Spectrophotometric Characterization of the Action of Tyrosinase on p

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Title: Spectrophotometric Characterization of the Action of Tyrosinase on p-Coumaric and Caffeic Acids. Characteristics of the o-Caffeoquinone. Antonio Garcia-Jimenez, Jose Luis Munoz-Munoz, Francisco GarcíaMolina, Jose Antonio Teruel-Puche, and Francisco Garcia-Canovas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00446 • Publication Date (Web): 07 Apr 2017 Downloaded from http://pubs.acs.org on April 9, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Title: Spectrophotometric Characterization of the Action of Tyrosinase on p-Coumaric and Caffeic Acids. Characteristics of the o-Caffeoquinone. AUTHORS Antonio Garcia-Jimeneza, Jose Luis Munoz-Munozb, Francisco Garcia-Molinaa, Jose Antonio Teruel-Puchec, Francisco Garcia-Canovasa* ADDRESSES a

GENZ-Group of research on Enzymology (www.um.es/genz), Department of

Biochemistry and Molecular Biology-A, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, E-30100, Espinardo, Murcia, Spain. b

Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle

upon Tyne, England, United Kingdom c

Group of Molecular Interactions in Membranes, Department of Biochemistry

and Molecular Biology-A, University of Murcia, E-30100, Espinardo, Murcia, Spain.

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ABSTRACT: New methods are proposed to determine the activity of tyrosinase

2

on caffeic and p-coumaric acids. Since the o-quinone from caffeic acid is

3

unstable in its presence, it has been characterized through spectrophotometric

4

measurements of the disappearance of coupled reducing agents, such as

5

NADH. It has also been characterized by a chronometric method, measuring

6

the time a known concentration of ascorbic acid takes to be consumed. The

7

activity on p-coumaric acid has been followed by measuring the formation of o-

8

quinone of caffeic acid at the isosbestic point originated between the caffeic

9

acid and the o-caffeoquinone, and by measuring the formation of o-quinone at

10

410 nm, which is stable in the presence of p-coumaric acid (both of them in the

11

presence of catalytic amounts of caffeic acid, maintaining the ratio between p-

12

CA and CAFA constant, R = 0.025). The kcat value of tyrosinase obtained for

13

caffeic acid was higher than that obtained for p-coumaric acid, while the affinity

14

was higher for p-coumaric acid. These values agree with those obtained in

15

docking studies involving these substrates and the oxytyrosinase.

16

17

KEYWORDS: tyrosinase, caffeic acid, p-coumaric acid, spectrophotometric

18

characterization, docking

19 20

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INTRODUCTION

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p-Coumaric acid (p-CA) is a phenolic acid widely distributed in fungi, fruits

23

(apples, grapes, tomatoes, etc.) and vegetables (beans, potatoes, onions,

24

etc.).1 It can be found alone or conjugated,2 both forms having a variety of

25

activities as an antioxidant,3 antimicrobial and antiviral,4 in antimutagenesis and

26

as an anti-cancer agent,5 or alleviator of diabetes6 and gout.7 It also has a

27

hypopigmenting

28

phenylpropanoid pathway (whose precursors are phenylalanine and tyrosine),

29

which is subsequent to the shikimic acid pathway that gives rise to lignin

30

formation.11-13 Moreover, it can be converted into phenolic acids, such as caffeic

31

acid (CAFA), ferulic acid, chlorogenic acid or sinapic acid.14

effect.8-10

This

compound

is

an

intermediate

of

the

32

For its part, CAFA is another hydroxycinnamic acid, and precursor of

33

phlorogenic acid in plants. It can be synthesized from p-CA, in a reaction that

34

can be carried out by polyphenol oxidase or tyrosinase from mushroom,15 frog

35

epidermis,16 and Vanilla planifolia.17 The hydroxylation of p-CA by spinach-beet

36

phenolase has been described following the addition of a reducer such as

37

NADH, dimethyltetrahydropteridine, caffeic acid or another o-diphenols.18-22

38

When the conversion of p-CA into CAFA catalyzed by tyrosinase was

39

studied through multivariate curve resolution, it was concluded that there were

40

three species in the reaction medium.23 Moreover, inhibitory effects of p-CA on

41

tyrosinase have been described.24 These include inhibition of human tyrosinase

42

in vitro; inhibition on the melanogenesis process in cells exposed to UVB;9

43

competitive and reversible inhibition of the diphenolase activity of mushroom

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tyrosinase, with an IC50 value of 0.50 mM;25 mushroom preservation26 and the

45

inhibition of polyphenol oxidase from blueberry (Vaccinium corymbosum).27

46

In a previous work, p-CA and CAFA were characterised as substrates of

47

tyrosinase from frog epidermis.16 The diphenolase activity was followed at 310

48

nm, recording the decrease in absorbance due to the conversion of CAFA into

49

o-caffeoquinone (o-CAFQ) (the chemical structures and the spectra of the three

50

compounds can be seen in Figure 1), while the monophenolase activity on p-CA

51

was followed at the isosbestic point (334 nm) originated between CAFA and o-

52

CAFQ (this point can be seen when low concentrations of CAFA (µM) are

53

oxidized by tyrosinase). Inhibition by excess of substrate was detected in both

54

cases.16 Moreover, spectrophotometric studies of the activities of mushroom

55

tyrosinase on p-CA and CAFA, using two other wavelengths (288 nm and 311

56

nm, respectively) to measure the consumption of these substrates are

57

described in the literature.28

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The objective of the research described herein was to design new

59

spectrophotometric methods to characterize this pair of natural substrates of

60

tyrosinase (p-CA and CAFA). In the first case, the activity of the enzyme on p-

61

CA was followed by measuring the formation of o-quinone at 410 nm (based on

62

the experimental demonstration that CAFQ is unstable in the presence of

63

CAFA, but stable in the presence of p-CA), or by following the conversion of p-

64

CA into CAFA and CAFQ at 334 nm (isosbestic point between the two

65

products), both of them in the presence of catalytic amounts of CAFA (the ratio

66

between p-CA and CAFA remained constant, R = 0.025).29 In the second case,

67

the activity of tyrosinase on CAFA was followed by measuring the consumption

68

of NADH, which acted as a coupled reagent, or using a chronometric method 4 ACS Paragon Plus Environment

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with ascorbic acid (AH2), measuring the time it takes to consume a given

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amount of AH2 in its reaction with CAFQ.

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MATERIALS AND METHODS

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Chemicals and Reagents. Mushroom tyrosinase (3130 U/mg), reduced

73

β-nicotinamide adenine dinucleotide (NADH), AH2, p-CA and CAFA were

74

supplied by Sigma (Madrid, Spain). The enzyme was purified as previously

75

described.30 The protein concentration was determined by Bradford’s method

76

using bovine serum albumin as standard.31 Stock solutions of p-CA and CAFA

77

were prepared in 0.15 mM phosphoric acid to prevent auto-oxidation. Milli-Q

78

system (Millipore Corp, Billerica, MA.) ultrapure water was used throughout.

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Spectrophotometric Assays. Spectrophotometric assays were carried

80

out with a double-beam PerkinElmer Lambda-35 spectrophotometer (using

81

water in the reference cuvette), online interfaced with a compatible PC 486DX

82

microcomputer controlled by UV-Winlab software, where the kinetic data were

83

recorded, stored, and analyzed.

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First, the diphenolase activity of tyrosinase on CAFA was characterized

85

by following the consumption of NADH, which reacts with CAFQ, or by

86

measuring the time taken to consume a given amount of AH2 due to its

87

oxidation by CAFQ.32,33 Secondly, the monophenolase activity on p-CA was

88

measured in the visible spectrum at 410 nm to follow the formation of CAFQ or

89

at the isosbestic point generated between CAFA and CAFQ at 334 nm (note

90

that the amount of CAFA is very low in this case, so that the CAFQ takes longer

91

to become unstable). The quantity of CAFA necessary to reach the steady state

92

at time t = 0 was added. In this way, the characteristic lag period of the

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monophenolase activity, which complicates the measurement of the V0, was

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eliminated. This quantity is given by the equation R = [CAFA]ss / [p-CA]ss = 6 ACS Paragon Plus Environment

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0.025, where [CAFA]ss and [p-CA]ss are the concentrations of o-diphenol

96

(CAFA) and monophenol (p-CA) in the steady state, respectively, with [p-CA]ss ≈

97

[p-CA]0.

98 99

All the assays were carried out at 25 °C, using 30 mM phosphate buffer at pH 7.0. Three repetitions of each experiment were made.

100

Kinetic Data Analysis. Initial rate values ( V0 ) were calculated at

101

different substrate concentrations and data were fitted by nonlinear regression

102

to the Michaelis−Menten equation using the Sigma Plot 9.0 program for

103

Windows

104

( K M ). The assays were carried out in saturating conditions of O2.35-37

34

, thus providing the maximum rate ( Vmax ) and the Michaelis constant

105

Computational Docking. p-CA and CAFA were docked into the catalytic

106

site of mushroom tyrosinase from Agaricus bisporus (PDB code: 2Y9W) using

107

AutoDock Vina,38 a program for predicting the most likely conformation of how a

108

ligand will bind to a macromolecule. The chemical structures of these ligands

109

were constructed with PyMOL 1.8.2.139 and their geometries were optimized to

110

their minimum energy with MOPAC2012 software40 and PM7 semiempirical

111

Hamiltonian. Rotatable bonds in the ligands were assigned by AutoDockTools4

112

program.41,42 The oxy form of tyrosinase was prepared as previously

113

described.43 AutoDock Vina parameters were as follows: receptor file; ligand

114

file; xyz centre coordinate of the pocket residue centred in the two copper ions;

115

search space in each dimension, 11.3 Å; exhaustiveness, 24; and generation

116

number of binding modes, 10.

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RESULTS AND DISCUSSION

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Figure 1 shows the chemical structures and spectra of p-CA, CAFA and CAFQ,

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the last was obtained by the oxidation of CAFA by sodium periodate in excess.

121

Note the cut off point between the spectra of CAFA and CAFQ at 334 nm, which

122

makes it possible to measure the monophenolase activity, as described below.

123

Assay of the Diphenolase Activity of Tyrosinase on CAFA. The

124

kinetic mechanism described for the diphenolase activity of tyrosinase is shown

125

in Figure 2.

126

First, the activity of tyrosinase on CAFA was analyzed, measuring the

127

formation of CAFQ at 410 nm. Previously, CAFA was oxidised with sodium

128

periodate in excess to test the stability of CAFQ. Moreover, this experiment

129

allows the λmax and ε = 2062 M-1 cm-1 to be determined (Figure 1), as described

130

previously.33 The initial rates shown in Figure 3 were obtained from the

131

spectrophotometric recordings in the presence of enzyme, where an apparent

132

inhibition by substrate excess can be observed.

133

When the measurement was made at 310 nm, absorbance decreased,

134

and the initial rates showed similar behaviour; however, the phototube reached

135

saturation, pointing to an apparent inhibition by excess of substrate (Figure 3

136

Inset). This similar behaviour would also have been due to a possible o-

137

quinone/substrate reaction after the oxidation of CAFA by sodium periodate in

138

default, as can be seen in Figure 4, indicating that CAFQ reacts with CAFA,

139

probably originating a dimer, as previously described.44,45 However, when CAFA

140

is oxidized with sodium periodate in excess (Figure 4 Inset, recording “a”) and

141

p-CA is added (Figure 4 Inset, recording “b”), the o-quinone remains stable, 8 ACS Paragon Plus Environment

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which is important and must be taken into account when designing methods to

143

measure the activity on p-CA.

144

Therefore, the above described experiments demonstrate that the

145

diphenolase activity should not be measured by monitoring the formation of

146

CAFQ at 410 nm or the consumption of CAFA at 310 nm, and that other

147

spectrophotometric methods such as those described for other substrates32

148

should be used.

Measurement of the Consumption of NADH at 380 nm. NADH

149

CAFA

150

reduces CAFQ to CAFA, so the initial rate ( V0

151

can be obtained by measuring the consumption of NADH ( V0

152

1520 M-1 cm-1). This method keeps the concentration of substrate constant due

153

to the reduction of the o-quinone by NADH. The mechanism is shown in Figure

154

5.

CAFA

37

the rate equation in the steady state is: CAFA Vmax [CAFA ]0

157

V0CAFA =

158

where

159

CAFA CAFA Vmax = 2kcat [ E ]0

160 161

= V0NADH ; ε380 =

Taking into account that the Michaelis constant for oxygen is very low,35-

155 156

) of the activity of tyrosinase

(1)

K MCAFA + [CAFA ]0

(2)

CAFA CAFA CAFA The kcat and KM values can be obtained by data analysis of V0

vs. [CAFA]0 (Figure 6) using nonlinear regression (see Table 1).

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CAFA Chronometric Method Using Ascorbic Acid. V0 can also be

163

determined by a chronometric method (Figure 7).32 In this case, the time for the

164

formation of o-quinone is taken as the time needed for AH2 to be completely

165

consumed through the reduction of the CAFQ. The mass balance is:

166

V0CAFA t = [CAFQ ] + [ AH2 ]

167

CAFA that is, the matter entering the system in the form of CAFQ, V0 t , consumes

168

the AH2 and, when no more AH2 remains, CAFQ is accumulated in the medium.

169

A spectrophotometric record at the wavelength at which CAFQ is absorbed

170

shows that there is a lag period (τ) since CAFQ is reduced to CAFA. The

171

prolongation of the accumulation of CAFQ intercepts the time axis at t = τ and,

172

according to eq 3, if [CAFQ] = 0, then

173

V0CAFA τ = [ AH2 ]0

174

Therefore, the expression of the enzyme activity rate according to the time

175

taken to consume an amount of AH2 is:

176

V0CAFA =

(3)

(4)

[ AH2 ]0

(5)

τ

177

CAFA where V0 is the formation rate of the o-quinone, [AH2] is the concentration of

178

AH2 and τ is the time taken to consume the AH2 (Figure 8). Data analysis of

179

CAFA CAFA V0CAFA vs. [CAFA]0 according to eq 1 allows Vmax and KM to be obtained

180

CAFA (Figure 8), where Vmax follows the expression indicated in eq 2 (see Table 1).

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Assay of the Monophenolase Activity on p-CA. The kinetic

181 182

mechanism describing the monophenolase activity is shown in Figure 9.

183

It is known that tyrosinase has a lag period ( τ ) when it acts on L-

184

tyrosine46 and that the system needs the accumulation of certain amount of o-

185

diphenol to reach the steady-state.29 The action of Eox (oxytyrosinase) on p-CA

186

generates CAFQ and CAFA. The enzyme hydroxylates p-CA to CAFA, which is

187

either oxidized to CAFQ and Ed (deoxytyrosinase) or released to the medium,

188

generating Em (mettyrosinase) (Figure 9)47 This is why three species (p-CA,

189

CAFA and CAFQ) are detected in the multivariate curve resolution.23

190

The action of tyrosinase on p-CA and the subsequent formation of

191

CAFQ, measured at the isosbestic point generated between CAFA and CAFQ

192

(at low concentrations) is shown in Figure 10. As can be seen, there was a lag

193

in the system, which did not reach the steady-state (recording “a”). In the

194

subsequent recordings, slightly increasing amounts of o-diphenol (CAFA) were

195

added to the medium to ascertain the minimum concentration of this compound

196

that would prevent the lag period, and, since the concentration of CAFA was

197

very low and the absorbance of p-CA at 334 nm is low, the phototube was not

198

saturated. This concentration allowed the ratio [CAFA]0/[p-CA]0 to be set as R =

199

CAFQ 0.025, which was used in the following assays. Finally, the V0 data were

200

fitted by nonlinear regression to eq 6 with respect to [p-CA]0 (Figure 11),

201

p−CA p −CA obtaining the Vmax and KM values (see Table 1). The concentration vs. time

202

values were obtained using the molar absorptivity of CAFQ at 334 nm, which

203

was calculated by taking into account the increase in absorbance (∆ε = 4400 M-

204

1

cm-1) due to the conversion of p-CA into CAFA and CAFQ.16

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p-CA Vmax [ p-CA ]0 p-CA K M + [ p-CA ]0

205

V0p-CA =

206

Where

207

p-CA p-CA Vmax = 4k cat [ E ]0

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(6)

(7)

208

The activity of the enzyme on p-CA can also be measured under the

209

same conditions (maintaining the ratio [CAFA]0 / [p-CA]0) in the visible spectrum

210

at 410 nm (Figure 12), since p-CA does not react with CAFQ and the compound

211

remains stable. The concentration vs. time values were obtained taking into

212

account the molar absorptivity of CAFQ at 410 nm (ε = 2062 M-1 cm-1).33

213

The above methods demonstrate that it is not necessary to measure

214

substrate consumption to characterize the catalytic activity of tyrosinase on p-

215

CA and CAFA as has been proposed previously.28 Indeed, measuring the

216

product or the reaction of the coupled reactive is a more sensitive method.48

217

Moreover, the activity of tyrosinase on CAFA originates two molecules of

218

CAFA CAFQ (Figure 2), and so the Vmax value (eq 3) is double. In the case of

219

monophenolase activity, in order to maintain the steady state, the enzyme must

220

make one turnover per diphenolase cycle (consuming two molecules of CAFA)

221

and two turnovers per monophenolase cycle (consuming two molecules of p-

222

CA), so, it originates four molecules of CAFQ and the value obtained must be

223

divided by four, according to eq 7.49 In our case, when the corrections were

224

made, the values obtained were in the range expected and in accordance with

225

their chemical structures.50 Note that the C-1 substituent influences the

226

monophenolase and diphenolase activities, mainly with regard to their kcat

227

values.50 12 ACS Paragon Plus Environment

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The KM values for the action of tyrosinase on p-CA and CAFA obtained in

229

this work are quite different from those described in the bibliography. The KM

230

values described were given as 33.20 µM for CAFA and 97.47 µM for p-CA,28

231

whereas we obtained KM values in the millimolar range for both substrates (0.71

232

± 0.05 mM and 0.64 ± 0.07 mM for CAFA, based on the consumption of NADH

233

and the chronometric method, respectively; and 0.37± 0.02 mM and 0.44 mM ±

234

0.06 for p-CA, measuring the formation of product at 334 and 410 nm

235

respectively). This discrepancy can be explained by the differences that exist

236

between the measurement methods used. Thus, if the consumption of substrate

237

is to be measured in the UV spectrum, the substrate concentration cannot be

238

increased because the phototube will become saturated. The experiments

239

described in the bibliography28 varied the concentrations of CAFA and p-CA

240

from 10 µM to 100 µM and from 12 µM to 100 µM, respectively. These ranges

241

are not wide enough to obtain an appropriate hyperbola. In fact, the highest

242

concentration in the case of p-CA was the same as KM (the appropriate range is

243

from KM / 5 to 5 KM). Moreover, when their Vmax values are standardized to the

244

p−CA CAFA same enzyme concentration, the Vmax value is higher than that of Vmax , and

245

p-CA CAFA so kcat > kcat .28 However, the kcat values obtained in this work (see Table 1)

246

confirm those obtained for other monophenol/diphenol couples.50 For its part,

247

the nucleophilicity of the oxygen from the OH group in C-4 is always higher for

248

p −CA an o-diphenol (CAFA) than a monophenol (p-CA).50 As shown in Table 1, kcat

249

CAFA values are lower than the values for kcat . Furthermore, these values reflect

250

the fact that the δ4 value for the monophenol (p-CA) is higher (lower

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nucleophilicity of the oxygen atom) than the δ4 (C-4) value for o-diphenol

252

(CAFA), as described in the bibliography (both being higher than the values

253

described for p-hydroxyphenyl propionic acid and 3,4-dihydroxyphenylpropionic

254

acid), so that the catalytic constant of p-CA is lower.50-52

255

Molecular Docking. It is known that the oxy form of tyrosinase

256

hydroxylates monophenols and oxidizes o-diphenols,49 so the this form was

257

selected for the docking study.

258

It was shown above that p-CA is hydroxylated by the oxy form of

259

tyrosinase, while CAFA is oxidized. To address this issue the oxy form of

260

tyrosinase was selected for docking studies.

261

The docking of p-CA at the binuclear copper active site of tyrosinase is

262

shown in Figure 13, with a dissociation constant of 0.3 mM. The hydrogen atom

263

of the hydroxyl group at C-4 is 2.9 and 3.0 Å from the oxygen atoms of the

264

peroxide ion and so hydrogen bond interactions are possible. The phenyl ring of

265

p-CA appears to be almost parallel to the imidazole ring of H263, while the

266

distance between both aromatic rings (4.2 Å) would allow π-π-interactions, thus

267

stabilizing the ligand bound to the active center.53 It is interesting to note that

268

the C3 atom of p-CA is located 3.4 Å from an oxygen atom of the peroxide ion,

269

which is close enough for the electrophilic attack of the oxygen atom to occur on

270

the carbon atom and leading to substrate hydroxylation (Figure 13).

271

The docking conformation of CAFA at the binuclear copper active site of

272

tyrosinase shows a dissociation constant of 1.18 mM (Figure 14). The phenyl

273

ring is almost perpendicular to the imidazole ring of H263, and, so

274

π-π-interactions must be excluded when trying to explain the larger dissociation 14 ACS Paragon Plus Environment

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constant found for CAFA. The hydrogen atom of the hydroxyl groups and the

276

oxygen atoms of the peroxide ion are located at 3.3 Å and 3.8 Å from the active

277

site (Figure 14), which is greater than in the case of p-CA, suggesting weaker

278

interactions and therefore a larger dissociation constant. Nevertheless, the

279

docking conformation of CAFA is arranged at a distance and orientation suitable

280

for oxidation by the peroxide ion and, therefore, to be initiated by the

281

diphenolase activity of the oxy form of tyrosinase.

282

This docking study shows that both molecules, p-CA and CAFA, can be

283

bound to the catalytic center of tyrosinase with dissociation constants close to

284

the experimental values. Besides, they adopt a suitable conformation to be

285

hydroxylated by the monophenolase activity (p-CA) or oxidized by the

286

diphenolase activity (CAFA), both catalytic activities residing in the oxy form of

287

tyrosinase.

288

In summary, various spectrophotometric methods are proposed for

289

characterizing the activities of tyrosinase on the natural substrates, p-coumaric

290

acid and caffeic acid. In the case of p-CA, the methods consist of directly

291

measuring the formation of the product, following the formation of CAFQ at 334

292

nm (isosbestic point between the CAFA and CAFQ), or at 410 nm. The most

293

important point is to add CAFA at a constant ratio ([CAFA]0/[p-CA]0 = 0.025). In

294

the case of CAFA, the method measures the consumption of a coupled reactive

295

(NADH), which reacts with product o-quinone, or the time taken to consume a

296

given amount of AH2 through its reaction with CAFQ (chronometric method),

297

avoiding the presence of CAFQ along with CAFA, which makes the

298

measurement impossible. Docking studies show that the arrangement of the

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299

ligands in the oxy form of tyrosinase could make possible the hydroxylation of p-

300

CA and the oxidation of CAFA..

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AUTHOR INFORMATION

302

Corresponding Author

303

*Phone: +34 868 884764; Fax: +34 868 883963; E-mail: [email protected]

304

Funding

305

This work was partially supported by grants from several Spanish

306

organizations: Projects 19545/PI/14, 19304/PI/14 and 19240/PI/14 (Fundación

307

Seneca,

308

CTQ2014-56887-P (MINECO, Madrid); Projects UMU15452 and UMU17766

309

(University of Murcia, Murcia); A. Garcia-Jimenez has a FPU fellowship from

310

University of Murcia.

311

Notes

312

The authors declare no competing financial interest.

313

ABBREVIATIONS USED

314

NADH: nicotinamide adenine dinucleotide reduced form; AH2: ascorbic acid;

315

CAFA: caffeic acid; CAFQ: caffeoquinone; p-CA: p-coumaric acid.

CARM,

Murcia,

Spain);

Projects

SAF2013-48375-C2-1-R

316 317

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(14) Brown, S. A. Lignins. Annu. Rev. Plant Physiol. 1966, 17, 223-244. (15) Satô, M. The conversion of phenolase of p-coumaric acid to caffeic acid with special reference to the role of ascorbic acid. Phytochemistry. 1969, 8, 353-362. (16) Carmona, F. G.; Pedreño, E.; Galindo, J. D.; Cánovas, F. G. A new spectrophotometric method for the determination of cresolase activity of epidermis tyrosinase. Anal. Biochem. 1979, 95, 433-435. (17)

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Lim, J. Y.; Ishiguro, K.; Kubo, I. Tyrosinase inhibitory p-Coumaric acid from Ginseng

leaves. Phytother. Res. 1999, 13, 371-375. (25) Shi, Y.; Chen, Q. X.; Wang, Q.; Song, K. K.; Qiu, L. Inhibitory effects of cinnamic acid and its derivatives on the diphenolase activity of mushroom (Agaricus bisporus) tyrosinase. Food Chem. 2005, 92, 707-712. (26) Hu, Y. H.; Chen, Q. X.; Cui, Y.; Gao, H. J.; Xu, L.; Yu, X. Y.; Wang, Y.; Yan, C. L.; Wang, Q. 4-Hydroxy cinnamic acid as mushroom preservation: Anti-tyrosinase activity kinetics and application. Int. J. Biol. Macromolec. 2016, 86, 489-495.

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(27) Siddiq, M.; Dolan, K. D. Characterization of polyphenol oxidase from blueberry (Vaccinium corymbosum L.). Food Chem. 2017, 218, 216-220. (28)

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oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates. Anal. Biochem. 2003, 312, 23-32. (29) Ros, J. R.; Rodriguez-Lopez, J. N.; García-Cánovas, F. Tyrosinase: kinetic analysis of the transient phase and the steady state. Biochim. Biophys. Acta. 1994, 1204, 33-42. (30) Rodriguez-Lopez, J. N.; Fenoll, L. G.; Garcia-Ruiz, P. A.; Varon, R.; Tudela, J.; Thorneley, R. N.; Garcia-Canovas, F. Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase. Biochemistry. 2000, 39, 10497-10506. (31) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. (32) Garcia-Molina, F.; Munoz, J. L.; Varon, R.; Rodriguez-Lopez, J. N.; Garcia-Canovas, F.; Tudela, J. A review on spectrophotometric methods for measuring the monophenolase and diphenolase activities of tyrosinase. J. Agric. Food Chem. 2007, 55, 9739-9749. (33) Munoz, J. L.; Garcia-Molina, F.; Varon, R.; Rodriguez-Lopez, J. N.; Garcia-Canovas, F.; Tudela, J. Calculating molar absorptivities for quinones: Application to the measurement of tyrosinase activity. Anal. Biochem. 2006, 351, 128-138. (34) Sigma Plot 9.0 for WindowsTM; Systat Software: San Jose CA, 2006. (35)

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(39) The PyMOL Molecular Graphics System, version 1.5.0.1; Schrödinger, LLC: 2010. (40)

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2016. (41) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785-2791. (42) Sanner, M. F. Python: A programming language for software integration and development. J. Mol. Graph. Model. 1999, 17, 57-61. (43) Maria-Solano, M. A.; Ortiz-Ruiz, C. V.; Munoz-Munoz, J. L.; Teruel-Puche, J. A.; Berna, J.; Garcia-Ruiz, P. A.; Garcia-Canovas, F. Further insight into the pH effect on the catalysis of mushroom tyrosinase. J. Mol. Catal. B-Enzym. 2016, 125, 6-15. (44)

Hotta, H.; Nagano, S.; Ueda, M.; Tsujino, Y.; Koyama, J.; Osakai, T. Higher radical

scavenging activities of polyphenolic antioxidants can be ascribed to chemical reactions following their oxidation. Biochim. Biophys. Acta. 2002, 1572, 123-132. (45) Hotta, H.; Ueda, M.; Nagano, S.; Tsujino, Y.; Koyama, J.; Osakai, T. Mechanistic study of the oxidation of caffeic acid by digital simulation of cyclic voltammograms. Anal. Biochem. 2002, 303, 66-72. (46) Molina, F. G.; Muñoz, J. L.; Varón, R.; López, J. N. R.; Cánovas, F. G.; Tudela, J. An approximate analytical solution to the lag period of monophenolase activity of tyrosinase. Int. J. Biochem. Cell Biol. 2007, 39, 238-252. (47) Rodriguez-López, J. N.; Fenoll, L. G.; Peñalver, M. J.; García-Ruiz, P. A.; Varón, R.; Martínez-Ortiz, F.; García-Cánovas, F.; Tudela, J. Tyrosinase action on monophenols: evidence for direct enzymatic release of o-diphenol. Biochim. Biophys. Acta. 2001, 1548, 238-256. (48) Segel, I. H. Kinetics of unireactant enzymes. In: Enzyme kinetics: Behaviour and analysis of rapid equilibrium and steady-state enzyme systems; Segel, I. H., Eds.; J. Wiley and Sons. London, United Kingdom, 1975, pp. 83. (49)

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Tyrosinase: a comprehensive review of its mechanism. Biochim. Biophys. Acta. 1995, 1247, 111.

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(50) Espín, J. C.; Varón, R.; Fenoll, L. G.; Gilabert, M. A.; García-Ruíz, P. A.; Tudela, J.; García-Cánovas, F. Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase. Eur. J. Biochem. 2000, 267, 1270-1279. (51) Salum, M. L.; Robles, C. J.; Erra-Balsells, R. Photoisomerization of ionic liquid ammonium cinnamates: One-pot synthesis−isolation of z-cinnamic acids. Org. Lett. 2010, 12, 4808-4811. (52) Cui, Q.; Lewis, I. A.; Hegeman, A. D.; Anderson, M. E.; Li, J.; Schulte, C. F.; Westler, W. M.; Eghbalnia, H. R.; Sussman, M. R.; Markley, J. L. Metabolite identification via the Madison Metabolomics Consortium Database. Nat. Biotech. 2008, 26, 162-164. (53) Janiak, C. A critical account on [π–π] stacking in metal complexes with aromatic nitrogencontaining ligands. J. Chem. Soc. Dalton Trans. 2000, 3885-3896.

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Figure 1. Chemical structures and UV-Vis spectra of CAFA; p-CA and CAFQ. The experimental conditions were [p-CA] = 100 µM, [CAFA] = 100 µM and [CAFQ] = 100 µM. CAFQ was obtained by the oxidation of CAFA (100 µM) by sodium periodate in excess (200 µM). Figure 2. Diphenolase activity of tyrosinase on CAFA. Em = mettyrosinase, Ed = deoxytyrosinase and Eox = oxytyrosinase. Figure 3. Oxidation of CAFA by tyrosinase. A. Representation of the initial rate values (expressed in ∆A/s units) of the action of tyrosinase on CAFA at 410 nm in the presence of different concentrations of substrate. The experimental conditions were [E]0 = 10 nM. Inset. Representation of the initial rate values (expressed in ∆A/s units) of tyrosinase on CAFA at 310 nm in the presence of different concentrations of substrate. Enzyme concentration was 6 nM. Figure 4. Reaction of CAFQ with CAFA. Different concentrations of CAFA were oxidized by sodium periodate in default (0.1 mM) and the evolution of CAFQ was recorded at 410 nm. The experimental conditions were [CAFA]0 (mM): a) 0.5, b) 0.75, c) 1, d) 1.75 and e) 2.25. Inset. Possible reaction of CAFQ with pCA. CAFA (0.25 mM) was oxidized by sodium periodate in excess (0.5 mM) and CAFQ was recorded at 410 nm (a). After adding p-CA (0.25 mM), the evolution of CAFQ was recorded at the same wavelength (b). Figure 5. Representation of the equations that express the oxidation of NADH coupled to the oxidation of CAFA by tyrosinase. Figure 6. Changes in absorbance at 380 nm during the reduction of the CAFQ (generated by the oxidation of CAFA by tyrosinase) in the presence of NADH 23 ACS Paragon Plus Environment

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(0.08 mM). Different concentrations of CAFA were used (mM): a) 0.15, b) 0.30, c) 0.60, d) 0.90, e) 1.2, f) 1.5, g) 2, h) 2.7 and i) 3. The rest of the experimental conditions were [E]0 = 6 nM. Inset. Representation of the initial rates obtained from the slopes of the recordings vs the different concentrations of CAFA used in the experiments of the main Figure. Figure 7. Equations that show the oxidation of AH2 coupled to the action of tyrosinase on CAFA. The method consists of measuring the time taken to consume a given amount of AH2. Figure 8. Changes in absorbance at 410 nm during the oxidation of CAFA by tyrosinase in the presence of AH2, which generates CAFQ, which, in turn, is accumulated in the medium, after the consumption of a given amount of AH2 (67 µM) in time τ. The rest of the experimental conditions were [E]0 = 7 nM and CAFA (µM): a) 60, b) 150, c) 300, d) 450, e) 600 f) 750, g) 900, h) 1200, i) 1500, j) 1700 and k) 2400. Inset. Representation of the initial rates obtained from the recordings of the figure, applying the eq 5 vs the different concentrations of CAFA used in the experiments of the main Figure. Figure 9. Monophenolase activity of tyrosinase on p-CA in the presence of catalytic amounts of CAFA. The enzymatic forms are the same as in Figure 2. Figure 10. Changes in absorbance at 334 nm (isosbestic point between CAFA and CAFQ) during the action of tyrosinase on p-CA (0.5 mM) in the presence of different concentrations of CAFA (µM): a) 0.25, b) 2.5, c) 7.5, d) 12.5 and e) 25. Enzyme concentration was 10 nM.

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Figure 11. Changes in absorbance at 334 nm during the action of tyrosinase on p-CA at 334 nm in the presence of catalytic quantities of CAFA. The experimental conditions were [E]0 = 70 nM, [p-CA]0 (mM): a) 0.1, b) 0.2, c) 0.4, d) 0.5, e) 0.75, f) 1.0 g) 1.5, h) 2.0, i) 3.0, and [CAFA]0 = 0.025 [p-CA]0. Inset. Representation of the initial rate values obtained from the recordings of the figure vs the different concentrations of p-CA used. Figure 12. Changes in absorbance at 410 nm during the action of tyrosinase on p-CA at 410 nm in the presence of catalytic quantities of CAFA. The experimental conditions were [E]0 = 40 nM, p-CA (mM): a) 0.1, b) 0.2, c) 0.4, d) 0.6, e) 1.0, f) 2.0, g) 3.0, and [CAFA]0 = 0.025 [p-CA]0. Inset. Representation of the initial rate values obtained from the recordings of the figure vs the different concentrations of p-CA used. Figure 13. Computational docking of p-CA. Lowest energy docked configuration of p-CA at the tyrosinase active site is shown as thick 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. Figure 14. Computational docking of CAFA. Lowest energy docked configuration of CAFA at the tyrosinase active site is shown as thick sticks. Color scheme as in Figure 13.

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Table 1. Kinetic Constants for the Action of Tyrosinase on CAFA and p-CA Obtained by Different Measurement Methods.

Compound

Consumption of NADH

Formation of product at

Formation of product

334 nm (UV)

410 nm (Visible)

Docking

Chronometric method

K M (mM)

kcat (s-1)

K M (mM)

kcat (s-1)

K M (mM)

kcat (s-1)

K M (mM)

kcat (s-1)

K d (mM)

CAFA

0.71 ± 0.05

403 ± 8.30

0.64 ± 0.07

381 ± 15.24

-

-

-

-

1.18

p-CA

-

-

-

-

0.37 ± 0.02

25 ± 0.34

0.44 ± 0.06

15 ± 0.48

0.3

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