Tyrosinase inactivation in its action on dopa

Article abstract of DOI:10.1016/j.bbapap.2010.02.015

Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by its physiological substrate l-dopa. Under aerobic conditions, this inactivation occurs through a process of suicide inactivation involving the fo

Full text of DOI:10.1016/j.bbapap.2010.02.015

Biochimica et Biophysica Acta 1804 (2010) 1467–1475
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbapap
Tyrosinase inactivation in its action on dopa
a
a
a
b
c
a
J.L. Muñoz-Muñoz , J.R. Acosta-Motos , F. Garcia-Molina , R. Varon , P.A. Garcia-Ruíz , J. Tudela ,
a,
a
F. Garcia-Cánovas ⁎, J.N. Rodríguez-López
a
GENZ: Grupo de Investigación Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biologia, Universidad de Murcia, E-30100, Espinardo, Murcia, Spain
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
QCBA: Grupo de Química de Carbohidratos y Biotecnología de Alimentos, Departamento de Química Orgánica. Facultad de Química, Universidad de Murcia, E-30100, Espinardo,
b
c
Murcia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by
its physiological substrate L-dopa. Under aerobic conditions, this inactivation occurs through a process of
suicide inactivation involving the form oxy-tyrosinase. Under anaerobic conditions, both the met- and deoxy-
tyrosinase forms undergo irreversible inactivation. Suicide inactivation in aerobic conditions is slower than
the irreversible inactivation under anaerobic conditions. The enzyme has less affinity for the isomer D-dopa
than for L-dopa but the velocity of inactivation is the same. We propose mechanisms to explain these
processes.
Received 10 November 2009
Received in revised form 1 February 2010
Accepted 23 February 2010
Available online 6 March 2010
Keywords:
o-Diphenol
Stereospecificity
Inactivation
Suicide
© 2010 Elsevier B.V. All rights reserved.
Tyrosinase
1
. Introduction
Tyrosinase or polyphenol oxidase (EC 1.14.18.1, o-diphenol:O
the results have been unequal and even contradictory [20–22]. The use
of a suitable measuring method to characterize monophenolase and
2
diphenolase activities, using the chromogenic nucleophile 3-methyl-2-
benzothiazolinone hydrazone (MBTH) [23,24], demonstrated that
tyrosinase shows stereospecificity in its affinity towards its substrate
and has a higher Michaelis constant for the isomers D (D-tyrosine
and D-dopa) than for the L (L-tyrosine and L-dopa). However, it
demonstrates the same velocity of catalysis for both isomers (L- and
D-dopa and L- and D-tyrosine), confirming that the velocity is related
with the charge density of the oxygen atoms of the hydroxyl groups
of the benzene ring, which, in turn, is related with the chemical shifts
oxidoreductase) is a copper protein that uses molecular oxygen to
catalyse the hydroxylation of monophenols to o-diphenols (mono-
phenolase activity) and the oxidation of o-diphenols to o-quinones
(
diphenolase activity) [1,2]. Tyrosinase from a variety of sources
undergoes an inactivation process when it reacts with its substrate
under aerobic conditions [3–5]. The study of enzymatic inactivation
by suicide substrates or mechanism-based inhibitors has grown in
importance because of possible pharmacological applications [6,7].
Furthermore, these compounds may be useful for studying enzymatic
mechanisms and for designing new pharmacological drugs [8].
The suicide inactivation of enzymes has been studied in several
works since Waley in 1980 [9]. Our own group has made several kinetic
studies of the suicide inactivation mechanism from a theoretical point
of view [10,11] and using mushroom tyrosinase [12,13], frog skin
tyrosinase [14,15] and peroxidase from different sources [16,17]. We
have also studied the suicide inactivation of an enzyme that can be
measured from coupled reactions [18] and published a review on the
experimental designs for a kinetic study of suicide inactivation [19].
Several monophenols and o-diphenols are chiral tyrosinase sub-
strates (L- and D-enantiomers). Some papers have reported the stereo-
selective action of several tyrosinases on these enantiomers, although
3 4
δ and δ . The same conclusion was reached using the immobilised
enzyme [25].
The suicide inactivation of tyrosinase from different sources
has been studied and many attempts have been made to find a
mechanism that explains the phenomenon [26,27]. Recently, our
group published a possible explication for this process [28]. In our
mechanism (Scheme ISM), the Eox form binds one molecule of
substrate (o-diphenol) (Step 6). In this case, the base (B), possibly a
histidine, is already protonated and cannot help the substrate bind
to the copper. The proton of the hydroxyl group of C-4 must be
transferred to the peroxide group, which acts as a base [29,30]. This
would be the slow step of the turnover, controlled by k71 (Step 7) [30].
The fact that the peroxide acts as a base and is involved in the proton
transfer from the hydroxyl group of C-4 and C-3 is important. The
protonated peroxide draws the copper atoms in the Eox form closer
together, since the distance in the unprotonated form, 3.6 Å, is too
great for the molecule of substrate to bind diaxially to them. However,
⁎
Corresponding author. Tel.: +34 868 884764; fax: +34 868 883963.
E-mail address: canovasf@um.es (F. Garcia-Cánovas).
1570-9639/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2010.02.015

1468
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1804 (2010) 1467–1475
D(L or D)
D(L or D)
E
for saturating substrate.
this would be possible in the protonated Eox form. With the substrate
axially bound to one copper atom, there are two possibilities in Step 8:
λ
λ
E
maximum value of λ
ox(max)
ox
D(L or D)
apparent constant of irreversible inhibition of E
d
by L- or
E
d
(
a) the oxygen of the hydroxyl group of C-3 may attack the other
D-dopa.
D(L or D)
d
⁎
copper atom, which is drawn closer following protonation of the
peroxide group, transferring the proton to the histidine, binding to the
substrate diaxially and co-planar with the copper atoms. In this way,
the concerted redox reaction in Step 9 can occur, releasing o-quinone
λ
E⁎
apparent constant of irreversible inhibition of E
D-dopa.
maximum value of λ d
maximum value of λE⁎d
d
by L- or
D(L or D)
k
k
k
iD(L or D)
E
for saturating substrate.
for saturating substrate.
⁎
D(L or D)
i
D(L or D)
⁎
and E
m
. (b) The hydroxyl group of C-3 may donate a proton to the
T
rate constant of the transition E
d
to E
d
.
−
i
protonated peroxide, remaining as C–O in Step 10. The co-planarity
of the substrate ring, the oxygen of C-4 and the copper atom favor
the simultaneous oxidation of the substrate to o-quinone and the
reduction of one Cu
copper (0) and hydrogen peroxide are released, and the enzyme is
inactivated as a consequence (Step 11) (Scheme ISM).
For its part, under anaerobic conditions it has been demonstrated
that a reductant like ascorbic acid inactivates the enzyme in its met-
tyrosinase and deoxy-tyrosinase forms [31]. The capacity to inacti-
vate the enzyme under both aerobic and anaerobic conditions
may have an industrial application for protecting juices and other
preparations from enzymatic browning: hence the interest in its
study and characterisation.
r
D
partition ratio for the diphenolase activity, r
D
=k 2
7
k
72
=
D
D
k
cat/λ
E
ox(max)
.
O
2
m
K
K
k
Michaelis constant of oxygen.
Michaelis constant of tyrosinase for L- or D-dopa.
catalytic constant of the diphenolase activity for L- or
D-dopa.
dissociation constant of the complex E
L- or D-dopa.
2
+
D(L or D)
m
atom to copper (0) through the ring, while
D(L or D)
cat
D(L or D)
K
E⁎
d
⁎D, where D is
d
D(L or D)
E
K
dissociation constant of the complex E
D-dopa.
d
D, where D is L- or
d
2. Experimental
The aims of this study were: to analyse the inactivation of tyrosinase
by its physiological substrate L-dopa under aerobic conditions (suicide
inactivation of oxy-tyrosinase form) and under anaerobic conditions
2.1. Materials
Mushroom tyrosinase or polyphenol oxidase (o-diphenol: O
2
(
irreversible inactivation of met-tyrosinase and deoxy-tyrosinase)
oxidoreductase, EC 1.14.18.1) (8300 U/mg) and β-NADH were supplied
by Sigma (Madrid, Spain). The enzyme was purified as previously
described in [32]. Protein concentration was determined by the Lowry
method [33]. The substrates used L- and D-dopa were purchased from
Sigma (Madrid, Spain). All other chemicals were of analytical grade.
Stock solutions of the diphenolic substrates were prepared in 0.15 mM
phosphoric acid to prevent autooxidation. Milli-Q system (Millipore
corp.) ultrapure water was used throughout.
and to study the effect of the stereospecificity, using the isomers
L- and D-dopa.
1
.1. Notation
For clarity and for the sake of brevity, the following additional
notations will be used in the text.
1
.1.1. Species and concentrations
2.2. Methods
[
E
E]
0
initial concentration of tyrosinase
active enzyme
instantaneous concentration of E
a
instantaneous concentration of met-tyrosinase
initial concentration of met-tyrosinase
instantaneous concentration of deoxy-tyrosinase
2.2.1. Spectrophotometric assays
a
These assays were carried out with a Perkin-Elmer Lambda-35
spectrophotometer, on line interfaced to a PC-computer, where the
kinetic data were recorded, stored and later analysed. The product of the
enzyme reaction o-dopaquinone is unstable and evolves towards
dopachrome, which is not stable at long assay times [34,35]. For this
reason, the reaction was followed by measuring the disappearance of
NADH at 340 nm, with ε=6230 M cm , which reduces o-dopaqui-
none to L-dopa [36]. The inactivation kinetics was studied in 30 mM
sodium phosphate buffer (pH 6.0), since o-dopaquinone evolves rapidly
to dopachrome at pH 7.0, while at lower pH the reaction slows down
and o-dopaquinone is reduced by NADH [34].
[
[
[
[
[
E
[
[
E
E
E
E
E
a
]
]
]
]
m
m
0
d
d
⁎
]
0
initial concentration of deoxy-tyrosinase
−
1
−1
⁎
d
deoxy-tyrosinase after of E
d
to E
d
transition
⁎
⁎
E
E
d
]
]
instantaneous concentration of E
d
⁎
⁎
d
0
initial concentration of E
d
E
[
D
i
inactive enzyme
E
i
]
instantaneous concentration of E
L- or D-o-diphenol
i
[D]
0
initial concentration of D
2.2.2. Kinetic data analysis
Q
o-quinone product of the enzymatic reaction
instantaneous concentration of Q
o-quinone concentration at t→∞
nicotinamin adenin dinucleotide
initial concentration of NADH
[
[
Q]
Q]
2.2.2.1. Diphenolase activity. The experimental data of time-based
assays for the disappearance of NADH (Fig. 1) follow the Eq. (1):
∞
NADH
D
ox
−
λE
t
[
[
[
[
NADH]
0
½NADHꢀ = ½NADHꢀ + ½NADHꢀ e
ð1Þ
f
∞
NADH] instantaneous concentration of NADH
NADH]
NADH]
NADH-value at t→∞
[NADH]
f
,[NADH]
∞
=[Q]
∞ E
and λD
can be obtained by non-linear
f
ox
∞
NADH consumed at the end of the reaction, t→∞, i.e.
regression [37]. There is always a slow spontaneous oxidation of o-
diphenol and NADH (Fig. 1), which should be computer corrected in
further NADH vs. time plots (in general this treatment is applied to all
the kinetic recordings). The experimental recordings obtained in the
∞ 0 f
[NADH] =[NADH] –[NADH]
1
V
.1.2. Kinetic parameters
following steps, therefore, fit Eq. (1), from which the corresponding
D(NADH)
0
D
initial rate of tyrosinase acting on D in the presence of
inactivation parameters, [NADH]
∞
=[Q]
∞
and λ
E
ox, can be determined.
NADH.
D(Q)
D(Q)
D(NADH).
V
λ
0
initial rate of tyrosinase acting on D, V
apparent constant of suicide inactivation of tyrosinase in
the presence of L- or D-dopa.
0
=V
0
2.2.3. Generation of Eox and E
To kinetically characterize the inactivation of the E
tyrosinase, E was generated from the native enzyme, adding
d
D(L or D)
E
d
form of
ox
d

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1804 (2010) 1467–1475
1469
3
. Results
The active site of tyrosinase is found in three forms in the catalytic
2+
2+
+
+
cycle: met-tyrosinase (Cu Cu ), deoxy-tyrosinase (Cu Cu ) and
oxy-tyrosinase (Cu2 Cu2+O
+
2−
2
) [39]. These enzymatic forms may
undergo a substrate-induced irreversible inactivation. Under aerobic
conditions, the enzyme undergoes a process of suicide inactivation,
localised in oxy-tyrosinase, while under anaerobic conditions the met-
tyrosinase and deoxy-tyrosinase forms undergo irreversible inactiva-
tion. These processes are studied below with o-diphenols (L- and
D-dopa), under both aerobic and anaerobic conditions.
3
3
.1. Tyrosinase action on o-diphenols
.1.1. Inactivation of tyrosinase in its action on o-diphenols under aerobic
conditions: suicide inactivation
The kinetic mechanism proposed to explain the suicide inactiva-
tion of tyrosinase acting on o-diphenols is based on the structural
mechanism proposed in Scheme ISM and can be outlined as follows in
Scheme I.
When [D] , [O ] NN[E] and [Q] bb [D] , [O ] , it is possible a
0 2 0 0 ∞ 0 2 0
derivation of the analytical expression which establishes the accumu-
lation of the product (o-quinone) with time, as is detailed in reference
[28].
Fig. 1. Experimental recordings of the disappearance of NADH in the suicide inactivation
of tyrosinase by L-dopa and curves corrected after applying the kinetic data analysis.
The variation of [Q] with time is given by Eq. (2) [28].
Conditions were 30 mM sodium phosphate buffer (pH 6.0), 0.2 mM NADH, 0.26 mM O
mML-dopa and enzyme concentrations were: (a) 0.7 nM and (c) 0.85 nM. Curves
b) and (d) are the corrected recordings from (a) and (c), respectively, subtracting the
2
,
ꢀ
ꢁ
D
ox
1
(
½Qꢀ∞ 1 ꢁ eꢁλE
t
½
½
Qꢀ
=
ð2Þ
ð3Þ
L-dopa and NADH spontaneous oxidation.
∞
When t→∞, [Q]=[Q] and, according to [28]:
2 2 m
micromolar concentrations (2 μM) of H O , so that, the E form passed
2k72
Qꢀ∞
=
½Eꢀ0
2
to Eox. Then, nitrogen was bubbled through the solution transforming all
the Eox to E (Eox ⇄ E + O2) [38,39].
i
k
7
d
d
The velocity of the suicide inactivation is regulated by the apparent
inactivation constant, λ ox, whose expression is given by Eq. (4). Taking
E
D
2
.2.4. Generation of E
The inactivation of E
from the native enzyme in two ways: (a) adding catalase 2 μM, so
that Eox ⇄ Em + H2O2; the catalase acts on the H displacing the
above equilibrium, 2H +catalase→O +2H O, so that all Eox is
transformed into E at the end of the reaction. (b) Adding 2 μM of
, so that all the enzyme passes to Eox and then adding 2 μM
catalase [38,39].
m
m
was characterized by first generating it
into account the saturation of tyrosinase by oxygen ([O
according to [28]:
2
]
→∞), thus
0
2 2
O
D
2
O
2
2
2
D
λ
EoxðmaxÞ
½Dꢀ0
λ
=
ð4Þ
Eox
D
m
K
+ ½Dꢀ0
m
2 2
H O
The partition ratio, r , between the catalytic and suicide inactiva-
D
tion pathways is:
2
.2.5. Evaluation of enzymatic species E
preparation of tyrosinase
It is known that in an enzymatic preparation of tyrosinase from any
source is found in three forms, E , E and Eox [39]. Several authors have
proposed spectrophotometric methods for evaluating these forms
39–41]. Here, we propose a kinetic method based on the fact that the
inactivation of these forms by 2-mercaptoethanol [42] occurs over a
m d
, E and Eox in an enzymatic
D
k
k
½Qꢀ
½NADHꢀ∞
2½Eꢀ0
72
cat
∞
rD
=
=
=
=
ð5Þ
i
D
k
λ
2½Eꢀ
0
7
2
EoxðmaxÞ
m
d
7
where k 2 is the substrate binding constant to the copper atom
i
[
through the C-3 hydroxyl (axial) and k
7
2
is the rate constant of
+
transfer of a H to the protonated peroxide (see Scheme ISM).
−
1
−5 −1
wide time range: inactivation constants of 0.014 s , 4×10
s
and
, respectively. Under aerobic conditions
at oxygen concentrations of 0.26 mM, practically the only forms ex-
Therefore, from Eq. (1–5), we can obtain the kinetic constants that
−
5
−1
D
1
×10
s
for Eox, E
m
and E
d
characterize the kinetic behaviour of a suicide substrate: r
D
, λ
E
ox(max)
,
D
m
and k Dc at. Note that if dopachrome or oxygen consumption is being
measured, Eqs. (1–5) are totally applicable and it is only necessary to
take into account the stoichiometric relations: 2Q:1 DC:1O and that
K
ox
m
m i i
isting are Eox and E [32]. Note that the difference between k and k
is three orders of magnitude, a difference that can be used to evaluate
these enzymatic forms. The experimental method was described in
2
Q and DC are originated in the medium by the action of the enzyme,
while the oxygen concentration falls.
[31].
1
3
2
.2.6. C-NMR assays
13C-NMR spectra of several substrates were obtained in a Varian
Unity spectrometer at 300 MHz. The spectra were obtained at pH=6.0
3.1.1.1. Experimental study of suicide inactivation. The study of the
suicide inactivation of tyrosinase was developed in three steps and the
o-diphenols used in this work were L-dopa and D-dopa.
Step 1. Fig. 1 shows the spectrophotometric recordings of the
disappearance of NADH, curves (a) and (c), and their corrections by
fitting to Eq. (1), after subtracting the NADH and o-diphenol sponta-
neous oxidation, Fig. 1, curves (b) and (d). These corrected recordings
are analysed as described in the Kinetic Data Analysis section. By means
2
by using H
2
O as solvent for the substrates. Chemical displacement (δ)
values were measured relative to those for tetramethylsilane (δ=0).
The maximum line breadth accepted in the 13C-NMR spectra was
0
.06 Hz. Therefore, the maximum accepted error for each peak of the
spectrum was ± 0.03 p.p.m.

1470
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1804 (2010) 1467–1475
Scheme I. Kinetic mechanism proposed to explain the suicide inactivation of tyrosinase acting on dopa enantiomers (see text and Scheme ISM for further details).
⁎
of these preliminary studies, the concentration of enzyme is optimised so
that [Q] bb[O ,[D]
Step 2. Variation in enzyme concentration. The [E]
while the substrate concentration is kept constant. The results
obtained are shown in Fig. 2A and B. The concentration of the
substrate in these experiments does not change because the o-quinone
The forms E
d
and E
d
show different affinities for o-diphenols and
D(L)
D(L)
∞
]
2 0
0
.
hence different activities (Km,E =0.52 mM and Km,E ⁎d = 0.99 mM in the
d
D(D)
D(D)
d
0
-value is varied
case of L-dopa, and Km,E =3.95 mM and Km,E⁎ =5.36 mM in the case of
d
⁎
D-dopa), so that the transition of E
the initial rate with L-dopa, as shown in Fig. 4. Analysis of the data of
Fig. 4 according to Eq. (6)
d
and E
d
can be followed by measuring
is reduced by NADH. However, the concentrations of O
2
and NADH do
is very small [43], which permits
an oxygen consumption of more than 10%. The NADH must be in
excess. Note how the values of [NADH] are directly proportional to
does not vary with [E] (Fig. 2A Inset and B Inset).
Fig. 2A Inset and B Inset show the dependence of the values ob-
tained for the product at the end of the reaction, [NADH] vs. [E] , from
whose slopes and, according to Eq. (5), r can be determined (Table 1).
O
2
change. In the case of tyrosinase, K
m
A
A0
½E ꢀ
−kTt
a
=
= α + βe
ð6Þ
½E ꢀ
d 0
∞
D(L or D)
[
E]
0
, while λ
E
0
ox
gives the apparent constant of the transition in the conditions de-
−
2
−1
T
scribed in Fig. 4 (k =(3.9± 0.4)×10 min ) and where A is the
∞
0
instantaneous enzymatic activity.
D
⁎
3
.1.2.1.1. E
the form E
the form E
d
inactivation by L-dopa and D-dopa. From the form Eox
,
Step 3. Variation in substrate concentration. The experimental
recordings for the disappearance of NADH during the action of tyros-
inase on L-dopa and D-dopa obtained by varying the concentration of
d
was generated by passing a nitrogen stream. After 90 min,
is transformed into E
conditions. In this section, we shall study the reaction of E
⁎
d
d
, which is stable under anaerobic
⁎
d
with L-dopa
substrate are shown in Fig. 3A and B, respectively. Fitting by non-linear
D(L or D)
and D-dopa.
regression to Eq. (1) gives the apparent inactivation constant λ
E
.
ox
D(L or D)
At t=0, a given concentration of L-dopa was added as indicated in
Fig. 5A. Aliquots were taken and, when the activity was followed with
time, an irreversible inactivation, which can be described kinetically
by Scheme IV and whose structural interpretation is depicted in
Scheme IISM, was observed.
The hyperbolic dependence of λ
E
vs. [D]
0
is shown in Fig. 3A Inset
ox
and B Inset. Fitting these data by non-linear regression according
D(L or D)
D(L or D)
to Eq. (4) gives λ
These values of λ
E
(max) and K
m
for each of the isomers (Table 1).
ox
D(L or D)
D(LorD)
E
ox
(max) and r (Table 1) permit us to obtain kcat
D
(Table 1).
The monoexponential behaviour observed in Fig. 5A (gradual loss
of activity) can be fitted to Eq. (7):
3
.1.2. Tyrosinase inactivation under anaerobic conditions: irreversible
inactivation
The three enzymatic forms of the turnover of tyrosinase are in
equilibrium, as shown by the following scheme (Scheme II).
DðLÞ
h
i
h
i
−λ
0e
t
⁎
d
⁎
d
E
⁎
E
=
E
d
ð7Þ
In the presence of oxygen (0.26 mM), the principal forms are E
m
and Eox, whose concentration in an enzymatic preparation was deter-
mined by measuring the inactivation of the native enzyme by
irreversible inhibition with 2-mercaptoethanol, as described in [31].
D(L)
where D(L) corresponds to L-dopa. The values of λE ⁎d vary with the
concentration of substrate used in the assays, according to Eq. (8):
⁎
iDðLÞ
3
.1.2.1. Inactivation of deoxy-tyrosinase by o-diphenols.
The form
k
½Dꢀ0
+ ½Dꢀ0
DðLÞ
Ed*
λ
=
:
ð8Þ
deoxy-tyrosinase was generated as described in Materials and
Methods sections [31]. Maintaining anaerobic conditions, E
slowly towards another enzymatic form that we denominate E
DðLÞ
K
Ed
*
d
evolves
⁎
d
[44],
The data analysis by non-linear regression shown in Fig. 5A Inset
as described in the Scheme III.
*
DðLÞ
according to Eq. (8) gives the values of ki
and K
shown in Table 2.
Through analogy with that observed in hemocyanin, it has been
DðLÞ
d
E*
⁎
proposed that the copper atoms in the enzymatic form E
separated than in E [44,45].
d
are more
When the same experiments were carried out with D-dopa, Fig. 5B
and B Inset, the data shown in Table 2 were obtained.
d

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1804 (2010) 1467–1475
1471
Fig. 2. Corrected recordings of the disappearance of NADH in the suicide inactivation of
tyrosinase by o-diphenol for different enzyme concentrations. (A) L-dopa, (B) D-dopa.
Conditions were: 30 mM sodium phosphate buffer (pH 6.0), 0.2 mM NADH, 0.26 mM
Fig. 3. Corrected recordings of the disappearance of NADH in the suicide inactivation of
tyrosinase by different substrate concentrations. (A) L-dopa, (B) D-dopa. Conditions
were 30 mM sodium phosphate buffer (pH 6.0), 0.26 mM
concentrations of NADH and tyrosinase were: 0.2 mM and 0.6 nM for L-dopa and
.2 mM and 0.2 nM for D-dopa, respectively. (A) L-dopa, the substrate concentrations
2
O and the initial
O
2 0
. (A). 1 mML-dopa and [E] (nM): (a) 0.09, (b) 0.19, (c) 0.28, (d) 0.38, (e) 0.48,
(
(
(
(
f) 0.57, (g) 0.67, (h) 0.70 and (i) 0.85. Inset. Representation of the values of [NADH]
∞
0
D(L)
) and λ
E
(■) vs. enzyme concentration for L-dopa. (B). 4 mM D-dopa and [E]
0
(nM):
D(L)
•
ox
were (mM): (a) 0.25, (b) 0.6, (c) 0.9, (d) 1.5, (e) 1.8, (f) 2.4 and (g) 3. Inset. Values of λEox
a) 0.05, (b) 0.10, (c) 0.15, (d) 0.20, (e) 0.22, (f) 0.28, (g) 0.30, (h) 0.35, (i) 0.38 and
j) 0.41. Inset. Representation of the values of [NADH]
for different L-dopa concentrations. (B) D-dopa, the substrate concentrations were
D(D)
∞
( ) and λ
•
E
ox
(■) vs. enzyme
D(D)
(mM): (a) 2, (b) 3, (c) 4, (d) 6, (e) 8, (f) 10, (g) 11 and (h) 12. Inset. Values of λEox for
concentration for D-dopa.
different D-dopa concentrations.
Table 1
Kinetic constants which characterize the suicide inactivation of tyrosinase by the dopa isomers and values of the chemical shifts of these compounds obtained by 13C-NMR for the C-3
and C-4 carbons at pH 6.0.
DE ox(max) ×102 (min−1
)
r=k cD at/λEox(max)
D
k
D
)
K
D
(mM)
K
O
2
m
(μM)
δ
3
(p.p.m.)
o-Diphenol
λ
4
δ (p.p.m.)
L-dopa
D-dopa
13.3± 0.6
12.9± 0.9
46133± 2306
44895± 2573
6156± 1152
5910± 1014
0.55± 0.08
3.98± 0.57
4.46± 0.89
4.28± 0.86
146.92
146.92
146.06
146.06

1472
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1804 (2010) 1467–1475
Scheme II. Equilibrium between Eox, E
m
and E
d
forms of tyrosinase.
⁎
Scheme III. Kinetic mechanism of the transition of E
d
to E
d
.
3
d d
.1.2.1.2. E inactivation by L-dopa and D-dopa. The form E
generated from Eox under the nitrogen stream was made to react
rapidly with different concentrations of L-dopa. The results (Fig. 6A)
can be explained kinetically by Scheme V and, structurally, by a
Scheme similar to Scheme IISM, although the distance between
the coppers in E
monoexponential behaviour was observed, which can be fitted to
Eq. (1SM), which is similar to Eq. (7), but for E , while the inactivation
constant values better fit an equation similar to Eq. (8), but for E
d
may be smaller, as occurs in [45]. Once again,
d
d
[
Eq. (2SM)].
D(L)
E
A fit, by non-linear regression, of the values of λ
to Eq. (2SM),
d
D(L)
Fig. 6A Inset, gives kiD(L) and K
E
(Table 2). When the same exper-
d
iments are carried out with D-dopa, Fig. 6B and B Inset, the values
D(D)
shown in Table 2 are obtained for kiD(D) and K
E
.
d
⁎
⁎
d
Fig. 5. Inactivation of the E
obtained by allowing the form E
d
form of tyrosinase by o-diphenols. The form E was
d
obtained as described in Fig. 4 to evolve under
⁎
anaerobic conditions for 90 min to obtain E
d
. The o-diphenol was added at t=0 and
aliquots were taken at different times to measure the residual activity with 2.5 mML-dopa
λ=475 nm). The values obtained were fitted to Eq. (1SM) and the apparent inactivation
constant was obtained at each substrate concentration. (A) L-dopa. The experimental
conditions were: 30 mM sodium phosphate buffer (pH 6.0), 25 °C, [E] =0.1 μM (see
Materials and Methods sections), [H =2 μM and L-dopa (μM): (a) 5 (●), (b) 10 (○),
c) 15 (■), (d) 20 (□), (e) 25 (▲), (f) 30 (△) and (g) 35 (▼). Inset. Representation of λE⁎d
vs. [L-dopa] . (B) D-dopa. The experimental conditions were the same as in (A) but D-dopa
μM): (a) 5 (●), (b) 10 (○), (c) 15 (■), (d) 20 (□), (e) 25 (▲), (f) 30 (△) and (g) 35 (▼).
(
0
2 2 0
O ]
D(L)
(
0
(
D(L)
0
Inset. Representation of λE ⁎d vs. [D-dopa] .
3.1.2.1.3. E
m
inactivation by L-dopa and D-dopa.
m
E was generated
from the native enzyme, as described in Materials and Methods
sections [31]. Under anaerobic conditions, this form is stable, but in
the presence of o-diphenols, it is inactivated, as described in kinetic
form in Scheme VI and structurally in Scheme IIISM.
⁎
Fig. 4. Kinetic study of the transition of E
d
to E
d
. The enzyme form E
=0.1 μM (see Materials
=2 μM. Aliquots were taken at different times and
d
was generated by
passing a stream of nitrogen through a preparation of Eox, [E]
and Methods sections) and [H
enzymatic activity was detected with 1 mML-dopa (λ=475 nm). The values obtained
were fitted by non-linear regression to Eq. (6).
0
2
O
2
]
0
E
m
was incubated with L-dopa and aliquots were taken at different
times and revealed with L-dopa, the results being similar to those

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1804 (2010) 1467–1475
1473
Scheme IV. Kinetic mechanism of E⁎
inactivation by o-diphenols.
d
obtained with the E
Scheme VI, the enzyme E
Scheme VI and Scheme IIISM). The values of the constants are shown
in Table 2.
d
. According to the mechanism described in
m
rapidly passes to E and is inactivated
d
(
4
. Discussion
This study reveals that the physiological substrate of tyrosinase,
L-dopa, and its iosomer, D-dopa, act as tyrosinase inactivators both
in aerobic and anaerobic conditions. Under aerobic conditions, L- or
D-dopa act as a suicide substrate of the enzyme basically through the
enzymatic species Eox and, under anaerobic conditions, they act as
irreversible inactivators of the enzyme in the enzymatic species E
m
,
⁎
E
d
and E
d
.
4
.1. Aerobic conditions: suicide inactivation
Kinetic characterisation of an enzyme acting on a suicide substrate
is determined from r (partition ratio), λ (apparent constant of suicide
inactivation) and K (Michaelis constant). In the action of tyrosinase
m
on L- or D-dopa these parameters are determined according to Eqs. (4)
and (5).
From an analysis of the data shown in Fig. 2A Inset and B Inset and,
according to Eq. (5), r
D
can be determined, giving practically the same
value for the two isomers (Table 1). From Eq. (5) and taking into
D(D)
D(L)
account that kcat ≈kcat [23], it is deduced that the inactivation
constant is approximately equal in both isomers (Table 1).
When the effect of substrate concentration is studied by analysing the
results shown in Fig. 3A and B, the corresponding apparent inactivation
D(L or D)
constants (λ
(
E
) are obtained, and their analysis according to Eq. (4)
ox
D
D
Fig. 3A Inset and B Inset) enables us to obtain λ
E
and K
m
(Table 1).
ox(max)
D
Note that the values of λ
E
ox(max)
are practically the same for L and D.
D(L)
D(D)
However, K
m
bK
m
, thus, the enzyme shows stereospecificity in its
D
substrate binding. These results agree with the values of kcat obtained
D(D)
D(L)
from a study of the steady state, kcat ≅kcat [23], since these values and
D
those of λ
E
are related with the strength of the nucleophilic attack of
ox(max)
the oxygens of the hydroxyls of C-3 and C-4, which, in turn, depends on δ
and δ (Table 1).
3
4
The inactivation mechanism described here agree with the
experimental result obtained by other authors [27], regarding to the
loss of a 50% of copper during the oxidation of catechol, and with
others [26] regarding to the impossibility of inactive enzyme
d d
Fig. 6. Inactivation of the E form of tyrosinase by o-diphenols. The form E was
obtained as is described in Fig. 4 and immediately incubated with o-diphenol, taking
aliquots at different times to measure the residual activity with 2.5 mML-dopa
2
+
reactivation after Cu
caddie” protein [29].
addition, perhaps indicating the need for a
(
λ=475 nm). The values obtained were fitted to Eq. (2SM) and the apparent
“
inactivation constant was obtained at each concentration of o-diphenol. (A) L-dopa.
The experimental conditions were: 30 mM sodium phosphate buffer (pH 6.0), 25 °C,
[
(
E]
0 2 2 0
=0.1 μM (see Materials and Methods sections), [H O ] =2 μM and L-dopa (μM):
Table 2
a) 5 (●), (b) 10 (○), (c) 15 ( ), (d) 20 (□), (e) 25 (▲) and (f) 30 (△). Inset.
▪
⁎
D(L)
Kinetic constants which characterize the inactivation of E
isomers.
d
, E
m
and E
d
by the dopa
Representation of λ d
E
vs. [L-dopa] . (B) D-dopa. The experimental conditions were the
0
same as in (A) but D-dopa (μM): (a) 5 (●), (b) 10 (○), (c) 15 ( ), (d) 20 (□), (e) 25 (▲),
(f) 30 (△) and (g) 40 (▼). Inset. Representation of λ
the same procedure was followed as with E
▪
D(L)
D(a)
iD ×103 (min−1
⁎D × 103 (min−1
)
E
d
vs. [D-dopa]
0
. In the case of E
m
,
Enzymatic form Substrate
K
E
(μM)
k
)
k
i
d
.
⁎
E
E
E
d
L-dopa
D-dopa
L-dopa
D-dopa
L-dopa
D-dopa
19.24± 2.12
28.88± 2.93
10.77± 1.12 0.37± 0.01
25.34± 2.24 0.37± 0.03
10.77± 1.12 0.37± 0.01
25.34± 2.24 0.37± 0.03
–
–
0.25± 0.01
0.26± 0.02
d
–
–
–
–
m
(
a) In KD
with E , the data obtained correspond to E
into E (Scheme VI).
E
, E corresponds to the enzymatic forms E⁎
, because of the rapid transformation of E
m
d d
or E . When the reaction is begun
m
d
d
d
Scheme V. Kinetic mechanism of E inactivation by o-diphenols.

1474
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1804 (2010) 1467–1475
m
Scheme VI. Kinetic mechanism of E inactivation by o-diphenols.
On the other hand, the physiological implication of the suicide
tion constant) in suicide inactivation (Table 1) is lower than the
⁎
inactivation of tyrosinase by its physiological substrate (L-dopa) is not
still clear, although it would result in a stop of action of the enzyme,
since the products of its reaction are highly cytotoxic for the cell [46,47].
inactivation constants of Em, Ed and E
d
under anaerobic conditions
(Table 2). This observation may have industrial applications since a
nitrogen stream passed quickly through a solution of a fruit extract
would prevent the enzyme from acting, the presence of o-diphenols
provoking its inactivation. In this way, browning would be avoided.
Besides, irreversible inactivation has the advantage over suicide
inactivation because there is no turnover, so the product would be
stoichiometric with the enzyme.
4
.2. Inactivation of deoxy-tyrosinase by o-diphenols
The inactivation of E⁎
, E and E was studied in anaerobic
d
d
m
conditions.
⁎
4
.2.1. E
d
inactivation by L-dopa and D-dopa
4.5. Conclusions
Analysis of the data of Fig. 5A and B according to Eq. (7) provides
⁎
the apparent inactivation constant of E
d
and its analysis according to
From a kinetic study of the inactivation of tyrosinase in the
⁎
⁎
⁎
Eq. (8) provides the values of k
i
and KE ⁎d . The values of k
approximately equal (Table 2), which agrees with the values of δ
i
and k
i
are
different forms involved in the catalytic cycle, Eox (aerobic condi-
D(L)
D(D)
⁎
3
and
tions), Em, Ed and E
d
(anaerobic conditions), it can be deduced that
D(L)
D(D)
δ
4
of the isomers (Table 1). However, the values of KE⁎ and KE ⁎d are
the enzyme shows stereospecificity in the binding of isomers but not
in catalysis or inactivation, since both processes are related with the
nucleophilic power of the oxygens of the hydroxyls in C-3 and C-4 of
the benzene ring. o-Diphenols inactivate the enzyme both under
aerobic conditions (suicide inactivation through the form Eox) and
d
D(L)
D(D)
not the same (Table 2), (KE ⁎d bKE⁎ ), which indicates that this
d
enzymatic form has more affinity for the L isomers.
4
.2.2. E
d
inactivation by L-dopa and D-dopa
by L- or D-dopa, analysis of
When we study the inactivation of E
d
under anaerobic conditions (irreversible inactivation through the
⁎
the data in Fig. 6A and B according to Eq. (1SM) provides the apparent
inactivation constant, and its analysis according to Eq. (2SM) gives the
forms E
m
, E
d
and E
d
).
values k
i
and K d
E
. The values of kiD(L) and kiD(D) are, approximately, equal
Acknowledgements
(
Table 2), since these values are directly related with the values of δ
3
D
E
D(L)
and δ
4
of both isomers, which are equal (Table 1). However, K d
E
bK
This paper was partially supported by grants from the Ministerio de
Educación y Ciencia (Madrid, Spain) Project BIO2006-15363 and, from
the Fundación Séneca (CARM, Murcia, Spain) Projects 08856/PI/08
and 08595/PI/08, from the Consejería de Educación (CARM, Murcia,
Spain) BIO-BMC 06/01-0004 and from FISCAM PI-2007/53 from the
Consejería de Salud y Bienestar Social de la Junta de Comunidades de
Castilla La Mancha. JLMM has a fellowship from the Ministerio de
Educación y Ciencia (Madrid, Spain) Reference AP2005-4721. FGM has
a fellowship from Fundación Caja Murcia (Murcia, Spain).
d
(
D)
,
which demonstrates the stereospecificity of the substrate binding.
⁎
Furthermore, the values of kiD(L) and kiD(D) are greater than those of k
and k
enzyme in the form E
the form E
i
D(L)
⁎
i
D(D)
, respectively (Table 2), indicating that the structure of the
⁎
d
transforms the substrate less efficiently than
+
⁎
d
, perhaps because the Cu atoms in E
d
are more separated
[
44]. As regards the affinity of these forms for the substrates, the E
d
D(L)
E
D(D)
E
⁎
form shows greater affinity (lower K
and K
) than E
d
(Table 2).
d
d
4
m
.3. E inactivation by L- or D-dopa
Appendix A. Supplementary data
When the inactivation of E
similar to those obtained in the study of the inactivation of E
m
by L- or D-dopa is studied, the results are
⁎
d
and E
d
by
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbapap.2010.02.015.
the same o-diphenols. According to the kinetic mechanism described in
Scheme VI and the structural mechanism described in Scheme IIISM, the
enzyme E
m d
rapidly passes to E and is inactivated (Scheme VI and
References
Scheme IIISM). The values of the constants are shown in Table 2.
[
1] A. Sanchez-Ferrer, J.N. Rodriguez-Lopez, F. Garcia-Canovas, F. Garcia-Carmona,
Tyrosinase: a comprehensive review of its mechanism, Biochim. Biophys. Acta
4
.4. Oxygen protects against the inactivation of tyrosinase
1247 (1995) 1–11.
[
2] S. Halaouli, M. Asther, J.C. Sigoillot, M. Hamdi, A. Lomascolo, Fungal tyrosinases:
new prospects in molecular characteristics, bioengineering and biotechnological
applications, J. Appl. Microbiol. 100 (2006) 219–232.
From the results obtained in this study and, especially, taking into
account the inactivation processes, suicide inactivation (under
aerobic conditions) occurs through Eox and irreversible inactivation
[3] L.L. Ingraham, J. Corse, B. Makower, Enzymatic browning of fruits. 3. Kinetics
of the reaction inactivation of polyphenol oxidase, J. Am. Chem. Soc. 74 (1952)
⁎
(
under anaerobic conditions) occurs through E
, E
m d
and E
d
, both with
2
623–2626.
o-diphenols. It is clear that the oxygen protects the enzyme from
[
4] Y. Tomita, A. Hariu, C. Mizuno, M. Seiji, Inactivation of tyrosinase by dopa, J. Invest.
D
inactivation, since the value of λ
E
(maximum apparent inactiva-
Dermatol. 75 (1980) 379–382.
ox(max)

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1804 (2010) 1467–1475
1475
[
5] K. Lerch, Neurospora tyrosinase: structural, spectroscopic and catalytic properties,
Mol. Cell. Biochem. 52 (1983) 125–138.
6] J. Zhong, W.C. Groutas, Recent developments in the design of mechanism-based
and alternate substrate inhibitors of serine proteases, Curr. Top. Med. Chem. 4
[27] C. Dietler, K. Lerch, Reaction inactivation of tyrosinase, in: T.E. King, H.S. Mason, M.
Morrison (Eds.), Oxidases and Related Redox Systems, Pergamon Press, NY, 1982,
pp. 305–317.
[28] J.L. Muñoz-Muñoz, F. Garcia-Molina, P.A. Garcia-Ruiz, M. Molina-Alarcon, J.
Tudela, F. Garcia-Canovas, J.N. Rodriguez-Lopez, Phenolic substrates and suicide
inactivation of tyrosinase: kinetics and mechanism, Biochem. J. 416 (2008)
413–440.
[29] Y. Matoba, T. Kumagai, A. Yamamoto, H. Yoshitsu, M. Sugiyama, Crystallographic
evidence that the dinuclear copper center of tyrosinase is flexible during catalysis,
J. Biol. Chem. 281 (2006) 8981–8990.
[
(2004) 1203–1216.
[
7] F. Ghanbari, K. Rowland-Yeo, J.C. Bloomer, S.E. Clarke, M.S. Lennard, G. Tucker, A.
Rostami-Hodjegan, A critical evaluation of the experimental design of studies of
mechanism based enzyme inhibition, with implications for in vitro–in vivo
extrapolation, Curr. Drug Metab. 7 (2006) 315–334.
[
8] R.B. Silverman, Mechanism-based enzyme inactivators, Meth. Enzymol. 249
(
1995) 240–283.
[30] L.G. 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.
[31] J.L. Munoz-Munoz, F. Garcia-Molina, P.A. Garcia-Ruiz, R. Varon, J. Tudela, F. Garcia-
Canovas, J.N. Rodriguez-Lopez, Stereospecific inactivation of tyrosinase by L- and
D-ascorbic acid, Biochim. Biophys. Acta 1794 (2009) 244–253.
[32] J.N. Rodriguez-Lopez, L.G. Fenoll, P.A. Garcia-Ruiz, R. Varon, J. Tudela, R.N. Thorneley,
F. Garcia-Canovas, Stopped-flow and steady-state study of the diphenolase activity
of mushroom tyrosinase, Biochemistry 39 (2000) 10497–10506.
[33] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the
Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.
[34] F. Garcia-Carmona, F. Garcia-Canovas, J.L. Iborra, J.A. Lozano, Kinetic study of the
pathway of melanization between L-dopa and dopachrome, Biochim. Biophys.
Acta 717 (1982) 124–131.
[35] F. Garcia-Canovas, F. Garcia-Carmona, J. Vera-Sanchez, J.L. Iborra-Pastor, J.A.
Lozano-Teruel, The role of pH in the melanin biosynthesis pathway, J. Biol. Chem.
257 (1982) 8738–8744.
[
9] S.G. Waley, Kinetics of suicide substrates, Biochem. J. 185 (1980) 771–773.
[
10] J. Tudela, F. Garcia Canovas, R. Varon, F. Garcia Carmona, J. Galvez, J.A. Lozano,
Transient-phase kinetics of enzyme inactivation induced by suicide substrates,
Biochim. Biophys. Acta 912 (1987) 408–416.
11] M.A. Moruno-Davila, C. Garrido-del Solo, M. Garcia-Moreno, B.H. Havsteen, F.
Garcia-Sevilla, F. Garcia-Canovas, R. Varon, Kinetic analysis of enzyme systems
with suicide substrate in the presence of a reversible competitive inhibitor, tested
by simulator progress curves, Int. J. Biochem. Cell Biol. 33 (2001) 181–191.
12] F. Garcia-Canovas, J. Tudela, C. Martinez-Madrid, R. Varon, F. Garcia-Carmona, J.A.
Lozano, Kinetic study on the suicide inactivation of tyrosinase induced by
catechol, Biochim. Biophys. Acta 912 (1987) 417–423.
13] J. Tudela, F. Garcia-Canovas, R. Varon, M. Jimenez, F. Garcia-Carmona, J.A. Lozano,
Kinetic characterization of dopamine as a suicide substrate of tyrosinase, J. Enzyme
Inhib. 2 (1987) 47–56.
14] J. Tudela, F. Garcia-Canovas, R. Varon, M. Jimenez, F. Garcia-Carmona, J.A. Lozano,
Kinetic study in the transient phase of the suicide inactivation of frog epidermis
tyrosinase, Biophys. Chem. 30 (1988) 303–310.
[
[
[
[
[
[
[
[36] F. Garcia-Molina, J.L. Muñoz, R. Varon, J.N. Rodriguez-Lopez, F. Garcia-Canovas, J.
Tudela, A review on spectrophotometric methods for measuring the mono-
phenolase and diphenolase activities of tyrosinase, J. Agric. Food Chem. 55 (2007)
9739–9749.
15] M. Garcia-Moreno, R. Varon, A. Sanchez-Gracia, J. Tudela, F. Garcia-Canovas, The
effect of pH on the suicide inactivation of frog epidermis tyrosinase, Biochim.
Biophys. Acta 1205 (1994) 282–288.
16] M.B. Arnao, M. Acosta, J.A. del Rio, R. Varon, F. Garcia-Canovas, A kinetic-study on
the suicide inactivation of peroxidase by hydrogen-peroxide, Biochim. Biophys.
Acta 1041 (1990) 43–47.
17] A.N. Hiner, J. Hernandez-Ruiz, M.B. Arnao, F. Garcia-Canovas, M. Acosta, A
comparative study of the purity, enzyme activity, and inactivation by hydrogen
peroxide of commercially available horseradish peroxidase isoenzymes A and C,
Biotechnol. Bioeng. 50 (1996) 655–662.
[37] Jandel scientific. Sigma Plot 9.0 for Windows™; Jandel scientific: Core Madera
2006.
[38] R.L. Jolley Jr., L.H. Evans, N. Makino, H.S. Mason, Oxytyrosinase, J. Biol. Chem. 249
(1974) 335–345.
[39] M. Jackman, M. Huber, A. Hajnal, K. Lerch, Stabilization of the oxy form of
tyrosinase by a single conservative amino acid substitution, Biochem. J. 282
(1992) 915–918.
[
18] J. Escribano, J. Tudela, F. Garcia-Carmona, F. Garcia-Canovas, A kinetic-study of
the suicide inactivation of an enzyme measured through coupling reactions: appli-
cation to the suicide inactivation of tyrosinase, Biochem. J. 262 (1989) 597–603.
19] F. Garcia-Canovas, J. Tudela, R. Varon, A.M. Vazquez, Experimental methods for
kinetic study of suicide substrates, J. Enzyme Inhib. 3 (1989) 81–90.
20] A.J. Winder, H. Harris, New assays for the tyrosine hydroxilase and dopa oxidase
activities of tyrosinase, Eur. J. Biochem. 198 (1991) 317–326.
21] D.E. Wilcox, A.G. Porras, Y.T. Hwang, K. Lerch, M.E. Winkler, E.I. Solomon,
Substrate analog binding to the coupled binuclear copper active site in tyrosinase,
J. Am. Chem. Soc. 107 (1985) 4015–4027.
[40] M. Beltramini, K. Lerch, Fluorescence properties of Neurospora tyrosinase,
Biochem. J. 205 (1982) 173–180.
[41] J. Deinum, K. Lerch, B. Reinhammar, An EPR study of Neurospora tyrosinase, FEBS
Lett. 69 (1976) 161–164.
[42] R. Aasa, J. Deinum, K. Lerch, B. Reinhammar, The reaction of mercaptans with
tyrosinases and hemocyanins, Biochim. Biophys. Acta 535 (1978) 287–298.
[43] L.G. Fenoll, J.N. Rodriguez-Lopez, F. Garcia-Molina, F. Garcia-Canovas, J. Tudela,
Michaelis constants of mushroom tyrosinase with respect to oxygen in the
presence of monophenols and diphenols, Int. J. Biochem. Cell Biol. 34 (2002)
332–336.
[
[
[
[
[
[
[
22] V. Kahn, S.H. Pomerantz, Monophenolase activity of avocado polyphenol oxidase,
[44] J.L. Munoz-Munoz, F. Garcia-Molina, P.A. Garcia-Ruiz, R. Varon, J. Tudela, F. Garcia-
Canovas, J.N. Rodríguez-Lopez, Some kinetic properties of deoxytyrosinase, J. Mol.
Cat. B: Enzymatic 62 (2010) 173–182.
Phytochemistry 19 (1980) 379–385.
23] J.C. Espin, P.A. Garcia-Ruiz, J. Tudela, F. Garcia-Canovas, Study of the stereospec-
ificity in mushroom tyrosinase, Biochem. J. 331 (1998) 547–551.
24] J.C. Espin, P.A. Garcia-Ruiz, J. Tudela, F. Garcia-Canovas, Study of stereospecificity in
pear and strawberry polyphenol oxidase, J. Agric. Food Chem. 46 (1998) 2469–2473.
25] M.E. Marin-Zamora, F. Rojas-Melgarejo, F. Garcia-Canovas, P.A. Garcia-Ruiz,
Stereospecificity of mushroom tyrosinase immobilized on a chiral and a nonchiral
support, J. Agric. Food Chem. 55 (2007) 4569–4575.
[45] M. Metz, E.I. Solomon, Dioxygen binding to deoxyhemocyanin: electronic
2
structure and mechanism of the spin-forbidden two-electron reduction of O ,
J. Am. Chem. Soc. 123 (2001) 4938–4950.
[46] K. Urabe, P. Aroca, K. Tsukamoto, D. Mascagna, A. Palumbo, G. Prota, V. Hearing,
The inherent toxicity of melanin precursors: a revision, Biochim. Biophys. Acta
1221 (1994) 272–278.
[
26] E.J. Land, C.A. Ramsden, P.A. Riley, The mechanism of suicide-inactivation of
[47] J.L. Munoz-Munoz, F. Garcia-Molina, R. Varon, J. Tudela, F. Garcia-Canovas, J.N.
Rodriguez-Lopez, Generation of hydrogen peroxide in the melanin biosynthesis
pathway, Biochim. Biophys. Acta 1794 (2009) 1017–1029.
tyrosinase: a substrate structure investigation, Tohoku J. Exp. Med. 212 (2007)
341–348.