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Discrimination between alternative substrates and inhibitors of tyrosinase Carmen Vanessa Ortiz-Ruiz, Maria del Mar Garcia-Molina, Jose Tudela Serrano, Virginia Tomas-Martinez, and Francisco García-Cánovas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5051816 • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 11, 2015

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Journal of Agricultural and Food Chemistry

Title and authorship Discrimination between alternative substrates and inhibitors of tyrosinase.

Carmen Vanessa Ortiz-Ruiz1, Maria del Mar Garcia-Molina1, Jose Tudela Serrano1, Virginia Tomas-Martinez2 and Francisco Garcia-Canovas1*

1

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, Spain. 2

Department of Analytical Chemistry, Regional Campus of International

Excellence "Campus Mare Nostrum", University of Murcia, Spain.

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

Abbreviated names: V. Ortiz-Ruiz; M. Garcia-Molina; J. Tudela; V. Tomas; F. Garcia-Canovas

1 - Environment ACS Paragon -Plus


1

Abstract

2

Many phenolic compounds have been described in the scientific literature

3

as inhibitors of tyrosinase. In this work a test is proposed that allows us to

4

distinguish whether a molecule is an enzyme inhibitor or substrate. The test has

5

several stages. First, the degree of inhibition of the studied molecule is

6

determined on the monophenolase activity (iM) and on the diphenolase activity

7

(iD). If iM = iD, it is an inhibitor. If iM ≠ iD, the molecule could be substrate or

8

inhibitor. Several additional stages are proposed to solve this ambiguity. The

9

study described herein was carried out using the following molecules: benzoic

10

acid, cinnamic acid, guaiacol, isoeugenol, carvacrol, 4-tert-butylphenol, eugenol

11

and arbutin.

12 13

Keywords

14

Tyrosinase, substrate, inhibitor, polyphenol oxidase, kinetic, degree of inhibition.


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15

Introduction

16

Tyrosinase is a copper monooxygenase widely distributed throughout the

17

phylogenetic scale: bacteria, fungi, plants and animals. This enzyme catalyzes

18

the hydroxylation of monophenols to o -diphenols, and the oxidation of the latter

19

to o -quinones with help of molecular oxygen. 1-3

20

The two enzymatic activities of tyrosinase are coupled to the non-

21

enzymatic reactions of the corresponding o-quinones. These non-enzymatic

22

reactions have been kinetically described, taking into consideration the main

23

regulatory role of the pH of the assay.4,5 The stability of the o-quinone is

24

important, since the activity of tyrosinase on a monophenol depends on a

25

sequence of chemical reactions related to the evolution of its o-quinone and the

26

probability that some of its corresponding o-diphenol is generated in the

27

reaction medium.3

28

The importance of the formation of an o-diphenol in the reaction medium

29

is essential for the enzyme to show its monophenolase activity when it acts on a

30

monophenol (see Figure 1). Many compounds, especially monophenols, have

31

been described in the bibliography as inhibitors of tyrosinase because after

32

testing them with the enzyme, it does not show any activity, although many of

33

them are tyrosinase substrates.6-8

34

The development of effective tyrosinase inhibitors has become

35

increasingly important in the cosmetic, medicinal and agricultural industries to

36

prevent hyperpigmentation.9 Such enzyme inhibition has been studied more

37

deeply in these last years.10-14

38

If a monophenol, which is assayed as tyrosinase substrate, gives rise to

39

an o-quinone that evolves chemically without originating an o-diphenol in the



40

medium, this compound would be undoubtedly classified as an inhibitor, as

41

there is no turnover by the enzyme. However, when investigators assay these

42

compounds considering their influence on the physiological substrates L-

43

tyrosine and L-dopa, according to the mechanism shown in Figure 1, the

44

monophenol under study can be used as substrate because the o-diphenol

45

necessary for the turnover is provided by the o-quinone (o-dopaquinone → L-

46

dopa + dopachrome) of the monophenol (L-tyrosine) which is used as substrate

47

in the monophenolase activity, or else, the o-diphenol itself (L-dopa), as

48

substrate in the diphenolase activity (Figure 2). However, in both cases

49

inhibition is observed on the monophenolase and diphenolase activity of the

50

enzyme, measured by the increase of absorbance at 475 nm. Such compounds

51

are classified as inhibitors.6

52

Considering the mechanism of action of the enzyme on monophenols

53

and o-diphenols (Figure 1 and Figure 2) and kinetic analysis of the two

54

activities, the analytical expressions of the degrees of inhibition are obtained,

55

and based on these, a test is proposed to differentiate a tyrosinase substrate

56

other than L-tyrosine and L- dopa from an inhibitor of the enzyme. At the same

57

time, the proper methodology is established to assay tyrosinase inhibition.

58 59


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60

Materials and Methods

61

Enzyme source. Mushroom tyrosinase (3130 U/mg) was purchased

62

from Sigma (Spain) and purified as previously described.15 Protein content was

63

determined by Bradford’s method.16 Reagents. 4-tert-butylphenol (TBP), 4-tert-butylcatechol (TBC), L-dopa,

64 65

L-tyrosine,

sodium

periodate,

3-methyl-2-benzothiazolinone

hydrazone

66

hydrochloride (MBTH), dimethylformamide (DMF), guaiacol, carvacrol, eugenol,

67

isoeugenol, arbutin, benzoic acid and cinnamic acid (see Figure 3) were

68

purchased from Sigma. Stock solutions of substrates were prepared in 0.15 mM

69

phosphoric acid to prevent auto-oxidation. Spectrophotometric assays. Absorption spectra were recorded in a

70 71

visible-ultraviolet

72

interfaced with a compatible PC 486DX microcomputer, with a 60 nm/s

73

scanning speed controlled by the UV-Winlab software. The temperature was

74

maintained at 25ºC using a Haake D16 circulating water bath with a

75

heater/cooler, and checked using a Cole-Palmer digital thermometer with a

76

precision of 0.1ºC. Kinetic assays were also carried out with the above

77

instruments by measuring the appearance of the products in the reaction

78

medium. All the assays were carried out under saturating conditions of

79

tyrosinase by molecular oxygen, 0.26 mM in the assay medium.17 The activity

80

on L-tyrosine and L-dopa was measured at 475 nm, which is the maximum

81

absorption wavelength of dopachrome. The activity on TBC was followed at 410

82

nm.

83

followed at wavelengths at which each generated product absorbed. All assays

84

were made, unless otherwise stated, using 30 mM phosphate buffer pH 7.0.

18-19

Perkin

Elmer

Lambda

35-spectrophotometer,

on-line

The assays of activity in the presence of hydrogen peroxide were



85 86

Simulation assays. The simulated progress curves were obtained by

87

numerical solution of the non-linear set of differential equations that describes

88

the mechanisms corresponding to Figures 1, 2, 5, 6, 1SI, 4SI and 2SI (see

89

Supplementary Information). The systems of differential equations were

90

solved numerically for particular sets of values of the rate constants and of initial

91

concentrations of the species involved in the reaction mechanism. The

92

numerical integration used a fourth order Runge-Kutta method and the

93

predictor-corrector Adams-Moulton algorithm, implemented on a PC-compatible

94

computer program (WES).20

95 96


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97

Results and Discussion

98

Tyrosinase shows two enzymatic activities, the hydroxylation of L-

99

tyrosine to L-dopa and the oxidation of the latter to o-dopaquinone. According to

100

the mechanism shown in Figure 2, the formation and accumulation of

101

dopachrome product is linear with time (data not shown). Figure 4 shows the

102

tyrosinase activity on L-tyrosine: notice the apparition of a lag in the

103

accumulation of dopachrome, curve (a). At the same time, this lag decreased

104

and was annulled when a certain amount of o-diphenol was added at the

105

beginning of the reaction, curve (b). The quantity of o-diphenol needed for the

106

system to reach the steady state is given by R = [D]ss/[M]ss.21 This equation

107

establishes the quantity of o-diphenol [D] in the steady state [D]ss needed so

108

that the monophenol concentration [M]ss reaches the steady state from t = 0.

109

When more diphenol than needed was added to fulfil the relation R, a burst

110

appeared at the beginning of the activity recording, curve (c). Figure 4 Inset

111

shows the simulated progress curves, (a)-(c), obtained through numerical

112

integration of the set of differential equations that describes the mechanism

113

shown in Figure 1 (see Supplementary Information).

114

The lag period observed at the beginning of the recording of the

115

monophenolase activity of tyrosinase represents a substantial difficulty when

116

making inhibitor assays.22 It is essential to determine when the steady state is

117

reached to measure the monophenolase activity. When an inhibitor is added to

118

the reaction medium, the reaction rate of the enzyme decreases and so does

119

the rate of accumulation of o-diphenol in the medium, and this means an

120

increase in the lag period and the steady state is reached later, which therefore

121

entails a longer measuring period. This has not usually been taken into account



122

when studying the inhibition of the monophenolase activity, meaning that

123

inexact values have been obtained for many studied inhibitors. 23-29

124

We propose that to know whether the target molecules act as substrates

125

or enzyme inhibitors, both the monophenolase and diphenolase activities must

126

be studied. The monophenolase activity will be studied by adding the amount of

127

o-diphenol necessary to avoid the lag period so that the steady state is reached

128

from the beginning of the reaction.22 In this case, the presence of an inhibitor in

129

the medium will not involve an increase in the lag period, even though it will

130

decrease the rate, so that the monophenolase activity can be measured from t

131

= 0, both in the presence and absence of different concentrations of a target

132

molecule.

133

In this work we study two classic inhibitors of tyrosinase for comparative

134

purposes (benzoic acid30 and cinnamic acid) and investigate whether a group of

135

target molecules (4-tert-butylphenol, eugenol, guaiacol, isoeugenol, carvacrol

136

and arbutin) act as tyrosinase substrates or inhibitors.

137

To determine whether a molecule of interest is a true substrate or an

138

enzyme inhibitor, we must first ascertain if the enzyme reacts with the

139

compound under study. In the case of target monophenols, this assay always

140

shows no activity (data not shown), because monophenols, as described below,

141

do not cause the chemical accumulation of o-diphenol in the medium.

142

Once this part of the test was completed, we determined the enzymatic

143

activity on L-dopa in the presence and absence of a target molecule, measuring

144

spectrophotometrically the accumulation of dopachrome at 475 nm. The

145

concentration of L-dopa used was K MD . After this, measurements were made at

146

different concentrations of the molecule of interest.


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147

Similarly, the enzymatic activity on L-tyrosine was studied in the

148

presence and absence of the target molecule, measuring the accumulation of

149

dopachrome at 475 nm. The concentration of L-tyrosine used was K MM . Also, a

150

concentration of L-dopa was added to fulfil R = [D]ss/[M]ss, reaching the steady

151

state at t = 0.31 After this, measurements were made at the same target

152

molecule concentrations, as in the case of the diphenolase activity. To develop

153

this kind of assay it is not necessary to use concentrations of monophenol and

154

o-diphenol similar to KM, but the relations [M]0 / K MM and [D]0 / K MD must be the

155

same.

156

Then, the degree of inhibition of the monophenolase (iM) and diphenolase

157

(iD) activities were calculated and were compared. In cases where the inhibition

158

degrees were the same (iM = iD), the molecule under study acted as an inhibitor.

159

These mechanisms are described in Figures 5 and 6 and the results are shown

160

in Table 2. The simulated progress curves obtained through numerical

161

integration of the set of differential equations that describe the above

162

mechanisms are shown in Figures 7A Inset and 7B Inset. If the degree of

163

inhibition differed (iM ≠ iD), the molecule in question could either be an

164

alternative substrate or an inhibitor (in which case it would bind to any of the

165

enzymatic forms of the diphenolase mechanism, and to one of the EmM or EoxM

166

complexes of the monophenolase mechanism). Possible mechanisms involved

167

are shown in Figures 1SI and 2SI and the results in Table 2. In Figures 3SI

168

and 3SI Inset the simulated progress curves obtained through numerical

169

integration of the set of differential equations that describe these mechanisms

170

are shown. In this case (iM ≠ iD) two types of additional assays had to be



171

undertaken to confirm if the target molecules were alternative substrates or

172

inhibitors (see below).

173

Figure 7A shows the recordings of the monophenolase activity of

174

tyrosinase on L-tyrosine at different concentrations of benzoic acid, obtained by

175

adding a given amount of o-diphenol from t = 0 ([D] = [D]ss), so as to satisfy the

176

relation R = [D]ss/[M]ss, and Figure 7B shows the diphenolase activity of

177

tyrosinase on L-dopa at the same concentrations of benzoic acid. Figures 7A

178

Inset and 7B Inset show the simulated progress curves obtained through

179

numerical integration of the sets of differential equations that describe the

180

mechanisms of the monophenolase and diphenolase activities of tyrosinase in

181

the presence of a competitive inhibitor (Figure 5 and Figure 6)30 (see

182

Supplementary information).

183

Figure 8 and Figure 8 Inset depict the kinetic recordings of the inhibition

184

of the monophenolase and diphenolase activities by cinnamic acid. From the

185

results obtained, both experimental and simulated, of the inhibition of the

186

monophenolase and diphenolase activities (Figures 7A and 7B, Figures 7A

187

Inset and 7B Inset, Figures 8 and 8 Inset, and Figures 3SI and 3SI Inset),

188

the degrees of inhibition were calculated and are shown in Table 1 and Table 2.

189

Note that the degrees of inhibition in both activities are the same, both for

190

the experimental and simulated cases ((Table 1 and Table 2 (a)), which agrees

191

with equations 4 A and 8 A (see Appendix).

192 193

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194

Investigating whether a phenol showing varying degrees of

195

inhibition of the monophenolase and diphenolase activities is a tyrosinase

196

substrate or inhibitor.

197

In cases in which the degree of inhibition of both activities was (iM ≠ iD),

198

two types of additional steps were made to confirm whether these molecules

199

were alternative substrates or inhibitors. Guaiacol, isoeugenol, carvacrol, TBP,

200

eugenol and arbutin

201

shown in Table 3. The recordings obtained with some of these molecules (TBP,

202

isoeugenol and guaiacol) are shown in Figures 9A-9C.

6, 32-38

showed this kind of behaviour (iM ≠ iD). Results are

203

Step 1. Measurement of the formation of the product originated in the

204

oxidation of an o-diphenol by tyrosinase until total consumption of oxygen. To

205

do this, TBC and L-dopa were chosen: in the case of TBC, the o-quinone, o-

206

tert-butylquinone, is very stable and in the case of L-dopa dopachrome is

207

formed, which is relatively stable. This step takes into account the stoichiometry

208

of the mechanism shown in Figure 2. For TBC 1O2: 2 o-quinone and for L-dopa

209

1O2:1 dopachrome. When all the oxygen is consumed in the absence or

210

presence of an inhibitor and, bearing in mind the stoichiometries, when

211

measuring the accumulation of o-tert-butylquinone, with ε = 1100 M-1cm-1, or

212

dopachrome, with ε=3600 M-1cm-1,

213

5SI depicts the simulated progress curves obtained through numerical

214

integration of the set of differential equations that describe the mechanism

215

shown in Figure 1SI. It can be seen that if the target molecule is an inhibitor,

216

the level of concentration of the final product is always the same. If the phenols

217

of interest are substrates, the level of concentration of the product is lower

218

because the target molecules consume oxygen. The simulations of the

18,19

the absorbances are constant. Figure



219

mechanism shown in Figure 4SI show this behaviour (Figure 5SI Inset). From

220

the experimental point of view, to ensure that the observed variation in

221

absorbance was due to the different coefficients of absorbtivity of the o-

222

quinones from the target compound and not to the interaction of this compound

223

with the o-quinones of the TBC or the L-dopa, prior to this assay, the stability of

224

these o-quinones was assayed. To test this, TBC or L-dopa substrates were

225

oxidized with sodium periodate in deficiency to generate o-tert-butylquinone and

226

dopachrome, respectively. Immediately after, the target phenols were added

227

and the reaction of the o-quinones was observed. In cases in which there was

228

not any variation in absorbance with time, the oxygen consumption step could

229

be used to help identify whether the compound under study is an inhibitor or an

230

alternative substrate.

231

In the reaction of dopachrome or o-tert-butylquinone with carvacrol or

232

guaiacol (Figure 10 and 10 Inset), the variation of absorbance with time was

233

evident. It was therefore not advisable to develop the oxygen consumption

234

assay with these molecules. However, when the arbutin was studied, the

235

absorbance did not change with time (Figure 11), and so, in this case, the test

236

could be run as described in Step 1 (Figure 11 Inset). The arbutin was seen to

237

act as an alternative substrate of tyrosinase, since total absorbance decreases

238

as the arbutin concentration increases (Figure 11 Inset).

239

When the experiment described in Step 1 was applied to true inhibitors

240

benzoic acid and cinnamic acid, oxygen consumption followed the typical

241

substrate-dependent stoichiometry (reaching the same absorbance value as in

242

the absence of the inhibitor). The apparent constant decreased when the


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243

inhibitor concentration increased, with both L-dopa (Figures 12A and 12A

244

Inset) and TBC (data not shown).

245

Step 2. Deviations of the absorbance, after carrying out the oxidation with

246

sodium periodate, indicated an interaction of the target molecule with the o-

247

quinones of TBC and L-dopa, and it would not be possible to carry out the step

248

1 correctly. In these cases we assayed the target phenol with enzyme and

249

hydrogen peroxide. It is known that hydrogen peroxide changes met-tyrosinase,

250

which is not active on monophenols, into oxy-tyrosinase, which is active. When

251

the target phenols in the presence of tyrosinase and hydrogen peroxide were

252

assayed, we observed a variation in absorbance in repeated scans. After

253

testing that this variation was not due to an attack of the hydrogen peroxide on

254

the molecules under study, we could conclude that they are alternative

255

substrates. This Step 2 was applied to isoeugenol and eugenol (Figure 13,

256

recordings a and b, respectively), TBP (Figure 13 Inset), arbutin and carvacrol

257

(Figure 14 recording a and b, respectively) and guaiacol (Figure 14 Inset),

258

measuring the increase of absorbance of the generated o-quinones. Note how

259

arbutin that fulfilled Step 1 of the test also fulfilled Step 2. These findings

260

confirm that all the studied molecules are tyrosinase substrates.

261

In conclusion, an experimental design is proposed that allows us to

262

establish whether a phenol of interest is an alternative substrate or inhibitor of

263

tyrosinase. The spectrophotometric method consists of several stages. If the

264

results of the degree of inhibition of the monophenolase and diphenolase

265

activities in the presence of the target molecule are the same, it can be deduced

266

that this compound is an inhibitor. However, if the degrees of inhibition of both

267

activities are different, it is still necessary to undertake Steps 1 and 2 to confirm



268

that the target molecule is really an alternative substrate or a tyrosinase

269

inhibitor. Also, a methodology is proposed to study the effect of inhibitors on the

270

monophenolase activity experimentally by adding the o-diphenol necessary to

271

reach the steady state at t = 0.


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Abbreviations used TBC

4-tert-butylcatechol

[TBC]0

initial concentration of 4-tert-butylcatechol

TBP

4-tert-butylphenol

MBTH

3-methyl-2-benzothiazolinone hydrazone hydrochloride

DMF

dimethylformamide

Em

met-tyrosinase

Ed

deoxy-tyrosinase

Eox

oxy-tyrosinase

EmD

met-tyrosinase/L-dopa complex

EoxD

oxy-tyrosinase/L-dopa complex

EmM

met-tyrosinase/L-tyrosine complex

EoxM

oxy-tyrosinase/L-tyrosine complex

EmI

met-tyrosinase/inhibitor complex

EoxI

oxy-tyrosinase/inhibitor complex

D

L-dopa

[D]0

initial concentration of L-dopa

[D]ss

L-dopa concentration in steady state

M

L-tyrosine

[M]0

initial concentration of L-tyrosine

[M]ss

L-tyrosine concentration in steady state

Q

o-quinone corresponding to L-dopa

[Q]

instantaneous concentration of Q

Cr

dopachrome

[Cr]

instantaneous concentration of dopachrome

I

inhibitor - 15Plus - Environment ACS Paragon


[I]0

initial concentration of inhibitor

V0D(Cr )

initial rate of tyrosinase acting on L-dopa (D), calculated by measuring Cr

D(Cr) Vmax

maximum rate of tyrosinase acting on L-dopa calculated by measuring Cr

V0D(Cr) (i)

initial rate of tyrosinase acting on L-dopa (D), calculated by measuring Cr in the presence of an inhibitor (I)

V0M(Cr)

initial rate of tyrosinase acting on L-tyrosine (M), calculated by measuring Cr

M(Cr) Vmax

maximum rate of tyrosinase acting on L-tyrosine (M), calculating by measuring Cr

V0M(Cr) (i)

initial rate of tyrosinase acting on L-tyrosine (M), calculated by measuring Cr, in the presence of an inhibitor (I)

iD

degree of inhibition of the diphenolase activity

iM

degree of inhibition of the monophenolase activity

R

ratio between [D]ss and [M]ss, R = [D]ss/[M]ss ≈ [D]ss/[M]0

ki

rate constants

K MD

Michaelis constant for (D)

K MM

Michaelis constant for (M)

K9

dissociation constant of the complex EmI

K10

dissociation constant of the complex EoxI

- 16Plus - Environment ACS Paragon

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APPENDIX

Analytical expressions for the degrees of inhibition of the diphenolase and monophenolase activities of tyrosinase. The rate of formation of dopachrome in the action of tyrosinase on o-diphenols is:

V0D(Cr) =

D(Cr) Vmax [D]0 D K M + [D]0

(1A)

In the presence of an inhibitor that binds to the enzymatic forms Em and Eox (Figure 7), it is:

V0(D(Cr) = i)

D(Cr) Vmax [D]0 k K + k K K MD (1 + 6 10 2 9 [I]0 ) + [D]0 k 2 K 9 K10

(2A)

The degree of inhibition is given by:

k6 K10 + k2 K 9 [I]0 ) − K MD k 2 K 9 K10 iD = x100 k K +k K K MD (1 + 6 10 2 9 [I]0 ) + [D]0 k 2 K 9 K10 K MD (1 +

(3A)

Working at [D]0 = K MD and considering that k2 >> k6, gives:

iD =

[I]0 x100 2 K10 + [I]0

(4A)



Page 18 of 48

A similar equation to Eq.(4A) can be obtained for the monophenolase activity:

V0M(Cr) =

M(Cr) Vmax [M]0 M K M + [M]0

(5A)

In the presence of an inhibitor that binds to the enzymatic forms Em and Eox (Figure 6), the rate of formation of dopachrome is:

V0(M(Cr) = i)

M(Cr) Vmax [M]0 k K + k K K MM (1 + 6 10 2 9 [I]0 ) + [M]0 k2 K 9 K10

(6A)

The degree of inhibition is given by:

  k K +k K K MM  1 + 6 10 2 9 [I]0  − K MM k 2 K 9 K10   iM =   k K +k K K MM  1 + 6 10 2 9 [I]0  + [M]0 k 2 K 9 K10  

(7A)

Working at [M]0 = K MM and considering that k2 >>k6, gives:

iM =

[I]0 x100 2 K10 + [I]0

(8A)

Thus, equations (4A) and (8A) show that, in the presence of the same concentration of inhibitor and the concentration of substrate corresponding to its respective KM, the degrees of inhibition of the monophenolase and diphenolase activities of tyrosinase are similar. - 18Plus - Environment ACS Paragon

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Acknowledgements This work was partially supported by grants from several Spanish organizations: Projects SAF2013-48375-C2-1-R (MINECO, Madrid), UMU15452 and UMU17766 (University of Murcia, Murcia), V. Ortiz-Ruiz has a MEC-FPU fellowship (AP20104300).

Supporting Information Description Supporting Information Available: Kinetic analysis of the proposed mechanisms and simulations of these mechanisms are shown in (Supporting Information). This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing financial interest.



References 1. Solomon, E.I.; Sundaram, U.M.; Machonkin, T.E. Multicopper oxidases and oxygenases. Chem. Rev. 1996, 96, 2563-2605. 2. Sanchez-Ferrer, A.; Rodriguez-Lopez, J.N.; Garcia-Canovas, F.; GarciaCarmona, F. Tyrosinase: a comprehensive review of its mechanism. Biochim. Biophys. Acta. 1995, 1247, 1-11. 3. Rodriguez-Lopez, J.N.; Fenoll, L.G.; Peñalver, M.J.; Garcia-Ruiz, P.A.; Varon, R.; Martinez-Ortiz, F.; Garcia-Canovas, F.; Tudela, J. Tyrosinase action on monophenols: evidence for direct enzymatic release of o-diphenol. Biochim. Biophys. Acta. 2001, 1548, 238-256. 4. Rodriguez-Lopez, J.N.; Tudela, J.; Varon, R.; Garcia-Canovas, F. Kineticstudy on the effect of pH on the melanin biosynthesis pathway. Biochim. Biophys. Acta 1991, 1076, 379-386. 5. Garcia-Moreno, M.; Rodriguez-Lopez, J.N.; Martinez-Ortiz, F.; Tudela, J.; Varon, R.; Garcia-Canovas, F. Effect of pH on the oxidation pathway of dopamine catalyzed by tyrosinase. Arch. Biochem. Biophys. 1991, 288, 427-434. 6. Garcia-Molina, M.M.; Muñoz-Muñoz, J.L.; Berna, J.; Rodriguez-Lopez, J.N.; Varon, R.; Garcia-Canovas, F. Hydrogen peroxide helps in the identification of monophenols as possible substrates of tyrosinase. Biosci. Biotechnol. Biochem. 2013, 77, 2383-2388. 7. Garcia-Molina, M.M.; Muñoz-Muñoz, J.L.; Martinez-Ortiz, F.; Martinez, J.R.; Garcia-Ruiz, P.A.; Rodriguez-Lopez, J.N.; Garcia-Canovas, F. Tyrosinase-catalyzed hydroxylation of hydroquinone, a depigmenting agent, to hydroxyhydroquinone: A kinetic study. Bioorg. Med. Chem. 2014, 22, 3360-3369. 8. Garcia-Molina, M.M.; Berna, J.; Muñoz-Muñoz, J.L.; Garcia-Ruiz, P.A.; Garcia-Moreno, M.; et al. Action of tyrosinase on hydroquinone in the presence of catalytic amounts of o-diphenol. A kinetic study. React. Kinet. Mech. Cat. 2014, 112, 305-320. 9. Kashima, Y.; Miyazawa, M. Synthesis, antioxidant capacity, and structureactivity relationships of tri-O-methylnorbergenin analogues on tyrosinase inhibition. Bioorg. Med. Chem. Lett. 2013, 23, 6580-6584. 10. Casañola-Martin, G.M.; Marrero-Ponce, Y.; Tareq Hassan Khan, M.; Torrens, F.; Perez-Gimenez, F.; Rescigno,A. Atom- and bond-based 2D TOMOCOMDCARDD approach and ligand-based virtual screening for the drug discovery of new tyrosinase inhibitors. J. Biomol. Screen. 2008, 13, 1014-1024. 11. Casañola-Martin, G.M.; Marrero-Ponce, Y.; Khan, M.T., Khan, S.B.; Torrens, F.; Perez-Jimenez, F.; Rescigno, A.; Abad, C. Bond-based 2D quadratic fringerprints in QSAR studies: virtual and in vitro tyrosinase inhibitory activity elucidation. Chem. Biol. Drug. Des. 2010, 76, 538-545. 12. Casañola-Martin, G.M.; Le-Thi-Thu, H.; Marrero-Ponce, Y.; Castillo-Garit, J.A.; Torrens, F.; Rescigno,A.; Abad, C.; Khan, M.T. Tyrosinase enzyme: 1. An overview on a pharmacological target. Curr Top. Med. Chem. 2014, 14, 1494-1501. 13. Sun, W.; Wendt, M.; Klebe, G.; Rohm, K.H. On the interpretation of tyrosinase inhibition kinetics. J. Enzyme Inhib. Med. Chem. 2014, 29, 92-99. 14. Le-Thi-Thu, H.; Casanola-Martin, G.M.; Marrero-Ponce, Y.; Rescigno, A.; Abad, C.; Khan, M.T. A rational workflow for sequential virtual screening of chemicals libraries on searching for new tyrosinase inhibitors. Curr. Top. Med. Chem. 2014, 14, 1473-1485.


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15. Rodriguez-Lopez, J.N.; Fenoll, L.G.; Garcia-Ruiz, P.A.; Varon, R.; Tudela, J.; Thorneley, R.N.F.; Garcia-Canovas, F. Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase. Biochemistry 2000, 39, 10497-10506. 16. M.M. Bradford. 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. 17. Fenoll, L.G.; Rodriguez-Lopez, J.N.; Garcia-Molina, F.; Garcia-Canovas, F.; Tudela, J. Michaelis constants of mushroom tyrosinase with respect to oxygen in the presence of monophenols and diphenols. Int. J. Biochem. Cell Biol. 2002, 34, 332-336. 18. Garcia-Molina, F.; Muñoz, J.L.; Varon, R.; Rodriguez-Lopez, J.N.; GarciaCanovas, 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. 19. Muñoz, J.L.; Garcia-Molina, F.; Varon, R.; Rodriguez-Lopez, J.N.; GarciaCanovas, F.; Tudela, J. Calculating molar absorptivities for quinones: application to the measurement of tyrosinase activity. Anal. Biochem. 2006, 351, 128-138. 20. Garcia-Sevilla, F.; Garrido-del Solo, C.; Duggleby, R.G.; Garcia-Canovas, F.; Peyro, R.; Varon, R. Use of a Windows program for simulation of the progress curves of reactants and intermediates involved in enzyme-catalyzed reactions. Biosystems. 2000, 54, 151-164. 21. Ros, J.R.; Rodriguez-Lopez, J.N.; Garcia-Canovas, F. Tyrosinase: kinetic analysis of the transient phase and the steady state. Biochim. Biophys. Acta. 1994, 1204, 33-42. 22. Garcia-Molina, F.; Muñoz, J.L.; Varon, R.; Rodriguez-Lopez, J.N.; GarciaCanovas, F.; Tudela, J. An approximate analytical solution to the lag period of monophenolase activity of tyrosinase. Int. J. Biochem. Cell Biol. 2007, 39, 238-252. 23. Chen, Q.X.; Ke, L.N.; Song, K.K.; Huang, H.; Liu,X.D. Inhibitory effects of hexylresorcinol and dodecylresorcinol on mushroom (Agaricus bisporus) tyrosinase. Protein J. 2004, 23, 135-141. 24. Huang, X.H.; Chen, Q.X; You, M.S.; Wang, Q.; Song, K.K.; Wang, J.; Sha, L.; Guan, X. Inhibitory effects of fluorobenzaldehydes on the activity of mushroom tyrosinase. J. Enzyme Inhib. Med. Chem. 2006, 21, 413-418. 25. Baek, Y. S.; Ryu, Y. B.; Curtis-Long, M. J.; Ha, T. J.; Rengasamy, R.; Yang, M. S.; Park, K. H. Tyrosinase inhibitory effects of 1,3-diphenylpropanes from Broussonetia kazinoki. Bioorg. Med. Chem. 2009, 17, 35-41. 26. Jeong, S. H.; Ryu, Y. B.; Curtis-Long, M. J.; Ryu, H. W.; Baek, Y. S.; Kang, J. E.; Lee, W. S.; Park, K. H. Tyrosinase Inhibitory Polyphenols from Roots of Morus Ihou. J. Agric. Food Chem. 2009, 57, 1195-1203. 27. Qiu, L.; Chen, Q.H.; Zhuang, J.X.; Zhong, X.; Zhou, J.J.; Guo, Y.J.; Chen, Q.X. Inhibitory effects of alpha-cyano-4-hydroxycinnamic acid on the activity of mushroom tyrosinase. Food Chem. 2009, 112, 609-613. 28. Zhuang, J.X.; Li, W.G.; Qiu, L.; Zhong, X.; Zhou, J.J.; Chen, Q.X. Inhibitory effects of Cefazolin and Cefodizime on the activity of mushroom tyrosinase. J. Enzyme Inhib. Med. Chem. 2009, 24, 251-256. 29. Lu, Y.H.; Chen, J.; Wei, D.Z.; Wang, Z.T.; Tao, X.Y. Tyrosinase inhibitory effect and inhibitory mechanism of tiliroside from raspberry. J. Enzyme Inhib. Med. Chem. 2009, 24, 1154-1160. 30. Conrad, J.S.; Dawso, S.R.; Hubbard, E.R.; Meyers, T.E.; Stronthkamp, K.G. Inhibitor binding to the binuclear active site of tyrosinase: temperature, pH, and solvent deuterium isotope effects. Biochemistry 1994, 33, 5739-5744. - 21Plus - Environment ACS Paragon


31. Rodriguez-Lopez, J.N.; Tudela, J.; Varon, R.; Garcia-Carmona, F.; GarciaCanovas, F. Analysis of a kinetic model for melanin biosynthesis pathway. J. Biol. Chem. 1992, 267, 3801-3810. 32. Parvez, S.; Kang, M.; Chung, H.S.; Cho, C.; Hong, M.C.; Shin, M.K.; Bae, H. Survey and mechanism of skin depigmenting and lightening agents. Phytother. Res. 2006, 20, 921-934. 33. Hu, Z.M.; Zhou, Q.; Lei, T.C.; Ding, S.F.; Xu, S.Z. Effects of hydroquinone and its glucoside derivatives on melanogenesis and antioxidation: biosafety as skin whitening agents. J. Dermatol. Sci. 2009, 55, 179-184. 34. Maeda, K.; Fukuda, M. Arbutin: mechanism of its depigmenting action in human melanocyte culture. J. Pharmacol. Exp. Ther. 1996, 276, 765-769. 35. Hori, I.; Nihei, K.; Kubo, I. Structural criteria for depigmenting mechanism of arbutin. Phytother. Res. 2004, 18, 475-479. 36. Satooka, H.; Kubo, I. Effects of thymol on mushroom tyrosinase-catalyzed melanin formation. J. Agric. Food Chem. 2011, 59, 8908-8914. 37. Satooka, H.; Kubo, I. Effects of Thymol on B16-F10 Melanoma Cells. J. Agric. Food Chem. 2012, 60, 2746-2752. 38. Garcia-Molina, M.M.; Munoz-Munoz, J.L.; Garcia-Molina, F.; Garcia-Ruiz, P.A.; Garcia-Canovas, F. Action of tyrosinase on ortho-substituted phenols: possible influence on browning and melanogenesis. J. Agric. Food Chem. 2012, 60, 64476453.


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Figure captions Figure 1. Action mechanism of tyrosinase on monophenols and o-diphenols.

Figure 2. Action mechanism of tyrosinase on o-diphenols.

Figure 3. Chemical structures of the compounds studied. (1) Guaiacol, (2) isoeugenol, (3) eugenol, (4) 4-tert-butylphenol, (5) arbutin, (6) carvacrol, (7) benzoic acid and (8) cinnamic acid.

Figure 4. Relevance of the relation (R) between the o-diphenol, [D]ss, and the monophenol, [M]ss, in steady state (R = [D]ss/[M]ss). Dopachrome accumulation with time of using [E]0 = 8.0 nM on L-tyrosine [M]0 = 0.25 mM. Curve (a): L-dopa is not added at the beginning of the reaction [D]0 = 0. Curve (b): L-dopa is added at the beginning of the reaction, [D]0 = 10.50 µM, in order to fulfil the relation R = 0.042. Curve (c): more L-dopa than the required to fulfil the relation R is added, [D]0 = 28.75 µM. Inset. Simulated progress curves of the mechanism shown in Figure 1. The conditions were: [E]0 = 22 nM, [Eox]0 = 0.3x[E]0, [Em]0 = 0.7x[E]0, [M]0 = 0.25 mM, (a) [D]0 = 0, (b) [D]0 = 10.50 µM and (c) [D]0 = 28.75 µM. The concentration of oxygen was 0.26 mM, and the rate constants were: k1 = 2x105 M-1s-1, k-1 = 10 s-1, k2 = 5x106 M-1s-1, k-2 = 10 s-1, k3 = 900 s-1, k4 = 2x104 M-1s-1, k-4 = 1 s-1, k5 = 5

S

-1

,k6 =

2.16x105 M-1s-1, k-6 = 10 s-1, k7 = 108 s-1, k8 = 2.3x108 M-1s-1, k-8 = 1.07x103 s-1 and k11 = 10 s-1.

Figure 5. Action mechanism of tyrosinase on monophenols and o-diphenols in the presence of an inhibitor that binds to the enzymatic forms Em and Eox.



Figure 6. Action mechanism of tyrosinase on o-diphenols in the presence of an inhibitor that binds to the enzymatic forms Em and Eox.

Figure 7. Action of benzoic acid on the diphenolase and monophenolase activities of tyrosinase A. Spectrophotometric recordings of the accumulation of dopachrome in the action of tyrosinase on L-tyrosine in the absence and presence of benzoic acid. The experimental conditions were: [M]0 = 0.25 mM, [E]0 = 7 nM, [D]0 = 10.50 µM and the benzoic acid concentrations were (mM): (a) 0, (b) 0.15, (c) 0.35, (d) 0.70 and (e) 1.40. A Inset. Simulated progress curves of the mechanism shown in Figure 5. The conditions were: [E]0 = 22 nM, [Eox]0 = 0.3x[E]0, [Em]0 = 0.7x[E]0, [M]0 = 0.25 mM and [D]0 = 10.50 µM. The concentration of oxygen was 0.26 mM and the benzoic acid concentrations were the same as in Figure 7A. The rate constants were the same as in Figure 4 Inset, and k9 = 103 M-1s-1, k-9 = 4.1 s-1, k10 = 104 M-1s-1 and k-10 = 4.1 s-1. B. Records of the accumulation of dopachrome in the action of tyrosinase on L-dopa in the absence and presence of benzoic acid. The experimental conditions were: [D]0 = 0.5 mM, [E]0 = 5 nM, and the benzoic acid concentrations were the same as in Figure 7A. B Inset. Simulation of the mechanism shown in Figure 6. The conditions were: [E]0 = 7.5 nM, [Eox]0 = 0.3x[E]0, [Em]0 = 0.7x[E]0; [D]0 = 0.5 mM. The concentration of oxygen was 0.26 mM and the

concentrations of the inhibitor the same as in Figure 7B. The rate constants were the same as in Figures 4 Inset and 7A Inset.

Figure 8 Action of cinnamic acid on the monophenolase and diphenolase activities of tyrosinase. Spectrophotometric recordings of the accumulation of dopachrome in the action of tyrosinase on L-tyrosine. The experimental conditions were: [M]0 = - 24Plus - Environment ACS Paragon

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0.25 mM, [E]0 = 7 nM and [D]0 = 10.50 µM and the cinnamic acid concentrations were (mM): (a) 0, (b) 0.2, (c) 0.4, (d) 1.0 and (e) 2.0. Inset. Spectrophotometric recordings of the accumulation of dopachrome in the action of tyrosinase on L-dopa in the presence of cinnamic acid. The experimental conditions were [D]0 = 0.5 mM and [E]0 = 5 nM. The cinnamic acid concentrations were the same as used in the monophenolase activity.

Figure 9. Action of the target molecules on the monophenolase and diphenolase activities of tyrosinase. A. Action of TBP on the monophenolase activity on Ltyrosine. The experimental conditions were: [M]0 = 0.25 mM, [E]0 = 7 nM and [D]0 = 10.50 µM. The concentrations of TBP were (mM): (a) 0, (b) 0.1 and (c) 0.4. A Inset. Action of TBP on the diphenolase activity on L-dopa. The experimental conditions were: [D]0 = 0.5 mM and [E]0 = 5 nM. The concentrations of TBP were the same as in Figure 9A. B. Action of isoeugenol on the monophenolase activity of tyrosinase. The experimental conditions were the same as in Figure 9A, and the concentrations of isoeugenol were (mM): (a) 0, (b) 0.9 and (c) 1.8. B Inset. Action of ioseugenol on the diphenolase activity of tyrosinase. The experimental conditions were the same as in Figure 9A Inset and the concentrations of isoeugenol were the same as in Figure 9B. C. Action of the guaiacol on the monophenolase activity of tyrosinase. The experimental conditions were the same as in Figure 9A and Figure 9B, and the concentrations of guaiacol were (mM): (a) 0, (b) 4 and (c) 8. C Inset. Action of guaiacol on the diphenolase activity of tyrosinase. The experimental conditions were the same as in Figure 9A Inset and Figure 9B Inset and the concentrations of guaiacol were the same as in Figure 9C.



Figure 10. Possible reaction of the o-quinones of L-dopa or TBC with the phenols under study. L-dopa 1mM was oxidized with sodium periodate in default (0.53 mM) and then the phenols were added (a) carvacrol 5.9 mM and (b) guaiacol 24 mM. The continuous recording (c) shows the stability of the dopachrome with time in the absence of the target molecules. Inset. TBC 1.6 mM was oxidized with sodium periodate in default (0.53 mM), and then the phenols were added (a) carvacrol 6.4 mM and (b) guaiacol 24.0 mM. The continuous recording (c) shows the stability of otert-butylquinone in the absence of the target molecules.

Figure 11. Stability of dopachrome and o-tert-butylquinone in the presence of arbutin. L-dopa 1mM was oxidized with sodium periodate in default (0.53 mM) and the stability of its o-quinone was recorded in the absence (a) and presence (b) of arbutin 18 mM. TBC was oxidized 1.6 mM with sodium periodate in default (0.53 mM) and the stability of the generated o-quinone was recorded in the absence (a) and presence (b) of arbutin 18 mM. Inset. Increase in absorbance in the action of tyrosinase [E]0 = 56 nM on L-dopa 1 mM, in the absence (a) and presence of arbutin (mM) (b) 0.6, (c) 3.0 and (d) 18.0, until all the oxygen was consumed.

Figure 12. Oxidation of L-dopa through tyrosinase in the presence of different concentrations of benzoic acid until all the oxygen was consumed. The conditions were: [D]0 = 1 mM, [E]0 = 56 nM and the concentrations of benzoic acid were (mM): (a) 0, (b) 1.25, (c) 2.5, (d) 3.75 and (e) 5.0. Inset. Oxidation of L-dopa by tyrosinase in the presence of cinnamic acid until all the oxygen was consumed. The experimental conditions were the same as in Figure 12, and the concentrations of cinnamic acid were (mM): (a) 0, (b) 0.6, (c) 1.2 and (d) 2.4.


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Figure 13. Action of tyrosinase on isoeugenol and eugenol in the presence of hydrogen peroxide (H2O2). Recordings of the variation of absorbance at 440 nm in the action on isoeugenol 1.75 mM (a) and eugenol 1.75 mM (b). The experimental conditions were: [E]0 = 130 nM and H2O2 10 mM. Inset. Action of the tyrosinase on TBP in the presence of H2O2. Recording of the variation of absorbance at 400 nm. The experimental conditions were: TBP 0.5 mM, [E]0 = 3 nM and H2O2 10 mM.

Figure 14. Action of tyrosinase on arbutin and carvacrol in the presence of hydrogen peroxide (H2O2). Variation of absorbance at 480nm. The experimental conditions were: (a) arbutin 5 mM, [E]0 = 45 nM and H2O2 5 mM and (b) carvacrol 1.4 mM, phosphate buffer 50mM pH 7.0, [E]0 = 0.16 µM, H2O2 0.05 mM, MBTH 4.5 mM and DMF 2%. Inset. Action of tyrosinase on guaiacol in the presence of H2O2. Variation of the absorbance at 337 nm. The experimental conditions were: guaiacol 7.5 mM, [E]0 = 85 nM and H2O2 10 mM.



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Tables Table 1. Degree of inhibition (i %) of the monophenolase (iM) and diphenolase (iD) activities of tyrosinase in the presence of different concentrations of two competitive inhibitors, that bind to the enzymatic forms Em and Eox.

Inhibitor

Concentration (mM)

iM

iD

Benzoic Acid

0.15

17.92 ± 0.10

16.36 ± 0.03

0.35

27.95 ± 0.12

29.35 ± 0.27

0.70

42.15 ± 0.19

44.87 ± 0.12

1.40

63.76 ± 0.45

62.90 ± 0.16

0.20

19.84 ± 0.08

19.44 ± 0.08

0.40

33.31 ± 0.19

37.03 ± 0.10

1.00

50.11 ± 0.28

47.61 ± 0.30

2.00

70.06 ± 0.70

72.14 ± 0.93

Cinnamic Acid


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Table 2. Degree of inhibition (i %) of the monophenolase (iM) and diphenolase (iD) activities of tyrosinase obtained through simulated recordings by numerical integration of the set of differential equations corresponding to: (a) the mechanisms shown in Figures 5 and 6, in which both activities are shown in the presence of different concentrations of an inhibitor that binds to the enzymatic species Eox and Em (Figures 7A Inset and 7B Inset); (b) the mechanisms shown in Figures 1SI and 2SI corresponding to the tyrosinase activity in the presence of an inhibitor that binds to the form Eox in the diphenolase activity and Eox and EmM in the monophenolase activity (Figures 3SI Inset and 3SI).

Inhibitor (a) iM = iD

(b) iM ≠ iD

Concentration (mM)

iM

iD

0.15

15.80

15.48

0.35

28.54

27.98

0.70

44.04

43.29

1.40

62.18

61.37

0.15

25.65

15.75

0.35

42.11

28.46

0.70

58.83

43.93

1.40

74.88

62.09

- 29 -Plus Environment ACS Paragon


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Table 3. Degree of inhibition (i %) of the monophenolase (iM) and diphenolase (iD) activities of tyrosinase in the presence of different concentrations of several target monophenols.

Target Monophenol

Concentration (mM)

iM

iD

Guaiacol

4.0

63.78 ± 1.07

26.26 ± 0.10

8.0

69.80 ± 0.90

39.35 ± 0.11

0.9

46.46 ± 0.31

22.72 ± 0.07

1.8

67.57 ± 0.92

40.96 ± 0.13

2.0

54.03 ± 0.63

14.15 ± 0.03

4.8

70.05 ± 1.25

45.41 ± 0.17

0.1

64.80 ± 0.57

14.88 ± 0.03

0.4

82.26 ± 1.76

33.52 ± 0.09

0.9

37.76 ± 0.24

17.74 ± 0.04

3.6

64.76 ± 0.49

58.74 ± 0.25

0.3

33.02 ± 0.29

13.99 ± 0.04

1.5

53.97 ± 0.76

27.59 ± 0.06

Isoeugenol

Carvacrol

TBP

Eugenol

Arbutin

- 30 -Plus Environment ACS Paragon

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Figure graphics

k1 EmM

M + Em + D

k-1

k2

EmD

k-2

Q

k3 k5

k7

Ed + O2 k-8 k8

EoxD

k6

D + Eox + M

k-6

k4 k-4

k11 2Q

D + Cr

Figure 1.


EoxM


Page 32 of 48

Q

Q

k2 Em + D

k-2

EmD

k3

Ed + O2

2Q

k8 k-8

k11

k6 Eox + D

D + Cr

Figure 2.


k-6

EoxD

k7

Em

Page 33 of 48


HO OH

OH

O

O

O

(1)

(2)

(3)

OH O HO HO

HO

O OH

(4) (5)

OH

O

O

HO OH

(6)

(7)

Figure 3.


OH

(8)


0.30

Page 34 of 48

Cr (M) x 105

6

0.25

0.20

5

c

4

b

3

a

c

2

b

1 0

A475

0

200

400

600

800

1000

time (s)

0.15

a

0.10

0.05

0.00 0

100

200

300 time (s)

Figure 4.


400

500

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EmI k-9

k9

EmM

I + M + Em + D

k1 k-1

k2

Q Ed + O2 k-8 EoxD

k-6

k3 k5

k7

k6

k8

D + Eox + M + I k-10

k4 k-4

k10

EoxI 2Q

EmD

k-2

k11

D + Cr

Figure 5.


EoxM


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Q

Q k2 Em + D +

k-2

EmD

k3

E d + O2

k8 k-8

I k-9

k6 Eox + D k-6 + I k-10 k10

k9

EoxI

EmI 2Q

k11

D + Cr

Figure 6.


EoxD

k7

Em

Page 37 of 48


16

Cr (M) x 106

0.06

A a

12

a

8 4

0.04

e

0

A475

0

100 200 time (s)

300

0.02 e

0.00 0

50

100 time (s)

Figure 7A.


150

200


B

10 Cr (M) x105

0.16

0.12

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a

8 6

a

4 2

e

A475

0 0

100 200 time (s)

300

0.08

0.04 e

0.00 0

50

100

150

Figure 7B.


200

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0.08 a

0.16

A475

0.12

0.06

a 0.08 0.04

A475

e 0.00 0

0.04

50

100

150

200

time (s)

0.02 e 0.00 0

50

100 time (s)

Figure 8.


150

200


Page 40 of 48

0.10

A

0.16

a 0.12 A475

0.08

b 0.08

a

c

0.04

0.06 A475

0.00 0

50

100

150

200

time (s)

0.04 b 0.02

c

0.00 0

50

100

150

200

time (s) Figure 9A.


250

300

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0.10

B

0.16

a b

0.12 A475

0.08

c

a

0.08 0.04

0.06 A475

0.00 0

50

100

150

200

time (s)

b

0.04

c 0.02

0.00 0

50

100

150

200

time (s)

Figure 9B.


250

300


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0.10

C

0.16

a 0.12 A475

0.08

b

0.08

a

c

0.04

0.06 A475

0.00 0

50

100 150 time (s)

200

0.04 b 0.02

c

0.00 0

50

100

150

200

time (s) Figure 9C.


250

300

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1.0

a c

0.8

b

0.8

a c

0.6

b A410

A475

0.6

0.4

0.4 0.2

0.2

0.0 0

50

100

150

200

250

time (s)

0.0 0

50

100

150

200

time (s) Figure 10.


250

300


Page 44 of 48

0.8

1.0

a b c

0.8

0.6

0.6

A410

a

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Figure 11.


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Figure 12.


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Figure 13.


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Figure 14.


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Discrimination between Alternative Substrates and Inhibitors of

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