and, so, have important applications in this respect [10–13]. In the case of Chinese olive, it was demonstrated that n-nonyl caffeate can prevent postharvest putrefaction because of its anti-browning and anti-bacterial effect [8]. Indeed, it was seen to attack Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Salmonella and Klebsiella pneumoniae, but not Pseudomonas aeruginosa or Agrobacterium tumefaciens. A kinetic analysis of this compound led the same authors to propose that n-nonyl caffeate is a reversible inhibitor of tyrosinase at low concentrations and an irreversible inhibitor at high concentrations [8]. It was this atypical behaviour of n-nonyl caffeate that led us to study the action of the enzyme on this compound based in the knowledge gained in our previous studies about the action mechanism of tyrosinase [7]. Therefore, n-nonyl caffeate and caffeic acid (CAFA), from which it is derived, were studied to understand the mechanism involved, while considering the possibility that such compounds, being substrates of tyrosinase, may give rise to unstable o-quinones.
∗ Corresponding author at: Departamento Bioquímica y Biología Molecular A, Facultad de Veterinaria, Campus de Espinardo, E-30100, Spain. E-mail address: [email protected] (F. Garcia-Canovas). 1 www.um.es/genz. https://doi.org/10.1016/j.ijbiomac.2017.10.151 0141-8130/© 2017 Published by Elsevier B.V.
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Fig. 1. Chemical structures of (a) CAFA, (b) n-nonyl caffeate, (c) DHPAA, (d) PCA and (e) DHPPA.
2. Materials and methods
ratio [21–24]. The corresponding analytic expression and data analysis provided r.
2.1. Materials
2.2. Methods 2.2.1. Synthesis method of n-nonyl caffeate Catalytic amounts of sulfuric acid were added to a solution of CAFA (100 mg, 0.6 mM) in nonanol (10 mL) and the reaction mixture was stirred at 100 ◦ C for 6 h. After this time, nonanol was removed by vacuum distillation, and the resulting solid was crystallized from ether, yielding the product of interest as a white solid. 2.2.2. Tyrosinase activity Spectrophotometric assays were carried out with a PerkinElmer Lambda-35 spectrophotometer (using water in the reference cuvette), online interfaced with a compatible PC 486DX microcomputer controlled by UV-Winlab software, where the kinetic data were recorded, stored, and analyzed. The diphenolase activity on Ldopa and the monophenolase activity on L-tyrosine were followed by measuring the increase of absorbance at = 475 nm led by the formation of dopachrome. For its part, the diphenolase activity on TBC (4-tert-butylcatechol) was measured at = 410 nm [16]. The suicide inactivation of tyrosinase acting on CAFA was followed by measuring the disappearance of NADH [17–20], originated by its oxidation by the o-quinone, at 380 nm. Finally, the suicide inactivation of tyrosinase acting on n-nonyl caffeate was followed by reacting the enzyme with this compound in the presence of AH2 and measuring the on-going residual activity of tyrosinase in a high concentration of TBC [17–20]. All the experiments were made in 30 mM phosphate buffer pH 7.0. In order to calculate the parameter r (the partition ratio or the turnover numbers made by one mol of the enzyme before its inactivation) when tyrosinase acts on n-nonyl caffeate, the enzyme was incubated with this compound while bubbling with air. After that, aliquots were taken at t → ∞ and the activity was assayed with TBC, providing the values of residual activity vs the substrate/enzyme
13 C
NMR assays NMR spectra of n-nonyl caffeate, CAFA and DHPPA were obtained on a Bruker Avance 300 MHz instrument. In the case of DHPAA and PCA, the instrument used was a Bruker Avance 400 MHz. In all the cases DMSO was used as solvent. The ␦ 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. 2.2.3.
Mushroom tyrosinase (3130 U/mg), reduced nicotinamide adenine dinucleotide (NADH), ascorbic acid (AH2 ), 3-(3,4-dihydroxyphenyl)propionic acid (DHPPA), 3,4dihydroxyphenylacetic acid (DHPAA), protocatechuic acid (PCA) and CAFA were purchased from Sigma (Madrid, Spain). The enzyme was purified as previously described [14] and the protein concentration was determined by Bradford’s method [15], using bovine serum albumin as the standard. Stock solutions of CAFA and n-nonyl caffeate were prepared in 0.15 mM phosphoric acid (aqueous solution) to prevent auto-oxidation and, this latter compound was also solubilised in DMSO (4% (v/v)) as cosolvent. Milli-Q system ultrapure was used throughout.
13 C
2.2.4. Computational docking The chemical structure of n-nonyl caffeate was constructed with PyMOL 1.8.4.0 [25] and its geometry was optimized with MOPAC2012 software [26] and PM7 semiempirical Hamiltonian. Rotatable bonds in the ligand were assigned by AutoDockTools4 program [27,28]. Oxytyrosinase was prepared as previously described [29]. Ligand was docked into the catalytic site of mushroom tyrosinase from Agaricus bisporus (PDB code: 2Y9W) using AutoDock Vina [30]. AutoDock Vina parameters were as follows: receptor file; ligand file; xyz centre coordinate of the pocket residue centred in the two copper ions; search space in each dimension, 11.3 Å; exhaustiveness, 24; and generation number of binding modes, 10. 2.2.5. Data analysis The experimental disappearance of NADH with time follows the equation:
[NADH] = [NADH]0 − [Q] = [NADH]0 − c1 1 − e
−c t
2
+ c3 + c4t
(1) where [Q ] is the concentration of the product of the enzymatic reaction, t is time and in which ci (i = 1–4) can be obtained by nonlinear regression [31]. The parameter c1 is equal to [Q ]∞ = [NADH]0 − [NADH]f (the concentration of product obtained at the end of the reaction) and c2 is equal to the corresponding . The coefficient c3 represents the uncertainty at zero time absorbance caused by the addition of the enzyme at the start of the reaction, while c4 corresponds to the slow spontaneous oxidation of o-diphenol and NADH. The effects of both experimental artefacts should be computer subtracted in further NADH against time. 3. Results and discussion CAFA and n-nonyl caffeate are ortho-diphenolic compounds (Fig. 1), and so can be chemically (by sodium periodate) [32] and enzymatically (by tyrosinase) oxidized [7].
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Fig. 2. A. Scan spectra of the evolution of CAFQ obtained by the oxidation of CAFA by NaIO4 in default. The experimental conditions were [CAFA]0 = 150 M and [NaIO4]0 = 50 M. Recordings were made every 2 min. Inset. Scan spectra (recordings “a–i” and spectrophotometric recording (“g”) at 550 nm of the evolution of the quinone of n-nonyl caffeate obtained by the oxidation of this compound by NaIO4 in default. The experimental conditions were [n-nonyl caffeate]0 = 200 M and [NaIO4 ]0 = 60 M. Recordings were made every 2 min. B. Scan spectra of the evolution of CAFQ obtained by the oxidation of CAFA by NaIO4 in excess. The experimental conditions were [CAFA]0 = 50 M and [NaIO4 ]0 = 400 M. Recordings were made every 2 min. Inset. Scan spectra of the evolution of the quinone of n-nonyl caffeate obtained by the oxidation of this compound by NaIO4 in excess. The experimental conditions were [n-nonyl caffeate]0 = 200 M and [NaIO4 ]0 = 600 M. Recordings were made every 2 min.
3.1. Characteristics of the o-quinones of the CAFA and n-nonyl caffeate Sodium periodate (NaIO4 ) is known to oxidize o-diphenols to oquinones [32]. Fig. 2A shows the spectrophotometric recordings of the oxidation of CAFA by NaIO4 in default. Note the presence of two imperfect isosbestic points due to the evolution of the o-quinone in the presence of o-diphenol and the accumulation of intermediate compounds as is represented in Fig. 1SM, which shows the possible set of reactions until the formation of a practically stable product [33]. The same experiment in the case of n-nonyl caffeate is shown in Fig. 2A Inset, where an increase of absorbance after an initial decay can be seen in the form of scans (recordings “a–i”) and as a spectrophotometric recording at 550 nm (recording “j”). When CAFA is oxidized by NaIO4 in excess, the isosbestic points are more clearly defined (Fig. 2B), since no intermediate products are accumulated in the medium (Fig. 2SM). The oxidation of n-nonyl caffeate by NaIO4 in excess generates an o-quinone, which evolves in the same way as that of CAFA (Fig. 2B Inset). 3.2. Action of tyrosinase on CAFA and n-nonyl caffeate The action of tyrosinase on CAFA and n-nonyl caffeate are represented in Fig. 3 Inset, respectively, the spectra being similar to those obtained with NaIO4 in default (Fig. 2A Inset). Thus, CAFA and n-nonyl caffeate are oxidized by tyrosinase, generating o-quinones. 3.3.
13 C
Fig. 3. A. Scan spectra of the action of tyrosinase on CAFA. The experimental conditions were [E]0 = 30 nM and [CAFA]0 = 50 M. Recordings were made every 2 min. B. Scan spectra of the action of tyrosinase on n-nonyl caffeate. The experimental conditions were [E]0 = 30 nM and [n-nonyl caffeate]0 = 50 M. Recordings were made every 2 min.
feate, DHPPA, DHPAA and PCA (the last three compounds shown for comparison). The ␦3 and ␦4 values are shown in Table 1.
NMR
The NMR spectra of CAFA and n-nonyl caffeate showed the chemical shifts of the carbon with the hydroxyl groups (C-3 and C-4), which indicated the potency of the nucleophilic attack on the copper atom of the active site by the oxygen of the hydroxyl group [34]. Hence, Fig. 3SM shows the NMR spectra of CAFA, n-nonyl caf-
3.4. Effect of CAFA and n-nonyl caffeate on the monophenolase and diphenolase activities of tyrosinase on L-tyrosine and L-dopa When the action of tyrosinase on L-tyrosine is assayed (Fig. 4A), the formation of dopachrome (recording “a”) shows a lag period, which corresponds to the time needed for the system to accumulate
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Fig. 4. A. Spectrophotometric recordings at 475 nm of the monophenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of CAFA (M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 100 nM and [L-tyrosine]0 = 0.5 mM. Inset. Spectrophotometric recordings at 475 nm of the monophenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of n-nonyl caffeate (M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 100 nM and [L-tyrosine]0 = 0.5 mM. B. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of CAFA (M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 40 nM and [L-dopa]0 = 0.5 mM. Inset. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of n-nonyl caffeate (M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 40 nM and [L-dopa]0 = 0.5 mM. Table 1 Values of the chemical shifts of the different suicide substrates obtained by 13 C NMR for the C-3 and C-4. Compound
␦4
␦3
DHPAA DHPPA CAFA n-nonyl caffeate PCA
145.7 145.9 149.0 149.2 150.8
144.8 144.3 146.5 146.4 145.6
ever, this set of data can also be explained by a suicide inactivation process, as is detailed in the following mechanism: The kinetic mechanism proposed to explain the suicide inactivation of tyrosinase is described in Fig. 7. The inactivation constant of tyrosinase ki72 is the lowest, so the enzymatic forms of the mechanism of Fig. 7 are in the steady state (ES ), which evolves through a transition phase towards the inactivation of the enzyme [17]. The variation of [ES ] and [Q] with time is given by: d [ES ] = −ki7 f(E ox − S)1 [E S ] 2 dt
a certain amount of o-diphenol in the steady state [35]. The addition of CAFA (Fig. 4A) or n-nonyl caffeate (Fig. 4A Inset) reduces the lag period and apparently activates the enzyme, since the rate is increased (recordings “b-f”). In the case of the diphenolase assay, the addition of CAFA (Fig. 4B) or n-nonyl caffeate (Fig. 4B Inset) apparently activates the enzyme when the formation of dopachrome is measured (recordings “b–f”). This happens because these compounds act as electron donors, converting Em to Ed , and the product formed in each case absorbs in the visible spectrum, the catalytic constant for these compounds being higher than for L-dopa (107.4 ± 3.1 s−1 ) and Ltyrosine (7.9 ± 0.3 s−1 ) [34].
and d[Q ] = 2k7 f(E ox − S)1 [E S ] 2 dt
(3)
where f (E ox − S)1 represents the fraction of the species (Eox − S)1 in the steady state (Es ). The integration of Eq. (2) and Eq. (3), considering [ES ] = [E]0 and [Q] = 0 at t = 0, yields: [Q] = [Q]∞ (1 − e−t )
(4)
with [Q]∞ =
3.5. Inactivation of tyrosinase by CAFA and n-nonyl caffeate Fig. 5A and B show the spectrophotometric recordings of the action of the enzyme on L-dopa in the presence of CAFA and n-nonyl caffeate, but starting the reaction with the addition of L-dopa. In this case, the previous contact of the enzyme with CAFA or n-nonyl caffeate inactivates it, which is particularly evident when the concentration of n-nonyl caffeate is increased (Fig. 6). For this reason, it was proposed that n-nonyl caffeate is a reversible inhibitor at low concentrations and irreversible at high concentrations [8]. How-
(2)
2k7 2kcat [E]0 = i 2 [E]0 = 2r[E]0 max k7
(5)
2
where the partition ratio (r) is r=
kcat max
(6)
the apparent inactivation constant () =
max [S]0 S + KM [S]0
(7)
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Fig. 5. A. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the absence of (a) and the presence of different concentrations of CAFA: b) 40, c) 120, d) 250 and e) 400; pre-incubating the assay with this compound and starting the reaction with L-dopa. The rest of the experimental conditions were [E]0 = 10 nM and [L-dopa]0 = 0.5 mM. B. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the absence of (a) and the presence of different concentrations of n-nonyl caffeate: b) 40, c) 120, d) 250 and e) 400; pre-incubating the enzyme with this compound and starting the reaction with L-dopa. The rest of the experimental conditions were [E]0 = 10 nM and [L-dopa]0 = 0.5 mM.
and the initial rate in the steady state gives V0 =
2kcat [S]0 [E]0
(8)
S + KM [S]0
with max =
k7i k3 k7 k7 2
1
3
k7 k7 k7 + k3 k7 k7 + k3 k7 k7 + k3 k7 k7 1
2
3
2
3
1
3
1
(9)
2
and kcat =
k7 k3 k7 k7 2
1
3
k7 k7 k7 + k3 k7 k7 + k3 k7 k7 + k3 k7 k7 1
2
3
2
3
1
3
1
(10)
2
The stoichiometry of Q when it is reduced by NADH is: [NADH] = [NADH]0 − Q
(11)
giving [NADH] = [NADH]f + [NADH]∞ e−t
(12)
with [NADH]f ([NADH]f value at t → ∞, [NADH]∞ = [NADH]0 − [NADH]f = [Q]∞ ), being (the apparent constant of suicide inactivation of tyrosinase in the presence of CAFA). The suicide inactivation can also be studied by measuring the residual activity of the enzyme, maintaining the concentration of substrate constant by means of a reductant, such as AH2 , which transforms the o-quinone into o-diphenol, and measuring the residual activity with a high concentration of TBC. Therefore, the variation of the active enzyme (Es ) corresponds to Eq. (2) and its expression with time is: −ki f (Eox −S)1 t
[ES ] = [E0 ]e
72
(13)
and the matter balance: [E 0 ] = [Es ] + [Ei ]
(14)
so [E s ] = [E0 ] − [Ei ] = [E0 ] e
−ki f (Eox −S)1 t 72
(15)
and Ar = A0 e−t
(16)
Fig. 6. Effect of the concentration of tyrosinase in its action on L-dopa (0.5 mM) in the absence (䊉) and the presence of different concentrations of n-nonyl caffeate (M): () 20 and (䊏) 60; pre-incubating the enzyme with this compound and starting the reaction with L-dopa.
where Ar is the residual activity corresponding to [E 0 ] − [Ei ] at time t and A0 is the initial activity. The apparent inactivation constant () depends on the concentration of substrate according to Eq. (7). 3.5.1. Suicide inactivation of tyrosinase acting on caffeic acid When tyrosinase acts on CAFA, an unstable o-caffeoquinone (CAFQ) is originated (Fig. 3). The spectrophotometric recordings made at short times allows the characterization of CAFA as substrate of the enzyme, as previously described [36]. In this way, the kinetic constants of the initial steady state are obtained: Michaelis CAFA constant (K CAFA M ), the catalytic constant (kcat ) and the catalytic efficiency (kcat /KM ) (Table 2). At long times, the initial steady state evolves through a transition phase towards the inactivation of the enzyme, in a process that allows new parameters to be determined: maximum apparent inactivation constant (max ), the inactivation efficiency (max /KM )
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Fig. 7. Kinetic mechanism proposed to explain the suicide inactivation of tyrosinase acting on o-diphenols. Em , metatyrosinase; Em S complex between Em and S; Ed , deoxytyrosinase; Eox , oxytyrosinase; Eox S, complex between Eox and S; (Eox -S)1 , axial binding complex of protonated S and Eox ; (Eox -S)2 , diaxial binding complex of deprotonated S and Eox ; (Eox -S)3 , axial binding complex of deprotonated S and Eox ; Ei , inactive enzyme.
Table 2 Kinetic constants which characterize the suicide inactivaction of tyrosinase by DHPPA, DHPAA, CAFA, n-nonyl caffeate and PCA. kcat
KM −1
(s DHPPA DHPAA CAFA n-nonyl caffeate PCA
)
(mM)
607.2 ± 25.1 433.1 ± 25.9 403 ± 8.30
0.70 ± 0.09 1.30 ± 0.10 0.70 ± 0.05
8.1 ± 0.3
0.07 ± 0.01
max x 103
kcat /KM (mM
−1 −1
s
)
867 ± 117 333 ± 34 576 ± 43 870 ± 110 116 ± 17
and the partition ratio r. In this way, the study of CAFA as substrate of tyrosinase is complete. 3.5.1.1. Effect of the concentration of substrate. The inactivation of tyrosinase at different concentrations of substrate (CAFA) was measured by following the disappearance of NADH (Fig. 8A) at 380 nm. The data analysis provides the apparent inactivation constant () if the spontaneous oxidation of CAFA and NADH are taken into account (see data analysis) and the curves are fitted to Eq. (12). The value of [NADH]∞ does not vary as expected in a suicide inactivation, while max and KM are obtained by means of an analysis of vs [S]0 (Fig. (8)A Inset) according to Eq. (7). This value of KM agrees with that obtained with the data for the steady state [36]. The parameter r can be derived from max and kcat , according to Eq. (6). 3.5.1.2. Effect of the concentration of enzyme. The concentration of tyrosinase was varied with a fixed concentration of substrate and adding AH2 to maintain the concentration of the substrate constant during each assay, since this compound reduces the o-quinone. The residual activity was measured with high concentrations of TBC (Fig. 8B). For its part, , which is obtained by analyzing these data according to Eq. (16) does not vary when the concentration of enzyme changes, as Eq. (7) predicted (Fig. 8B Inset). Therefore, the characterization of CAFA as suicide substrate is complete and all the parameters can be gathered, as in Table 2: kcat , KM , kcat /KM (catalytic efficiency), max , max /KM (inactivation efficiency) and r. 3.5.2. Suicide inactivation of tyrosinase acting on n-nonyl caffeate The low solubility and the broad spectra make a kinetic study of the suicide inactivation of tyrosinase in its action on n-nonyl caffeate difficult. To make the characterization, the residual activity of the enzyme was measured with high concentrations of TBC, maintaining the concentration of substrate constant in each experiment by the presence of AH2 , which reduces de o-quinone to n-nonyl caffeate. 3.5.2.1. Effect of the concentration of substrate. The effect of the concentration of substrate is shown in Fig. 9A and the data are fitted
−1
(s
)
6.93 ± 0.27 6.89 ± 0.37 12.7 ± 0.8 0.85 ± 0.03
max /KM x 103
r
Ref.
87646 ± 1315 62838 ± 1187 34300 ± 645 27200 ± 536 10186 ± 224
[17] [17] This paper This paper [17]
(mM−1 s−1 ) 9.9 ± 1.3 5.3 ± 0.5 18.1 ± 1.7 32.0 ± 4.0 12.1 ± 1.8
to Eq. (16), providing the apparent inactivation constant. Moreover Fig. 9A Inset show the dependence of vs [S]0 , whose slope corresponds to max /KM (inactivation efficiency), according to Eq. (7).
3.5.2.2. Effect of the concentration of enzyme. The effect of the concentration of tyrosinase is shown in Fig. 9B, while the data analysis according to Eq. (16) allows us to obtain the value of the apparent inactivation constant, whose independence with respect to the concentration of the enzyme is depicted in Fig. 9B Inset, which agrees with Eq. (7).
3.5.2.3. Deduction and calculation of parameter r. The determination of r for CAFA was made applying Eq. (6). In the case of n-nonyl caffeate, it was obtained measuring the residual activity at a constant concentration of enzyme after consumption of the different concentrations of substrate: Step 1: the enzyme is pre-incubated with increasing concentrations of substrate and bubbling with air in the medium to maintain constant the concentration of oxygen. Step 2: the residual activity (Ar ) at t → ∞ is determined with a high concentration of TBC. Step 3: the Ar /A0 is represented vs [S]0 /[E]0 , which give rises to a straight line (Fig. 10). The points at which the straight line cuts the axis, provide r. Step 4: deduction of the analytic expression of r and calculation of its value. Fig. 7 indicates that the action of tyrosinase on o-diphenols causes the suicide inactivation of the enzyme, but releases the product o-quinone (Q), both in the catalytic and the suicide pathways. In this way, matter balance: S0 = S + Q
(17)
where S0 is the initial concentration of o-diphenol (n-nonyl caffeate in this case), S is the concentration of substrate after the inactivation and Q is the product.
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Fig. 8. A. Effect of the concentration of substrate on the suicide inactivation during the action of tyrosinase on caffeic acid using NADH as reductant. The disappearance of NADH was measured at 380 nm. The experimental conditions were [E]0 = 20 nM, [NADH]0 = 2 mM and [CAFA]0 (mM): a) 0.1, b) 0.2, c) 0.25, d) 0.35, e) 0.4, f) 0.6, g) 0.8 and h) 1. Inset. Variation of the apparent inactivation constant () with the concentration of CAFA. The experimental conditions were the same as in the main figure. B. Effect of the concentration of the enzyme on the suicide inactivation caused by the action of tyrosinase on CAFA using AH2 as reductant. Different concentrations of tyrosinase (nM): a) 10, b) 20, c) 30 and d) 50; were pre-incubated in the presence of AH2 (0.7 mM) and CAFA (0.5 mM). Subsequently, various aliquots (10% of the total on the medium assay) were taken to test the activity of tyrosinase on TBC (3 mM) after different times of pre-incubation. Inset. Variation of the apparent inactivation constant () with the concentration of enzyme. The experimental conditions were the same as in the main figure.
Fig. 9. A. Effect of the concentration of substrate on the suicide inactivation caused by the action of tyrosinase on n-nonyl caffeate using AH2 as reductant. Tyrosinase (30 nM) was pre-incubated in the presence of ascorbic acid (0.7 mM) and different concentrations of n-nonyl caffeate (M): a) 10, b) 20, c) 30 and d) 50. Subsequently, various aliquots (10% of the total on the medium assay) were taken to test the activity of tyrosinase on TBC (3 mM) after different times of pre-incubation. Inset. Variation of the apparent inactivation constant () with the concentration of n-nonyl caffeate. The experimental conditions were the same as in the main figure. B. Effect of the concentration of the enzyme on the suicide inactivation caused by the action of tyrosinase on n-nonyl caffeate using AH2 as reductant. Different concentrations of tyrosinase (nM): a) 10, b) 20, c) 30 and d) 50; were pre-incubated in the presence of ascorbic acid (0.7 mM) and n-nonyl caffeate (30 M). Subsequently, various aliquots (10% of the medium assay) were taken to test the activity of tyrosinase on TBC (3 mM) after different times of pre-incubation. Inset. Variation of the apparent inactivation constant () with the concentration of enzyme. The experimental conditions were the same as in the main figure.
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turnovers of the enzyme, giving rise to o-quinones. Hence Eq. (24) becomes: 2 [S0 ] Ar [ES ] =1− = A0 2(r + 1) [E 0 ] [E0 ]
(25)
and the cut point on the X axis (Ar /A0 = 0) is: [S0 ] =r+1 [E0 ]
(26)
or [S0 ] −1 [E0 ]
r=
(27)
In the case of n-nonyl caffeate, the catalytic efficiency (kcat /KM ) (Table 2) can be obtained from r (Fig. 10) and the inactivation efficiency (max /KM ) (Fig. 9A), resulting: r=
kcat /KM kcat = max max /KM
(28)
Hence, the catalytic efficiency is: Fig. 10. Determination of parameter r by means of the sensivity of tyrosinase to inactivation at different molar ratios of n-nonyl caffeate/enzyme. Plot of residual activity against the [n-nonyl caffeate]/[tyrosinase] ratio. Residual activity was measured with TBC as substrate after incubations. During the assay, air was bubbled through to maintain the concentration of oxygen saturating. The line was fitted and the value of the intercept at the X-axis was used to calculate r.
The number of turnovers realized by the enzyme before its inactivation (r) is: [Q] − 2 [E i ] /2 r= [Ei ]
(18)
since two molecules of Q are released in each turnover, even in the inactivation pathway. The expression of Q can be obtained from Eq. (18): [Q] = 2(r + 1) [E i ]
(19)
The expression of [Ei ] can be obtained according to: [S0 ] = [S] + 2(r + 1) [E i ]
(20)
[S0 ] − [S] [E i ] = 2(r + 1)
(21)
The matter balance of the enzyme is: [E S ] = [E0 ] − [Ei ]
(22)
where [ES ] is the active enzyme. [ES ] [S0 ] − [S] =1− 2(r + 1) [E 0 ] [E0 ]
(23)
If the substrate is consumed ([S] = 0), Eq. (23) becomes: Ar [ES ] [S0 ] = =1− A0 2(r + 1) [E 0 ] [E0 ]
(24)
Taking into account the evolution of the quinone from n-nonyl caffeate (see Fig. 1BSM), the oxidation of the compound by the enzyme is analogous to the oxidation by NaIO4 in default. Assuming that the reaction starts with two molecules of n-nonyl caffeate, one of them is oxidized to o-quinone, which reacts with the other molecule of substrate, giving rise to a dimmer, which, in turn, changes to the leuco form. The leuco form is oxidized by the enzyme, giving rise to another quinone, which also becomes leuco. Finally, this latter compound is oxidized by tyrosinase, giving rise to an o-quinone, which becomes a diquinone by means of another enzymatic oxidation. Therefore, one initial molecule is oxidized three times and the other is oxidized only once, which means two
kcat = r max KM KM
(29)
as is shown in Table 2. In this way, the kinetic characterizations of CAFA and n-nonyl caffeate as suicide substrates of tyrosinase is complete. 3.6. Molecular docking The docking of n-nonyl caffeate at the binuclear copper active site of oxytyrosinase is shown in Fig. 11 with a dissociation constant of 0.5 mM, which is lower than that reported for CAFA (1.18 mM) [36]. The hydrogen atom of the C1-OH phenolic group is 2.7 Å from the oxygen atom of the amide bond of N260 and 3.9 Å from the oxygen atom of the carboxamide group of N260. The hydrogen atom of the C2-OH phenolic group is 2.9 Å from an oxygen atom of the peroxide ion and 3.8 Å from the oxygen atom of the carboxamide group of N260. Therefore, hydrogen bonds interactions between the phenolic groups of the ligand and the protein are possible. Besides, the aliphatic chain of the nonyl group is 3.6 Å from the aliphatic chain of V283, contributing to the stabilization of the docking configuration by hydrophobic interactions (Fig. 11). The disposition of the phenyl ring and the phenolic groups of n-nonyl caffeate is similar to that reported for CAFA previously [36]. The presence of the long alkyl chain of the nonyl group makes the phenyl ring rotates in its plane and only one hydroxyl group is bonded to the peroxide atom instead of two, as in the case of CAFA [36]. However, this configuration would allow electrostatic interactions with N260, hydrophobic interactions with V283 and, therefore, a lower Kd value than that of CAFA. The docking of n-nonyl caffeate to deoxytyrosinase has already been reported [8]. This docking proposed interactions with N260 and V283, nevertheless, the oxygen atoms of the phenolic groups interact with copper ions. In our study, the phenolic groups are located further from the copper ions because of the presence of the peroxide ion in the oxytyrosinase. Thus, the phenolic group would interact with the peroxide ion of oxytyrosinase and with copper ions of deoxytyrosinase. However, the docking to oxytyrosinase is the most significant, since the oxygen saturates the enzyme (deoxytyrosinase) [37] and, so, the substrate does not bind when the studied concentrations are used, as indicated in Fig. 11. In summary, the results obtained from the docking studies show that the dissociation constant for n-nonyl caffeate binding to oxytyrosinase (0.5 mM) is lower than that for CAFA (1.18 mM). These data could explain the fact that the catalytic efficiency and the inactivation efficiency are higher for n-nonyl caffeate, which may be
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Fig. 11. Computational docking of n-nonyl caffeate. Lowest energy AutoDock-Vina of n-nonyl caffeate at the tyrosinase active site is shown as green sticks. Distances (Å) are shown by dotted yellow lines. The atom colors are as follows: red = oxygen, blue = nitrogen, brown = copper, carbon = green and white = hydrogen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
associated with a lower KM , since the ␦4 values are similar (Table 1) and, so, the kcat values should be similar too. It is proposed that the hydrophobic chain of n-nonyl caffeate could lead to a high affinity for the enzyme, since the hydrophobic repulsion with the medium pushes the compound into the active site. This indicates that the n-nonyl caffeate is a more potent suicide substrate for tyrosinase than CAFA.
4. Conclusion When the inhibition of tyrosinase by CAFA and n-nonyl caffeate was studied in this work, both compounds were seen to act as suicide substrates of the enzyme due to their o-diphenolic structure. The inhibitory mechanism is the same at low and high concentrations, but the inactivation rate varies. The irreversible inactivation (suicide kinetic) is slow at low concentrations, and fast at high concentrations. n-Nonyl caffeate has a higher catalytic efficiency (870 ± 110 mM−1 s−1 ) and inactivation efficiency (32.0 ± 4.0 mM−1 s−1 ) than the other studied o-diphenols with a similar structure (DHPPA, DHPAA, CAFA and PCA). Since the ␦4 values for CAFA and n-nonyl caffeate are similar, the catalytic constants might be expected similar too, so the differences in the catalytic efficiency (1.5 times) and the inactivation efficiency (1.8 times) could be caused by the lower KM value for n-nonyl caffeate, which, in turn, is due to the presence of a hydrophobic chain. This behaviour as suicide substrate could explain its antibrowning effect and antibacterial activity against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Salmonella and Klebsiella pneumoniae.
Conflict of interest The authors declare no conflict of interest and funds.
Acknowledgements This work was supported by the Fundación Seneca (CARM, Murcia, Spain) under Projects 19545/PI/14, 19304/PI/14 and 19240/PI/14; MINECO under Projects SAF2016-77241-R and CTQ2014-56887-P (Co-financing with Fondos FEDER); and University of Murcia, Murcia under Projects UMU15452 and UMU17766. A. Garcia-Jimenez has a FPU fellowship from the University of Murcia.
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