Catalytic oxidation of o-aminophenols and aromatic amines by mushroom tyrosinase

Article abstract of DOI:10.1016/j.bbapap.2011.07.015

The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied. The catalytic constants of aromatic monoamines and o-diamines were both low, these results are consistent with our previous mechanism in which the slow step is the transfer of a proton by a hydroxyl to the peroxide in oxy-tyrosinase (Fenoll et al., Biochem. J. 380 (2004) 643-650). In the case of o-aminophenols, the hydroxyl group indirectly cooperates in the transfer of the proton and consequently the catalytic constants in the action of tyrosinase on these compounds are higher. In the case of aromatic monoamines, the Michaelis constants are of the same order of magnitude than for monophenols, which suggests that the monophenols bind better (higher binding constant) to the enzyme to facilitate the π-π interactions between the aromatic ring and a possible histidine of the active site. In the case of aromatic o-diamines, both the catalytic and Michaelis constants are low, the values of the catalytic constants being lower than those of the corresponding o-diphenols. The values of the Michaelis constants of the aromatic o-diamines are slightly lower than those of their corresponding o-diphenols, confirming that the aromatic o-diamines bind less well (lower binding constant) to the enzyme.

Full text of DOI:10.1016/j.bbapap.2011.07.015

Biochimica et Biophysica Acta 1814 (2011) 1974–1983
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Catalytic oxidation of o-aminophenols and aromatic amines by mushroom tyrosinase
Jose Luis Muñoz-Muñoz a, Francisco Garcia-Molina a, Pedro Antonio Garcia-Ruiz b, Ramon Varon c,
Jose Tudela a, Jose N. Rodriguez-Lopez a, Francisco Garcia-Canovas a,
⁎
a
GENZ: Grupo de Investigación Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100, Espinardo, Murcia, Spain
QCPAI: Grupo de Química de Carbohidratos, Polímeros y Aditivos Industriales, Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100, Espinardo,
b
Murcia, Spain
c
Departamento de Química-Física, Escuela de Ingenieros Industriales de Albacete, Universidad de Castilla la Mancha, Avda, España s/n, Campus Universitario, E-02071, Albacete, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 May 2011
Received in revised form 12 July 2011
Accepted 13 July 2011
Available online 22 July 2011
The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied. The
catalytic constants of aromatic monoamines and o-diamines were both low, these results are consistent with
our previous mechanism in which the slow step is the transfer of a proton by a hydroxyl to the peroxide in
oxy-tyrosinase (Fenoll et al., Biochem. J. 380 (2004) 643–650). In the case of o-aminophenols, the hydroxyl
group indirectly cooperates in the transfer of the proton and consequently the catalytic constants in the action
of tyrosinase on these compounds are higher. In the case of aromatic monoamines, the Michaelis constants are
of the same order of magnitude than for monophenols, which suggests that the monophenols bind better
(higher binding constant) to the enzyme to facilitate the π–π interactions between the aromatic ring and a
possible histidine of the active site. In the case of aromatic o-diamines, both the catalytic and Michaelis
constants are low, the values of the catalytic constants being lower than those of the corresponding o-
diphenols. The values of the Michaelis constants of the aromatic o-diamines are slightly lower than those of
their corresponding o-diphenols, confirming that the aromatic o-diamines bind less well (lower binding
constant) to the enzyme.
Keywords:
Tyrosinase
Aromatic amine
Aromatic o-diamines
o-aminophenols
Catalytic constant
Michaelis constant
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
The source of tyrosinase used by these authors [10] was Neurospora
crassa. Moreover, 3-hydroxyanthranilic acid has been shown to act as an
Tyrosinase (TYR, EC 1.14.18.1) is a copper-containingenzymewidely
distributed in nature. It catalyses two types of reactions: (a) the ortho-
hydroxylation of monophenols to o-diphenols (monophenolase activ-
ity) and (b) the oxidation of o-diphenols to o-quinones (diphenolase
activity). Both types of reaction require molecular oxygen as the second
substrate of the enzyme [2,3].
The catalytic centre of tyrosinase possesses a binuclear copper
similar to that of hemocyanin and catechol oxidase [3]. The catalytic
cycle of tyrosinase has three enzymatic forms: Em (met-tyrosinase) with
the copper as Cu2+Cu2+, Ed (deoxy-tyrosinase) with the copper as
Cu+Cu+ and Eox (oxy-tyrosinase) with copper in the form Cu2+Cu2+
O22-[4]. In the structures of hemocyanin [3,5], catechol oxidase [6] and
tyrosinase from Streptomyces castaneoglobisporus[7] and from Bacillus
megaterium[8,9], each copper ion is coordinated by three histidines.
Apart from its natural substrates (monophenols and o-diphenols),
tyrosinase is also capable of oxidising a variety of aromatic monoamines
and o-aminophenols, such as 4-aminotoluene, or several aminophe-
nolic derivatives of benzoic acid, such as 3-amino-4-hydroxybenzoic
acid, 4-amino-3-hydroxybenzoic acid and 3,4-diaminobenzoic acid[10].
activator of the monophenolase activity of mushroom tyrosinase [11],
reducing the lag period for reactions with non-cyclising phenolic
substrates (4-tert-butylphenol/4-tert-butylcatechol). In another report,
the same authors described the oxidation of 3-hydroxykynurenine, a
tryptophan catabolite, by mushroom tyrosinase [12]. On the other hand,
3-amino-L-tyrosine has been described as a potent competitive inhibitor
of the monophenolase activity and does not serve as a substrate. It was
proposed that this aminophenol acts as a complexing agent of the
copper ions in the oxy form of tyrosinase [13].
In a recent work [14], the interaction of mushroom tyrosinase with
several aromatic monoamines, o-diamines and o-aminophenols was
described using different spectrophotometric and oxymetric tech-
niques. Consequently, 3-amino-L-tyrosine was shown to be a real sub-
strate of tyrosinase, contrary to the findings published by Maddaluno
and Faull [13].
In all the works above [11–14], o-aminophenols and aromatic
monoamines and o-diamines were seento act assubstrates of tyrosinase
because they are all structural analogues of the natural substrates of the
enzyme (o-diphenols and monophenols).
The kinetic characterisation of aromatic monoamines, o-diamines
and o-aminophenols is difficult because the product of the reaction,
o-quinoneimine, is unstable [10,11]. Kinetic studies have been carried
out using oxymetric techniques, which have provided quantitative
⁎
Corresponding author. Tel.: +34 868 884764; fax: +34 868 883963.
E-mail address: canovasf@um.es (F. Garcia-Canovas).
1570-9639/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2011.07.015

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1814 (2011) 1974–1983
1975
information [10]. However, spectrophotometric techniques [10,14] do
not provide such information because the product evolves. In a recent
work [15], we presented a revision of the spectrophotometric tech-
niques used for measuring tyrosinase activity. In the present work, we
widen the use of these methods to the characterisation of the sub-
strates above, applied especially to the case of o-aminophenols and
aromatic o-diamines, using ascorbic acid, the concentration of which
decays and is followed spectrophotometrically. We also study the
possibility that some o-quinoneimines may give rise to an isosbestic
point during their evolution. Oxymetric techniques have also been
used [16], especially to characterise monophenols and aromatic amines,
permitting kinetic parameters to be reliably obtained for this series
of compounds.
Bearing in mind the above, the aim of this study was to take a
deeper look at the tyrosinase mechanism, considering its action on
several substrates: aromatic monoamines, o-diamines and o-amino-
phenols. The results obtained are compared to those obtained for o-
diphenols and monophenols. Based on the findings, the mechanism of
tyrosinase is discussed and the decisive steps of the catalysis are
characterised.
substrate concentration was varied as indicated in each figure legend,
and the reaction was started by adding enzyme (TYR). The coupled
reagent concentration (AH2, NADH or MBTH) is indicated in each figure
legend.
2.4. Oxymetric assays
Oxygen consumption was measured with a Clark-type electrode
coupled to a Hansatech Oxygraph (King's Lynn, Norfolk, UK). The
equipment was calibrated using the TYR/4-tert-butylcatechol method
[16]. Nitrogen was bubbled through the reaction medium to remove the
oxygen when necessary. The cuvette (final volume 2 mL) contained
30 mM sodium phosphate buffer (pH 7.0) and the substrate concentra-
tion wasvaried. In every case, AH2 was added tothe medium to maintain
the substrate concentration constant. The reaction was started by
adding substrate. The medium was stirred constantly and the temper-
ature was kept at 25 0.1 °C using a Haake D16 circulating water-bath.
The substrates characterised by this method were: 3-aminobenzoic acid,
4-aminobenzoic acid, 2-aminotoluene, 2-aminoanisole, 3-aminoanisole,
aniline, 3,4-diaminobenzoic acid, 3-amino-2-hydroxybenzoic acid,
2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic
acid, 2-methylphenol (o-cresol), 4-methylphenol (p-cresol), 3-hydro-
xyanisole, phenol, 2,3-dihydroxybenzoic acid and 3,4-dihydroxyben-
zoic acid. In the cases of aromatic monoamines and monophenols, the
corresponding o-diphenol or 4-tert-butylcatechol (when the corre-
sponding o-aminophenols or o-diphenol was not available commer-
cially, e.g. 2-aminotoluene, 2-aminoanisole and 3-aminoanisole)
were added to the medium to avoid the lag phase of the mono-
phenolase activity of tyrosinase ([D]ss/[M]ss =R) [20].
2. Material and methods
2.1. Reagents
Mushroom tyrosinase (TYR, o-diphenol:O2 oxidoreductase, EC
1.14.18.1) (1530 U/mg), was obtained from Sigma Chemical (St. Louis,
MO). All substrates and L-ascorbic acid (AH2) were obtained from
Sigma Chemical (St. Louis, MO). Stock solutions of the reducing sub-
strates were prepared in 0.15 mM phosphoric acid to prevent auto-
oxidation. Milli-Q system (Millipore Corp.) ultrapure water was used
throughout this research. The buffer used was 30 mM sodium
phosphate (pH 7.0).
2.5. Kinetic analysis
For TYR, the initial rate values (V0) were calculated from triplicate
measurements at each reducing substrate concentration, and V0vs.
[S]0 data were adjusted to the Michaelis–Menten equation [Eq. (1)]
through the Sigma Plot 9.0 program for Windows [21], obtaining the
maximum rate (Vmax) and Michaelis constant for each substrate (Km).
From these values and according to Eq. (2), the catalytic constant
(kcat) can be calculated.
2.2. Enzyme source
Tyrosinase (1530 U/mg; TYR) was purified according to [17].
Protein concentration was determined by Bradford's method using
bovine serum albumin as standard [18].
2.3. Spectrophotometric assays
3. Results and discussion
Absorption spectra were recorded in a visible–ultraviolet Perkin-
Elmer Lambda 35-spectrophotometer interfaced on-line with a PC
compatible Intel-Pentium microcomputer, ata scanningspeed of 60 nm/
s controlled by the UV-Winlab software. The temperature was
maintained at 25 °C using a Haake D16 circulating water bath with a
heater/cooler, and checked using a Cole-Parmer digital thermometer
with a precision of 0.1 °C. Kinetic assays were also carried out with the
instruments above, monitoring AH2 disappearance (2,3-diaminobenzoic
acid, 3,4-diaminotoluene, 4-methoxy-1,2-phenylendiamine, 4-amino-
3-hydroxybenzoic acid, 3-amino-L-tyrosine, 2-aminophenol and 3-
methylcatechol), NADH disappearance (catechol), the formation
of dopachrome (L-tyrosine and L-dopa), the appearance of an adduct
between o-quinoneimine and substrate (2-aminobenzoic acid, 3-
aminotoluene, 4-aminotoluene, 2,3-diaminotoluene, 1,2-diaminoben-
zene, 2-amino-3-hydroxybenozic acid, 3-amino-4-hydroxybenzoic
acid, 3-amino-4-hydroxytoluene, 4-amino-3-hydroxytoluene, 3-hydro-
xytoluene, 2-hydroxyanisole, 4-methylcatechol and 3-methoxycate-
chol) or the appearance of an adduct between MBTH, a nucleophile
reagent, and the substrate (4-aminoanisole, 4-aminophenylalanine and
4-hydroxyanisole). The measuring wavelength (λ) and the molar
absorbtivity coefficient (ε) for each substrate are summarised in Table
1SM (Supplementary material). The cuvettes (final volume of 1 mL)
contained 30 mM sodium phosphate buffer (pH 7.0) and O2 at a
concentration which saturates the corresponding enzymes [19]. The
In recent years much effort has been dedicated to the kinetics of the
action mechanism of tyrosinase acting on its physiological substrates,
monophenols and o-diphenols [2,3,22–24]. Besides these physiological
substrates, other types of non-phenolic substrate, such as NADH [25],
ascorbic acid [26], tetrahydropterines or tetrahydrofolic acid [27,28],
have been characterised. Moreover, to characterise kinetically aromatic
monoamines and o-diamines and o-aminophenols, several oxymetric
and spectrophotometric measuring methods have been designed [15].
3.1. Oxymetric methods
Generally speaking, all substrates of tyrosinase can be characterised
according to their consumption of oxygen, although, due to the low
activity shown by the enzyme towards some of them, such as aromatic
monoamines and o-diamines and o-aminophenols, spectrophotometric
methods are preferred, especially when the disappearance of a coupled
reagent with a high coefficient of molar absorptivity can be measured.
3.2. Spectrophotometric methods
When possible, ascorbic acid (AH2) was used, since it reduces the
o-quinoneimine or o-diimine to the corresponding o-aminophenol or
aromatic o-diamine, respectively. When the spectrum of the substrate
overlaps that of the AH2, a problem arises with the saturation of the

1976
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1814 (2011) 1974–1983
2.0
0.10
140
120
100
80
phototube. In this case, the oxymetric method is used or, if possible,
depending on the substrate, the visible zone is measured, taking
advantage of the formation of adducts between the o-quinoneimine
and the substrate (Scheme 1), or between the o-quinoneimine and a
coupled/bound reagent, such as MBTH.
A
B
(h)
0.08
0.06
0.04
0.02
0.00
60
1.5
1.0
0.5
0.0
(a)
40
3.3. Oxidation of o-aminophenols and aromatic o-diamines by periodate
in deficiency
20
0
0
20
40
60
80
100
0
2
4
6
[3-Hydroxyanthranilic acid]
t (s)
(mM)
0
The oxidation of o-aminophenols and aromatic o-diamines by perio-
date in deficiency gives rise to the formation of o-quinoneimine
(Scheme 1), which suffers a nucleophilic attack on the part of the
amino group of the substrate, originating an adduct which is oxidised
again by another molecule of o-quinoneimine. The new quinone suffers a
nucleophilic attack, giving rise to a new leuco compound, which, in turn,
is oxidised by another molecule of o-quinoneimine to form a p-quinone.
Fig. 1 depicts the appearance of an isosbestic point during the evolution
of the o-quinoneimine as a result of the oxidation of 3-hydroxyantr-
hanilic acid by periodate in deficiency. Note that a stoichiometry of 3
o-quinoneimine/1 p-quinoneimine is accomplished. The existence of
an isosbestic point permits the oxidation of 3-hydroxyanthranilic
acid to be followed at this wavelength.
300
350
400
450
500
550
600
λ (nm)
Fig. 1. Spectrophotometric scans of the 3-hydroxyanthranilic acid oxidation by
periodate in deficiency. The experimental conditions were 30 mM sodium phosphate
buffer (pH 7.0), 25 °C, initial concentrations of 3-hydroxyanthranilic acid and periodate,
[3-hydroxyanthranilic acid]0 and [IO4−]0, were 0.6 mM and 60 μM, respectively. The
spectrophotometric scans were made every 3 min. Inset A. Spectrophotometric
recordings of the oxidation of 3-hydroxyanthranilic acid by TYR at a wavelength of
398 nm. The experimental conditions were 30 mM sodium phosphate buffer (pH 7.0),
25 °C, initial concentration of enzyme, [E]0, was 0.61 μM and the initial substrate
concentrations (mM) were: (a) 0.5, (b) 1, (c) 1.5, (d) 2, (e) 3, (f) 4, (g) 5 and (h) 6. Inset
B. Representation of the steady-states rates (V0) vs. [3-hydroxyanthranilic acid]0.
3.4. Formation of chromophoric adducts with MBTH
In order to study the characteristics of the MBTH-o-quinoneimine
adducts, the oxidation of various aromatic monoamines by O2
catalysed by tyrosinase was carried out in the presence of MBTH
(Fig. 2). The pigment formed with 4-aminophenylalanine and 4-
aminoanisole had an absorbance maximum which ranged from 497 to
498 nm (Table 1SM). The pH affected the solubility and stability of the
adducts. The solubilisation of these compounds was achieved by
adding 2% (v/v) dimethylformamide (DMF) to the reaction medium
[29]. At pH 7.0, the adducts were unstable but showed an isosbestic
point. The MBTH is a potent nucleophile through its amino group,
which shows different degrees of protonation–deprotonation,
depending on the pH. The ionisation constant for this group was
determined by both potentiometric and spectrophotometric methods
(pK=5.8 0.4) [29]. Depending on its stability, each o-quinoneimine
needs a different [MBTH]sat to complete the formation of the adduct
(see the legends of the figures).
0.45
1.6
1.2
0.8
0.4
0.0
30
25
20
15
10
5
B
A
(h)
(a)
0.40
0.35
0.30
0.25
0.20
3.5. Oxidation of o-aminophenols by TYR and comparison with o-diphenols
0
0
400
800
1200
0.0 0.1 0.2 0.3 0.4 0.5 0.6
[4-AminoAnisole]
t (s)
(mM)
0
We studied the oxidation of several o-aminophenols by mushroom
tyrosinase using different measuring methods (see Supplementary
material), which enabled us to characterise kinetically these sub-
strates (Table 1).
Fig. 1 depicts the oxidation of 3-hydroxyanthranilic acid (2-amino-
3-hydroxybenzoic acid) by periodate in deficiency measuring the
gradual appearance of the product. An adduct is formed between the
HOOC
COOH
HOOC
H
N
NH2
OH
NH
O
OH
N
460
480
500
520
540
+
COOH
λ (nm)
NH2
OH
COOH
HOOC
Oxidation
Fig. 2. Spectrophotometric scans of the 4-aminoanisole oxidation by TYR in the
presence of MBTH. The experimental conditions were 30 mM sodium phosphate buffer
(pH 7.0), 25 °C, [4-aminoanisole]0 =0.5 mM, [E]0 =0.9 μM, [MBTH]0 =2.5 mM and
[DMF]0 =2%. The spectrophotometric scans were made every 15 min. Inset A.
NH2
OH
O
COOH
COOH
COOH
COOH
Spectrophotometric recordings of the oxidation of 4-aminoanisole by TYR at
a
H
N
NH2
OH
N
O
NH2
O
wavelength of 498 nm. The experimental conditions were 30 mM sodium phosphate
buffer (pH 7.0), 25 °C, [E]0 =10 nM, initial concentration of MBTH and DMF, [MBTH]0
and [DMF]0, were 2.5 mM and 2%, respectively and the initial substrate concentrations
(mM) were (a) 0.05, (b) 0.1, (c) 0.15, (d) 0.2, (e)0.3, (f) 0.4, (g) 0.5 and (h) 0.6. Inset B.
Representation of the initial steady-state rates (V0) vs. [4-aminoanisole]0. The
experimental conditions are the same as in Fig. 2.
Oxidation
O
Scheme 1. Mechanism for evolution of 3-hydroxyanthranilic acid´s o-quinoneimine in
the presence of an excess of o-aminophenol.

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1814 (2011) 1974–1983
1977
Table 1
Values of the kinetic constants characterising the oxidation of aminophenols and diphenols by TYR.
Aminophenols
kcat (s−1
)
Km (mM)
Diphenols
kcat (s−1
)
Km (mM)
2-amino-3-hydroxybenzoic acid
3-amino-2-hydroxybenzoic acid
3-amino-4-hydroxybenzoic acid
4-amino-3-hydroxybenzoic acid
3-amino-4-hydroxytoluene
4-amino-3-hydroxytoluene
3-amino-L-tyrosine
0.13 0.01
0.13 0.01
0.45 0.01
0.50 0.01
29.42 1.58
26.12 2.13
1.35 0.15
60.57 2.84
2.27 0.22
0.36 0.04
0.11 0.01
0.15 0.01
2.73 0.56
2.36 0.43
0.90 0.18
0.78 0.08
2,3-dihydroxybenzoic acid
0.37 0.04
8.11 0.31
1.02 0.12
0.07 0.01
0.10 0.01
3,4-dihydroxybenzoic acid
4-methylcatechol
842.12 26.11
L-dopa
Catechol
102.62 19.21
874.10 30.21
0.55 0.08
0.16 0.01
2-aminophenol
substrate and product of the reaction. This adduct is rapidly oxidised
to form cinnabarinic acid, giving rise to an isosbestic point at 398 nm
[10,11]. Fig. 1 inset A shows the experimental recordings of the
oxidation of different initial concentrations of 3-hydroxyanthranilic
acid, [S]0, by TYR. The initial rates of these oxidation processes with
respect to [S]0 are shown in Fig. 1 inset B. Non-linear regression gives
Vmax and Km according to the equation:
for the measurement, we first obtained the absorption spectra of AH2
(recording a, Fig. 3) and of 2-aminophenol (recording b, Fig. 3), choosing
in this case 265 nm, which represents the absorption maximum of AH2
(ε=11170 M−1 cm−1) [30]. Fig. 3, Inset A depicts the experimental
recordings of the gradual disappearance of AH2. Fig. 3, Inset B shows the
initial rates of TYR vs. [2-aminophenol]0. Non-linear-fitting of the values
of V0, in accordance with Eq. (1), gives Vmax and Km (see Table 1). In the
case of 3-amino-2-hydroxybenzoic acid (3-aminosalicylic acid), the
consumption of oxygen was measured (see Table 1).
Table 1 shows the kinetic constants obtained for the different
substrates and, where possible, these are compared with those ob-
tained for the corresponding o-diphenols. Note how the o-aminophe-
nols have the same or similar kcat values when positions 2 and 3 or 3
and 4 are compared for the amino or hydroxyl group; for example, the
value of kcat for 2-amino-3-hydroxybenzoic acid is approximately the
same as that of 2-hydroxy-3-aminobenzoic acid (0.13 0.01 s−1) and
the kcat of 3-amino-4-hydroxybenzoic acid is approximately the same
as that of 3-hydroxy-4-aminobenzoic acid (Table 1). The catalytic
constant increases from the ortho/meta position to the meta/para
position and the same occurs if the substituent in C-1 is an electro-
donating (―CH3) or electrowithdrawing (―COOH) group. A similar
situation exists for o-diphenols (see Table 1). The same applies when a
chain exists in C-1, as in the case of 3-amino-L-tyrosine and L-dopa,
while the values of kcat are much lower than in the case of 2-
aminophenol and catechol.
V max½Sꢀ0
V0
=
ð1Þ
ð2Þ
ð3Þ
Km + ½Sꢀ0
where:
V max = 2kcat½Eꢀ0
and
kcat
k6
Km
=
kcat being the catalytic constant, [E]0 the initial enzyme concentration
and k6 the substrate binding constant to the form oxy-tyrosinase.
In the case of 2-aminophenol, as shown in Fig. 3, ascorbic acid (AH2)
was used as reductant. In order to choose the most suitable wavelength
2.8
8
B
A
The Michaelis constants (see Table 1) and Eq. (3) are related with the
catalytic constants and, in the case of o-aminophenols, which have low
kcat values, the Km values are also low. Note that the ortho position of the
amino group of 2-amino-3-hydroxybenzoic acid has a higher Km, which,
according to Eq. (3), indicates that its binding constant k6 is lower than
that of 3-amino-2-hydroxybenzoic acid, which might be explained by
the steric hindrance of the amino group ortho position. The correspond-
ing o-diphenol (2,3-dihydroxybenzoic acid) has a lower Km value than
2-amino-3-hydroxybenzoic acid but it is three times higher than the
value corresponding to 2-hydroxy-3-aminobenzoic acid, which agrees
with the higher kcat. In the case of 3-amino-4-hydroxybenzoic acid
and 3-hydroxy-4-aminobenzoic acid, their values are similar to that of
3,4-dihydroxybenzoic acid (Table 1). The Km values of 3-amino-4-
hydroxytoluene and 3-hydroxy-4-aminotoluene (2.73 0.56 and
2.36 0.43 mM, respectively) are almost identical but, because the
kcat is higher than in the case above, the Km is also higher. When these
values are compared with those of their corresponding o-diphenols
(Table 1), the values of kcat are much lower in the case of o-
aminophenols and the Km values are in the same range, meaning that
the o-diphenols bind better to the enzyme (higher k6). The binding of
the o-diphenols to the enzyme may be more favoured than in the case
of the o-aminophenols because the π–π interaction between the
substrate and a possible histidine of the active site is favoured in the
case of o-diphenols and hindered in the case of aminophenols
(Table 1).
120
80
40
0
6
4
2
0
2.4
2.0
1.6
1.2
0.8
0.4
0.0
(a)
(i)
0
20
40
60
80
100
0
1
2
3
[2-aminophenol] (mM)
0
t (s)
(a)
(b)
240
260
280
300
320
λ (nm)
Fig. 3. Spectrophotometric scan of the 2-aminophenol and AH2. The experimental
conditions were 30 mM sodium phosphate buffer (pH 7.0), 25 °C, initial concentration of
ascorbic acid and 2-aminophenol, [AH2]0 and [2-aminophenol]0, were 0.12 mM (a) and
0.6 mM (b), respectively. Inset A. Spectrophotometric recordings of the 2-aminophenol
oxidation by TYR at a wavelength of 265 nm. The experimental conditions were 30 mM
sodium phosphate buffer (pH 7.0), 25 °C, [AH2]0 =0.13 mM and [E]0 =60 nM and the
initial substrate concentrations (mM) were: (a) 0.075, (b) 0.225, (c) 0.45, (d) 0.6, (e) 1.2,
(f) 1.5, (g) 2, (h) 2.5 and (i) 3. Inset B. Representation of the initial steady-state rates (V0)
vs. [2-aminophenol]0.
If an electrodonating group exists on C-1, the benzene ring will be
more electron-dense, binding will be less strong (due to the greater
repulsion between the rings) and therefore, the k6 value will be lower
since the π–π interactions are hindered. If an electrowithdrawing

1978
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1814 (2011) 1974–1983
Table 2
Values of the kinetic constants characterising the oxidation of aromatic diamines and o-diphenols by TYR.
Aromatic diamines
kcat (s−1
)
Km (mM)
Diphenols
kcat (s−1
)
Km (mM)
2,3-diaminobenzoic acid
3,4-diaminobenzoic acid
2,3-diaminotoluene
3,4-diaminotoluene
4-methoxy-1,2-phenylendiamine
1,2-diaminobenzene
0.05 0.01
0.08 0.01
0.11 0.01
0.51 0.04
1.51 0.13
0.53 0.05
0.28 0.04
0.15 0.02
0.96 0.11
0.06 0.01
0.36 0.05
0.46 0.05
2,3-dihydroxybenzoic acid
3,4-dihydroxybenzoic acid
3-methyl Catechol
4-methyl Catechol
3-methoxycatechol
Catechol
0.37 0.04
8.11 0.31
104.2 4.7
842.12 26.11
17.83 0.53
874.10 30.21
1.02 0.12
0.07 0.01
1.10 0.14
0.10 0.01
1.26 0.13
0.16 0.01
group exists on C-1 the benzene ring will be of lower electron density,
which will result in better substrate binding (higher k6).
3.7. Oxidation of aromatic monoamines by TYR and comparison with
monophenols
3.6. Oxidation of aromatic o-diamines by TYR and comparison with their
respective o-diphenols
TYR oxidises o-diphenols to o-quinones because of its diphenolase or
catecholase activity. However, it also shows monophenolase or cre-
solase, whereby it hydroxylates monophenols to o-diphenols. In the
same way as it oxidises o-aminophenols and aromatic o-diamines to
their corresponding o-quinoneimine and diimines, TYR can also
hydroxylate aromatic monoamines and convert them into o-amino-
phenols and o-quinoneimines. Using the measuring methods described
in this study, the hydroxylation of several aromatic monoamines with
different groups in their side chains (in C-1) was characterised.
Fig. 5 depicts how the oxidation of 3-amino-4-hydroxytoluene by
periodate in deficiency gives rise to an isosbestic point at λ=318 nm.
Fig. 5 inset A shows the spectrophotometric recordings at 318 nm,
obtained by measuring the appearance of oxidised adduct. Raising the
initial concentration of 3-aminotoluene (the aromatic monoamine
corresponding to 3-amino-4-hydroxytoluene) increases the reaction
rate, and when these initial rates are represented with respect to [S]0 and
fitted by non-linearregression to Eq. (1), Vmax and Km can be obtained for
this aromatic monoamine, as shown in Fig. 5 inset B (see Table 2).
The oxidation of 4-aminoanisole by tyrosinase in the presence of
MBTH gives an isosbestic point at λ=498 nm (Fig. 2), which permits
the molar absorptivity (ε) to be calculated at this point. Fig. 2 inset A
Table 2 shows the results obtained when the oxidation of aromatic
o-diamines was studied. The values of the catalytic constants of the
aromatic o-diamines are low, the 2,3-diamines having, as expected, a
lower kcat than the o-diamines substituted in 3,4. Furthermore, if the
molecule in C-1 has an electrowithdrawing group, the values of kcat
are lower than when an electrodonating group is present. The same
applies in the case of o-diphenols, although the catalytic constants are
greater than in the case of the corresponding aromatic o-diamines.
The values of Km are greater in the case of substrates with their
substituents in ortho position with respect to C-1 and, in general, fulfil
Eq. (3). Although the kcat values of the o-diphenols are much greater
than those of the aromatic o-diamines, the value of the binding
constant (k6) to the form Eox must be high, so that, according to
Eq. (3), the Km values must be in the same range as those of the
aromatic o-diamines. A possible explanation for this is that, in the case
of o-diphenols, π–π interactions between the aromatic ring and a
possible histidine of the active site are favoured.
Fig. 4 shows the recordings of the oxidation of 3,4-diaminobenzoic
acid by TYR and Fig. 4 inset shows the values of V0vs. the initial
concentration of 3,4-diaminobenzoic acid, [3,4-diaminobenzoic acid]0,
which enables Vmax and Km to be calculated, by means of Eq. (1).
A
B
2.8
2.4
2.0
1.6
1.2
0.8
0.4
0.0
0.3
0.2
0.1
0.0
(g)
1.2
0.8
0.4
0.0
260
255
(a)
0
10
20
30
40
50
60
0
1
2
3
4
[3-aminotoluene]
(mM)
t (s)
0
250
1.2
245
240
235
0.8
0.4
0.0
(a)
(g)
250
300
350
400
450
500
0.0
0.2
0.4
0.6
0.8
1.0
λ (nm)
[3,4-Diaminobenzoic acid] (mM)
0
Fig. 5. Spectrophotometric scans of the 3-amino-4-hydroxytoluene oxidation by
periodate in deficiency. The experimental conditions were 30 mM sodium phosphate
buffer (pH 7.0), 25 °C, [3-amino-4-hydroxytoluene]0 =0.6 mM and [IO4−]0 =60 μM.
The spectrophotometric scans were made every 3 min. Inset A. Spectrophotometric
recordings of the oxidation of 3-aminotoluene by TYR at a wavelength of 318 nm. The
experimental conditions were 30 mM sodium phosphate buffer (pH 7.0), 25 °C,
[E]0 =0.45 μM and the initial substrate concentrations (mM) were: (a) 0.25, (b) 0.75,
(c) 1.0, (d) 1.5, (e) 2.5, (f) 3 and (g) 3.5. Inset B. Representation of the initial steady-
state rates (V0) vs. [3-aminotoluene]0.
0.0
0.5
1.0
1.5
2.0
t (min)
Fig. 4. Oxymetric recordings of the oxidation of 3,4-diaminobenzoic acid by TYR. The
experimental conditions were 30 mM sodium phosphate buffer (pH 7.0), 25 °C,
[AH2]0 =0.2 mM, [E]0 =0.25 μM and the initial substrate concentrations (mM) were:
(a) 0.05, (b) 0.1, (c) 0.15, (d) 0.3, (e) 10.5, (f) 0.7 and (g) 0.9. Inset. Representation of
the initial steady-state rates (V0) vs. [3,4-diaminobenzoic acid]0.

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1814 (2011) 1974–1983
1979
Table 3
Values of the kinetic constants characterising the oxidation of aromatic monoamines and monophenols by TYR.
Aromatic monoamines
kcat (s−1
)
Km (mM)
Phenols
kcat (s−1
)
Km (mM)
Aniline
0.45 0.03
0.64 0.09
2.36 0.34
0.28 0.02
0.14 0.01
2.93 0.38
1.26 0.07
1.04 0.12
0.26 0.03
0.15 0.02
0.09 0.01
0.42 0.05
Phenol
10.81 1.51
0.046 0.002
0.64 0.03
1.70 0.07
7.37 0.40
7.51 0.65
16.12 1.53
5.47 0.21
46.87 2.06
184.20 6.10
8.12 1.44
0.61 0.04
2.62 0.18
2.35 0.32
0.22 0.03
1.48 0.18
0.62 0.09
0.38 0.05
5.40 0.60
2.53 0.26
0.08 0.01
0.27 0.03
2-aminobenzoic acid
3-aminobenzoic acid
4-aminobenzoic acid
2-aminotoluene
3-aminotoluene
4-aminotoluene
2-aminoanisole
3-aminoanisole
4-aminoanisole
4-aminophenylalanine
(0.13 0.01)×10−3
(0.33 0.01)×10−3
(5.04 0.32)×10−3
0.33 0.02
2-hydroxybenzoic acid
3-hydroxybenzoic acid
4-hydroxybenzoic acid
2-methylphenol
3-methylphenol
4-methylphenol
2-hydroxyanisole
3-hydroxyanisole
4-hydroxyanisole
4-hydroxyphenylalanine
0.46 0.04
1.01 0.03
0.09 0.01
0.12 0.01
1.24 0.21
0.01 0.01
depicts the oxidation of 4-aminoanisole by TYR, measuring the
appearance of oxidised adduct formed between the o-quinoneimine
and a powerful nucleophile like MBTH [15,29]. By representing V0vs.
[S]0 (Fig. 2 inset B), Vmax and Km can be calculated by non-linear
regression fitting with respect to Eq. (1) (Table 3).
As in the case of monophenols, the natural substrates of TYR,
aromatic monoamines, have very low catalytic constants compared
with o-aminophenols, although the constants are higher in aromatic
monoamines with electrodonating groups or with H in their side
chain on C-1 than in those with electrowithdrawing groups. When
aromatic monoamines substituted in 2, 3 or 4 position of the benzene
oxygen of the hydroxyl, giving (Eox −S)1; subsequently, the oxygen
from the OH attacks the copper and the hydrogen is transferred to
the histidine (Eox −S)2 (Step 7). The electrowithdrawing effect of the
CuB may facilitate the transfer of the proton from the amino group to
the peroxide group of the active site, since the base is already
protonated (Eox −S)3. (Step 8). Since the o-aminophenol is bound
diaxially to Eox, a concerted oxidation/reduction action gives rise to Em
and o-quinoneimine (Step 9), and the cycle starts again.
As regards the hydroxylation of the aromatic monoamines, as
occurs with monophenols, the system starts with the form Eox (Step
10) since the Em form is inactive towards monophenols (Steps 16
and 17) and aromatic monoamines, which is the reason for the
characteristic lag period of TYR [2,32,33]. The NH2 group cedes
the H+ to the peroxide group of the active site of Eox, while the
remaining NH binds to the CuB of the enzyme (Step 11). Now the
peroxide attacks the benzene ring, forming the complex (Em −A)4
(Step 12), which may undergo one of two possible reactions: (i) the
NH group is protonated to become NH2+ (Step 13) and the complex
(Em −S)5 is formed, while the binding of the oxygen to the copper of
(Em −S)5 is broken, producing the complex (Em −S)1 (Step 15).
This complex can yield (Em −S)0, releasing o-aminophenol or
initiates the diphenolase cycle or (ii) the substrate is released from
the enzyme by the OH group, forming the complex (Em −S)2 (Step
14), the rest of the cycle continuing as normal.
ring are compared, those with the amino group in 4 show a higher kcat
,
suggesting that the enzyme hydroxylates substrates substituted in
para position more readily than those substituted in meta and ortho
positions because of the lower steric hindrance [31] (Table 3).
The Michaelis constants (Km) follow the order orthoNmetaNpara
for both aromatic monoamines and phenols and, moreover, fulfil
Eq. (3). Note the very little difference that frequently exists between
the Michaelis constants of the aromatic monoamines and o-diamines
(Tables 2 and 3).
3.8. Proposed mechanisms for the oxidation of aromatic monoamines
and o-diamines and aminophenols by TYR
Note that, as with the monophenolase activity of TYR, which needs
an o-diphenol to act, in the case of aromatic monoamines the presence
of the o-aminophenol is necessary for the enzyme to act. This is
formed in Steps 1 and 15 of Scheme 1.
As regards the aromatic o-diamines, the structural mechanism
proposed (Scheme 3)isthesameasthatproposedfortheo-aminophenols
(Scheme 2) but, in this case, since two NH2 groups exist, the NH2 which
attacks first will be that with a lower chemical shift (δ)—which, with
electrodonating groups in C-1, will be the amino group in carbon 4 (δ4).
Schemes 2 and 3 represent the proposed structural mechanism
to explain the oxidation of aromatic monoamines and o-diamines
and o-aminophenols by TYR. Scheme 2 refers to the oxidation of
o-aminophenols (diphenolase activity) and aromatic monoamines
(monophenolase activity). Starting with the enzymatic form Em, its
binding to the o-aminophenol in Step 1 is seen to form the complex
(Em −S)0. Once bound to the substrate, a proton from the NH2 group
is transferred to a group of the enzyme, which acts as a base, and
then the nitrogen of the substrate binds to the CuB of the active site
of the enzyme, forming the complex (Em −S)1. The amino group of
the substrate is always the first to bind because it has a greater
nucleophilic power than the hydroxyl (Step 2). In Step 3, the
hydroxyl group transfers a proton to a histidine of the CuA of the
enzyme active site [7]. The o-aminophenol is thus bound diaxially to
the enzyme and the concerted oxidation/reduction generates the
quinoneimine and the deoxygenated form of the enzyme Ed (Step 4).
In Step 5, this form, Ed, binds to O2 to form Eox, while in Step 6,
another molecule of the substrate (o-aminophenol) binds to the
enzyme (Eox −S)0. Then, the nitrogen from the substrate binds to
CuB of the enzyme because it is a more potent nucleophile than the
4. Discussion on the kinetic constants of Tables 1–3
As a general commentary concerning Tables 1–3, the o-amino-
phenols and aromatic o-diamines are oxidised, while the aromatic
monoamines are hydroxylated with catalytic constants that are much
lower than those of the corresponding o-diphenols or monophenols.
In the case of o-aminophenols and o-diphenols (Table 1), the
difference between the molecules with the same structure is one
o − diphenols
order of magnitude (k
Nkcaamtinophenols). The similarity of the
cat
Km values indicates, according to Eq. (3), that the o-diphenols bind
Scheme 2. Structural mechanism to explain the oxidation of o-aminophenols and aromatic monoamines by TYR. Em, met-tyrosinase; (EmS)0, met-tyrosinase/o-aminophenol complex;
(Em–S)1, met-tyrosinase/o-aminophenol complex axially bound to a Cu atom; (Em–S)2, met-tyrosinase/o-aminophenol complex axially bound to a Cu atom with the proton transferred to
the base B; (Em–S)3, met-tyrosinase/o-aminophenol complex axially bound to the two Cu atoms with the proton of the OH group transferred to the histidine; Ed, deoxy-tyrosinase; Eox, oxy-
tyrosinase; (EoxS)0, oxy-tyrosinase/o-aminophenol complex; (Eox–S)1, oxy-tyrosinase/o-aminophenol complex axially bound to a Cu atom; (Eox–S)2, oxy-tyrosinase/o-aminophenol
complex axially bound to the two Cu atoms; (Eox–S)3, oxy-tyrosinase/o-aminophenol complex axially bound to the two Cu atoms with the proton transferred to the peroxide; (EoxA)0, oxy-
tyrosinase/aromatic monoamine complex; (EoxA)1, oxy-tyrosinase/aromatic monoamine complex axially bound to a Cu atom with the proton transferred to the peroxide; (Em–S)4, met-
tyrosinase/o-aminophenol complexaxial-equatoriallybound to thetwo Cuatoms; (Em–S)5, met-tyrosinase/o-aminophenol complex axial-equatorially bound to the twoCuatoms, binding
to the base BH through a hydrogen bond; (EmA)0, met-tyrosinase/aromatic monoamine complex; (EmA)1, met-tyrosinase/aromatic monoamine complex axially bound to a Cu atom.

1980
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1814 (2011) 1974–1983
better to the enzyme (higher k6) than the o-aminophenols, probably
because a π–π interaction with a possible histidine is favoured. Note
that the substituent in C-1 affects the values of kcat and Km—when the
substituent is a methyl group (electrodonating), the kcat for 3-
hydroxy-4-aminotoluene or 3-amino-4-hydroxytoluene are high
(slightly higher in the latter) and the Km values, in accordance with
R
R
Aromatic monoamine
hydroxylase cycle
k31
B
B
OH
N
OH
N
H2N
HN
Step 15
H
R
k-3
N
N
N
N
N
N
N
N
1
k-5
Cu
2+
Cu
2+
Cu
2+
Cu
2+
4
O
H
O
N
N
R
B
H
H
k54
(
Em-S)0
Step 1
(Em-S)1
NH
N
O
O
N
N
N
N
Step 2
k2 k-2
Cu
2+
Cu
H2N
OH
R
2+
N
H
H
Death
B
pathway
BH
(Em-S)5
R
O
N
HN
N
H
N
N
N
Cu
2+
Cu
k-5
N
N
N
N
N
5
O
H
2+
k53
k-5
3
Cu
2+
Cu
2+
N
O
H2N
N
H
Step 9
Em
k55
(Em-S)2
H2O
Step 16
k73
k32
k-3
Step 3
Step 14
Step 13
R
2
R
R
R
R
B
BH
BH
BH
NH
H2N
HN
NH
O
HN
O
HN
N
HN
O
N
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Cu
2+
Cu
2+
Cu
2+
Cu
Cu
2+
Cu
2+
Cu
2+
Cu
2+
O
N
O
H
2+
O
O
N
O
N
H
N
N
Step 8
H
H
H
(Em-S)3
(Em-S)4
(Eox-S)3
(
Em-A)0
k72
k33
Step 17
Step 12
H2O
Step 4
R
R
R
BH
BH
N
BH
BH
NH
N
N
N
HN
Cu
Cu
+
N
HN
O
HN
Aminophenol
oxidase
N
N
N
N
+
N
H
Cu
2+
Cu
N
N
N
N
Cu
2+
Cu
O
H
2+
N
N
N
N
O
O
N
N
2+
N
O
O
cycle
Cu
2+
Cu
2+
N
H
N
Ed
(Eox-A)1
O2
(Eox-S)2
Step 7
(
Em-A)1
Step 11
O2
Step 5
k51 k-5
k71
k-7
k-8
k8
1
1
R
R
R
k-6
k6
BH
BH
BH
BH
N
OH
N
HN
OH
N
HN
H2N
O
H
N
N
N
H
Cu
2+
Cu
2+
R
R
N
O
N
N
N
N
N
O
N
N
N
N
N
N
O
N
N
O
O
Cu
2+
Cu
2+
N
Cu
2+
Cu
2+
Cu
Cu
2+
O
2+
O
N
N
N
H2N
Eox
H2N
OH
(Eox-S)1
(Eox-S)0
(EoxA)0
Step 10
Step 6

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1814 (2011) 1974–1983
1981
R
R
B
k31
B
NH2
N
H2N
NH2
N
HN
H
N
N
N
N
Cu
2+
Cu
2+
N
N
N
N
k-3
Cu
2+
O
H
Cu
2+
1
N
O
N
H
(Em-S)0
Step 1
(
Em-S)1
Step 2
k-2 k2
R
R
B
H2N
NH2
BH
NH2
N
HN
N
N
N
N
S
Cu
2+
Cu
N
N
N
N
N
O
H
2+
Cu
2+
N
Cu
2+
O
H
N
Step 9
(Em-S)2
H2O
k-3
k32
2
Em
k73
R
Step 3
R
R
BH
BH
NH
NH
NH
HN
NH
HN
N
N
N
N
N
N
N
N
Cu
2+
Cu
2+
Cu
2+
Cu
2+
HN
NH
O
O
O
H
N
N
H
(Em-S)3
Step 4
(Eox-S)3
k33
k72
k-7
Step 8
2
H2O
R
BH
N
Aromatic diamine
oxidase cycle
BH
NH
N
N
Cu
Cu
+
NH
HN
N
N
+
H
N
N
N
O
O
Cu
2+
Cu
2+
N
N
N
(Eox-S)2
Ed
Step 7
k71
k-7
O2
O2
1
k-8
k8
Step 5
R
R
k-6
k6
BH
BH
BH
N
R
NH2
N
NH2
N
HN
H2N
O
N
N
N
H
Cu
2+
Cu
2+
N
O
N
N
N
N
N
N
N
N
O
O
O
N
Cu
2+
Cu
2+
Cu
2+
Cu
2+
H2N
NH2
O
N
N
S
(Eox-S)0
Eox
Step 6
(Eox-S)1

1982
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1814 (2011) 1974–1983
Eq. (3), increase (slightly more so in the case of 3-amino-4-
hydroxytoluene). When an electrowithdrawing group exists on C-1
(such as a carboxyl group), the kcat values fall (slightly more so for
3-amino-4-hydroxybenzoic acid), as do the Km values (Table 2). Note
that the kcat is highest when there is no substituent in C-1 (see
Table 2).
In the case of the aromatic o-diamines (Table 2), except 2,3-
diaminobenzoic acid and 2,3-dihydroxybenzoic acid, in which steric
hindrance phenomena dominate the process, the catalytic constants
In conclusion, we have demonstrated that mushroom TYR oxidises
aromatic monoamines and o-diamines and o-aminophenols. For this
oxidation process, we propose structural mechanisms that agree with
the obtained experimental results and which show a certain
parallelism to the mechanisms proposed for monophenolase and
diphenolase activities of TYR [1,36,37]. In addition, we have kinetically
characterised a wide number of substrates.
Acknowledgements
are two orders of magnitude lower than for their respective o-
o − diphenols
diphenols (k
≫kcdaiatmines). However, the Km values are in the
cat
This paper was partially supported by grants from: Ministerio de
Educación y Ciencia (Madrid, Spain) project BIO2009-12956, Funda-
ción Séneca (CARM, Murcia, Spain) projects 08856/PI/08 and 08595/
PI/08, andConsejería de Educación (CARM, Murcia, Spain) BIO-BMC
06/01-0004. JLMM and FGM hold two fellowships from Fundación
Caja Murcia (Murcia, Spain).
same range because the o-diphenols bind better to the enzyme than
the aromatic o-diamines. When the amino group is in 2,3 position, the
steric hindrance effect is greater than the electronic effect and the kcat
values fall, contrary to that which occurs with Km.
Such behaviour is similar in the case of aromatic monoamines
(Table 3). The values of the catalytic constants are two orders of
magnitude greater than for the monophenols. Note that the kcat values
of the substrates with the group in meta position with respect to C-1
are always greater than those with the group in ortho position, since
the steric hindrance is stronger than the electronic effect in ortho.
It has been demonstrated that TYR of N. crassa can oxidise several
types of o-aminophenols and aromatic amines [10]. The same authors
demonstrated the oxidation of these compounds using oxymetric
methods, revealing also that the catalytic constants of TYR towards
these compounds are three orders of magnitude lower than those of
their phenolic analogues.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.bbapap.2011.07.015.
References
[1] L. Fenoll, M.J. Peñalver, J.N. Rodriguez-Lopez, P.A. Garcia-Ruiz, F. Garcia-Canovas, J.
Tudela, Deuterium isotope effect on the oxidation of monophenols and o-
diphenols by tyrosinase, Biochem. J. 380 (2004) 643–650.
In a more recent work [11], mushroom TYR was demonstrated to
oxidise an o-aminophenol substituted in 2 and 3 of the benzene ring,
e.g. 3-hydroxy-2-aminobenzoic acid (3-hydroxyanthranilic acid, see
Scheme 1), in the same way as N. crassa TYR. Similarly, the authors
mention the influence of this compound on the monophenolase and
diphenolase activities of mushroom TYR. A different type of effect was
seen for o-aminophenol (3-hydroxykynurenine) on mushroom TYR,
when it acted as a substrate/activator [12].
As in the case of N. crassa TYR, mushroom TYR shows catalytic
constants that are two orders of magnitude lower for the oxidation of
2-aminobenzoic acid (0.0001 s−1) than for the oxidation of 2-hydro-
xybenzoic acid (0.046 s−1), the same being true for 4-aminoanisole
(1.24 s−1) compared with 4-hydroxyanisole (184.20 s−1) (Table 3).
As regards the proposed reaction mechanism, regardless of the R
group or the position that it occupies, the NH2 group will always attack
before the OH group in the case of o-aminophenols, while in the case of
aromatic o-diamines, the amino group with the lowest chemical shift
will attack first. In these mechanisms (Schemes 1 and 2), Steps 7 and
8 are the slowest since they involve a double migration of a proton.
Electrodonating groups such as methyl favour to the para position,
and, as a consequence, the oxygen of the hydroxyl group in this
position is more nucleophilic, while in electrowithdrawing groups
(e.g. the carboxyl group), the para position is not favoured and so the
oxygen of the hydroxyl group is less nucleophilic. Lastly, deprotona-
tion of the aromatic o-diamines is much more difficult, which greatly
slows down the oxidation process (Table 2).
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Scheme 3. Structural mechanism to explain theoxidation of aromatic o-diamines by TYR. Em, met-tyrosinase; (EmS)0, met-tyrosinase/aromatico-diamine complex; (Em-S)1, met-tyrosinase/
aromatic o-diamine complex axially bound to a Cu atom with the base forming a hydrogen bridge; (Em-S)2, met-tyrosinase/aromatic o-diamine complex axially bound to a Cu atoms with
protonated base; (Em-S)3, met-tyrosinase/aromatic o-diamine complex axially bound to the two Cu atoms; Ed, deoxy-tyrosinase; Eox, oxy-tyrosinase; (EoxS)0, oxy-tyrosinase/aromatic o-
diamine complex; (Eox-S)1, oxy-tyrosinase/aromatic o-diamine complex axially bound to a Cu atom; (Eox-S)2, oxy-tyrosinase/aromatic o-diamine complex axially bound to thetwo Cu atoms
and with protonated base; (Eox-S)3, oxy-tyrosinase/aromatic o-diamine complex axially bound to the two Cu atoms with the proton transferred to the peroxide.

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