Indirect inactivation of tyrosinase in its action on 4-tert-butylphenol

Article abstract of DOI:10.3109/14756366.2013.782298

Under anaerobic conditions, the o-diphenol 4-tert-butylcatechol (TBC) irreversibly inactivates met and deoxytyrosinase enzymatic forms of tyrosinase. However, the monophenol 4-tert-butylphenol (TBF) protects the enzyme from this inactivation. Under aerobic conditions, the enzyme suffers suicide inactivation when it acts on TBC. We suggest that TBF does not directly cause the suicide inactivation of the enzyme in the hydroxylase activity, but that the o-diphenol, which is necessary for the system to reach the steady state, is responsible for the process. Therefore, monophenols do not induce the suicide inactivation of tyrosinase in its hydroxylase activity, and there is a great difference between the monophenols that give rise to unstable o-quinones such as L-tyrosine, which rapidly accumulate L-dopa in the medium and those like TBF, after oxidation, give rise to a very stable o-quinone.

Full text of DOI:10.3109/14756366.2013.782298

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–9
2013 Informa UK Ltd. DOI: 10.3109/14756366.2013.782298
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ORIGINAL ARTICLE
Indirect inactivation of tyrosinase in its action on 4-tert-butylphenol
1
1
1
2
Jose Luis Mu n˜ oz-Mu n˜ oz , Mar ´ı a del Mar Garc ´ı a-Molina , Francisco Garc ´ı a-Molina , Ram o´ n Varon , Pedro Antonio
3
1
1
Garc ´ı a-Ruiz , Jose Neptuno Rodr ´ı guez-L o´ pez , and Francisco Garc ´ı a-C a´ novas
1
GENZ: Grupo de Investigaci o´ n Enzimolog ´ı a, Departamento de Bioqu ´ı mica y Biolog ´ı a Molecular-A, Facultad de Biolog ´ı a, Universidad de Murcia,
2
Espinardo, Murcia, Spain, Departamento de Qu ´ı mica-F ´ı sica, Escuela de Ingenieros Industriales de Albacete, Universidad de Castilla la Mancha,
3
Albacete, Spain, and QCPAI: Grupo de Qu ´ı mica de Carbohidratos, Pol ´ı meros y Aditivos Industriales, Departamento de Qu ´ı mica Org a´ nica, Facultad
de Qu ´ı mica, Universidad de Murcia, Espinardo, Murcia, Spain
Abstract
Keywords
Under anaerobic conditions, the o-diphenol 4-tert-butylcatechol (TBC) irreversibly inactivates
met and deoxytyrosinase enzymatic forms of tyrosinase. However, the monophenol 4-tert-
butylphenol (TBF) protects the enzyme from this inactivation. Under aerobic conditions, the
enzyme suffers suicide inactivation when it acts on TBC. We suggest that TBF does not directly
cause the suicide inactivation of the enzyme in the hydroxylase activity, but that the
o-diphenol, which is necessary for the system to reach the steady state, is responsible for
the process. Therefore, monophenols do not induce the suicide inactivation of tyrosinase in its
hydroxylase activity, and there is a great difference between the monophenols that give rise to
unstable o-quinones such as L-tyrosine, which rapidly accumulate L-dopa in the medium and
those like TBF, after oxidation, give rise to a very stable o-quinone.
Irreversible inhibition, suicide inactivation,
TBC, TBF, tyrosinase
History
Received 14 December 2012
Revised 21 January 2013
Accepted 28 February 2013
Published online 8 April 2013
Introduction
Then, through non-enzymatic reactions, the o-dopaquinone
evolves towards dopachrome.
b) The distal phase begins with the chemical decarboxylation of
Tyrosinase (EC 1.14.18.1) is
a copper-containing mono-
(
oxygenase widely distributed throughout the phylogenetic scale.
Tyrosinase catalyses two types of reactions on phenolic substrates,
in which molecular oxygen intervenes: (a) the hydroxylation of
monophenols to o-diphenols (monophenolase activity) and (b) the
oxidation of o-diphenols to o-quinones (diphenolase activity),
dopachrome towards 5,6-dihydroxyindole (DHI) or with an
enzymatic tautomerization of the dopachrome towards
1,6
,6-dihydroxyindole-2-carboxylic acid (DHICA) . These
5
compounds then polymerize giving rise to melanins.
1
Due to the wide specificity of tyrosinase, several types of
substrate can be considered according to the stability of the
corresponding quinone.
which, in turn, are polymerized to brown, red or black pigments .
Tyrosinase has two copper atoms in its active site, which may
2þ
2þ
be in three coordination states or forms: (i) Cu –Cu ,
2þ
2þ
2ꢀ
S
A
: Substrates yielding stable o-quinone. The clearest
example is 4-tert-butylcatechol (TBC), whose o-quinone
o-TBQ) is very stable. In this case, the o-quinone can be
met-tyrosinase, (ii) Cu –O –Cu with a peroxide group in its
1þ
2
active site, oxy-tyrosinase or (iii) Cu –Cu , deoxy-tyrosinase
1þ
2,3
.
Melanogenesis is the principal enzymatic pathway responsible
(
7
4
for browning in fruits and vegetables , and it also causes
measured and V0 can be calculated accurately . The diphenolase
8
activity can be determined, but not the monophenolase activity .
pigmentation of the hair, eyes and skin in mammals and other
,5
1
S : Substrates yielding a stable coupling product.
B
animals . This pathway can be divided into a proximal and distal
phase as follows:
Substrates which produce a very unstable o-quinone, but that
evolve to a stable product through a first-order reaction. An
example is 3,4-dihydroxymandelic acid (DOMA), whose
(a) The proximal phase begins with the hydroxylation of a
monophenol (L-tyrosine) to its corresponding o-diphenol
9
o-quinone evolves to 3,4-dihydroxybenzaldehyde (DOBA) .
(L-dopa) and the oxidation of this o-diphenol to its corres-
ponding o-quinone (o-dopaquinone). These two reactions
correspond to those described as monophenolase
6
and diphenolase activities of tyrosinase, respectively .
Other examples of type SB substrate are L-dopa, dopamine,
L-a-methylnoradrenaline, isoproterenol, epinine and L-dopa
methyl ester, whose o-quinone evolves to aminochrome .
10
SC: Substrates yielding a stable nucleophile-adduct. The
oxidation of this type of substrate gives rise to an unstable
o-quinone which suffers from the attack of a potent nucleophilic
reagent (N) and produces a chromophoric adduct (NQ) with a
clear stoichiometry. Among the nucleophilic reagents used are
Address for correspondence: Francisco Garc ´ı a-C a´ novas, GENZ: Grupo
de Investigaci o´ n Enzimolog ´ı a, Departamento de Bioqu ´ı mica y Biolog ´ı a
Molecular-A, Facultad de Biolog ´ı a, Universidad de Murcia, Campus de
Excelencia Internacional ‘‘Mare Nostrum’’, E-30100, Espinardo,
Murcia, Spain. Tel: +34 868 884764. Fax: +34 868 883963. E-mail:
canovasf@um.es
1
0
11–13
10
L-proline (Pro) , MBTH
and L-cysteine (Cys) . The
accumulation of the chromophoric product permits us to charac-
terize the monophenolase and diphenolase activities (L-tyrosine,

2
J. L. Mu n˜ oz-Mu n˜ oz et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
tyramine, 4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic (maximum apparent inactivation constant), r (a partition ratio
acid, L-dopa, dopamine, 3,4-dihydroxyphenylacetic acid, between the catalytic and the inactivation pathways), kcat
1
,4-dihydroxyphenylpropionic acid) .
SD: Substrates measured by means of ascorbic acid. strate)
The substrates give rise to a very unstable o-quinone that evolves
with no clear stoichiometry, even after MBTH attack. These and o-diphenols has been the subject of several studies, and
0
S
3
(catalytic constant) and K (Michaelis constant for the sub-
m
2
1,25,29–31
.
The kinetic mechanism for tyrosinase acting on monophenols
3
6–39
substrates can only be characterized through a reduction by various structural mechanisms have been proposed
ascorbic acid (AH ), either directly or spectrophotometrically substrates which give highly unstable o-quinones, such as
, e.g.
2
2
8,33,40
following the disappearance of ascorbic acid or using chrono- L-tyrosine, L-dopa or dopamine
metric methods that measure the time necessary for the ascorbic that o-diphenol accumulation in the medium through chemical
7
acid to be consumed due to the reaction with o-quinone . In this reactions is responsible for suicide inactivation when the enzyme
. These studies showed
case, measurement by reference to the reduction of the o-quinone acts on monophenols.
by ascorbic acid makes it possible to characterize the diphenolase
Here, we study the action of tyrosinase in aerobic and
anaerobic conditions on monophenols and o-diphenols which,
7
,8
but not the monophenolase activity
A large number of studies have been made of the action of after oxidation, give rise to very stable o-quinones, as in the case
.
tyrosinase on different types of monophenols such as highly of TBF and TBC. In the case of TBC, the enzyme is inactivated
8,10,15
1
0,14
unstable o-quinones
and slightly unstable o-quinones
.
As indicated above, tyrosinase is the main enzyme involved in inactivation) conditions. However, TBF protects the enzyme in
both in aerobic (suicide inactivation) and anaerobic (irreversible
melanization, but is also responsible for the loss of quality of fruit both conditions because the o-quinone is stable and cannot
4
and vegetables during post-harvest handling and processing . regenerate o-diphenol in the medium to inactivate the tyrosinase.
1
6
In addition, tyrosinase may cause disorders in pigmentation . But when catalytic concentrations of TBC are added to the
In insects, the enzyme is associated with three processes: cuticle reaction medium to follow the monophenolase activity, suicide
schlerotization, defensive encapsulation and melanization of inactivation occurs due to the action of the enzyme on the
1
7
foreign organisms, and wound healing . Therefore, inhibitors added o-diphenol. We try to demonstrate experimentally and
of this enzyme, which can be isolated from natural sources or be confirm by computer simulations that tyrosinase does not suffer
synthesized chemically, have been sought due to the many possible suicide inactivation when it acts on monophenols, but does so
1
8–20
applications
. Moreover, the enzyme may be inactivated when when acting on the o-diphenol that accumulates in the medium
it acts on a series of substrates, which act as suicide substrates; see and which is necessary for the steady state to be reached.
2
the work by Mu n˜ oz-Mu n˜ oz for a revision. The study of reversible The stability of o-quinones, then, plays a fundamental role in
1
and irreversible inhibitors and of suicide substrates is an interesting melanogenesis during the formation of melanins and the suicide
option for controlling the activity of this enzyme.
inactivation of the enzyme.
Both 4-tert-butylphenol (TBF) and 4-tert-butylcatechol (TBC)
have been described as depigmenting agents since they act as
Notation
2
2
tyrosinase inhibitors . The same compounds have been described
as apoptosis inducers in human melanocytes even without the
For clarity and for the sake of brevity, we will use the following
notations in the text.
2
3,24
tyrosinase activity of human melanocytes
compounds can act as alternative tyrosinase substrates, following
. These phenolic
a pathway other than the melanin biosynthesis pathway. At the Species and concentrations
same time, the o-diphenol TBC can act as a suicide substrate for
E, Tyrosinase; ½Eꢁ, instantaneous concentration of E; ½Eꢁ , initial
2
1,25
0
this enzyme
.
concentration of E; Ea, active enzyme; ½Eaꢁ, instantaneous
Tyrosinase undergoes an inactivation process when it reacts
with its o-diphenolic substrates, a phenomenon that has long been
known in the case of enzymes from a variety of natural sources,
concentration of Ea; Em, met-tyrosinase ½Emꢁ, instantaneous
concentration of met-tyrosinase; ½Emꢁ , initial concentration of
0
R
d
R
met-tyrosinase; E , Deoxy-tyrosinase relaxed form; ½E ꢁ, instant-
2
1
d
including fungi, plant and animals . The study of enzymatic
inactivation by suicide substrates or mechanism-based inhibitors
is of growing importance because of possible pharmacological
R
d 0
aneous concentration of deoxy-tyrosinase relaxed form; ½E ꢁ ,
T
initial concentration of deoxy-tyrosinase relaxed form; E , deoxy-
d
T
T
tyrosinase tense form after the transition of Ed to E ; ½E ꢁ,
2
6,27
d
d
applications
based enzyme inactivators may be useful for studying enzymatic
. In addition, suicide substrates and mechanism-
T
d
T
d
0
instantaneous concentration of E ; ½E ꢁ , initial concentration of
T
E ; Ei, inactive enzyme; ½Eiꢁ, instantaneous concentration of Ei;
2
7
d
TBC, 4-tert-butylcatechol; [TBC], instantaneous concentration of
mechanisms and designing new drugs . Given that tyrosinase
participates in different physiological processes, such as fruit and
1
vegetable browning and pigmentation in animals , the suicide
inactivation process of this enzyme is of even more interest.
Our group has made several kinetic studies of the suicide
TBC; [TBC] , initial concentration of TBC; D, o-diphenol (TBC);
0
½
Dꢁ, instantaneous concentration of D; ½Dꢁ , initial concentration
0
of D; Q, o-quinone product of the enzymatic reaction; ½Qꢁ,
instantaneous concentration of Q; ½Qꢁ , o-quinone concentration
2
1,25
1
inactivation of tyrosinase using mushroom tyrosinase
, frog
and peroxidases from different
. We have also studied the suicide inactivation of an
produced at the end of the reaction, t ! 1; TBF, 4-tert-
butylphenol; M, monophenol (TBF); ½Mꢁ, instantaneous concen-
2
8
epidermis tyrosinase
sources
2
9,30
tration of monophenol; ½Mꢁ , initial concentration of monophenol;
3
1
0
enzyme that can be measured from coupled reactions and have
published an experimental study, which used several experimental
3
designs to kinetically study suicide inactivation .
In the mechanism proposed by our research group, the
bifurcation of the catalytic and suicide inactivation pathways is
considered to occur through the Eox form
have also proposed that the suicide inactivation occurs through
O2, molecular oxygen; ½O2ꢁ , initial concentration of oxygen;
0
½
O2ꢁ, instantaneous concentration of oxygen; ½O2ꢁ , oxygen-
2
f
value at t ! 1; ½O2ꢁ , oxygen consumed at t ! 1,
1
i.e. ½O2ꢁ1 ¼ ½O2ꢁ ꢀ ½O2ꢁ .
0
f
2
1,25,33
. Other groups
Kinetic parameters
3
4,35
DðQÞ
DðQÞ
the same form, Eox
.
V
The main aim of kinetic studies with suicide substrates is initial rate of tyrosinase acting on D, measuring O2; ꢀ , apparent
, initial rate of tyrosinase acting on D, measuring Q; V
M
,
0
0
Eox
their kinetic characterization through the parameters: ꢀmax constant of suicide inactivation of tyrosinase in the

DOI: 10.3109/14756366.2013.782298
Indirect inactivation of tyrosinase
3
M
M
Eox
presence of monophenol; ꢀ
DðMÞ
, maximum value of ꢀ for electrode with a 12.5 mm Teflon membrane. The sample was
continuously stirred during the experiment and maintained at
EoxðmaxÞ
, apparent constant of irreversible
saturating substrate; ꢀEd
R
ꢂ
25 C. The zero oxygen level for the calibration and experi-
inhibition of ER by D in the presence of the monophenol ments was obtained by bubbling oxygen-free nitrogen through
d
4
2
DðMÞ
, apparent constant of irreversible inhibition of ET by
the sample for 10 min . TBF was studied by means of this
method, adding the amount of TBC necessary to reach the steady
state at t ! 0.
M; ꢀ
T
E
d
d
Q
D
D in the presence of the monophenol M; r , partition ratio for the
O2
diphenolase activity measuring Q; rD , partition ratio for the
O2
diphenolase activity measuring O2; rM , number of total turnovers
Kinetic data analysis of monophenolase and diphenolase
(in both cycles, hydroxylase and oxidase) made by one molecule activities
of enzyme acting on M before its suicide inactivation, obtained by
O2
MðOÞ
the oxidase cycle made by one molecule of enzyme acting on
The experimental recordings of oxygen consumption in the action
of tyrosinase on monophenol follow Equation (1):
measuring the oxygen consumed; r
, number of turnovers in
M
Eox
ꢀ
ꢀ
t
M before its suicide inactivation, measuring oxygen consumed;
O2
½O2ꢁ ¼ ½O2ꢁ þ ½O2ꢁ e
whose parameters can be obtained by a non-linear regression .
Thus, the experimental recordings obtained follow Equation (1),
ð1Þ
f
1
r
, number of turnovers in the hydroxylase cycle made by one
43
MðHÞ
molecule of enzyme acting on M before its suicide inactivation,
M
m
obtained by measuring the oxygen consumed; K , Michaelis
from which the corresponding inactivation parameters, ½O2ꢁ
f
constant of tyrosinase for M; k cMa t, catalytic constant of (oxygen remaining at the end of the reaction), ½O ꢁ (oxygen
2 1
DðMÞ
M
consumed by the end of the reaction) and ꢀ
inactivation constant), can be determined.
(apparent
the monophenolase activity for M;
K
T
E
d
,
dissociation
Eox
T
constant of the complex E D in the presence of the monophenol;
DðMÞ
d
The experimental recordings of Q accumulation in the action
, dissoci- of tyrosinase on monophenol fit Equation (2):
M
T
d
KE
T
d
, dissociation constant of the complex E M; K
R
d
E
R
d
D or M
ation constant of the complex E D in the presence of the
ꢀꢀEox
t
½
Qꢁ ¼ ½Qꢁ ð1 ꢀ e
Þ
whose parameters can be obtained by a non-linear regression .
ð2Þ
1
monophenol; KM
, dissociation constant of the complex E M.
E
d
d
R
R
43
The experimental recordings obtained fit Equation (2), from
M
Material and methods
which the corresponding inactivation parameters, ½Qꢁ and ꢀ
,
1
Eox
Reagents
can be determined.
In the case of the diphenolase activity, the experimental data of
time-based assays for Q accumulation in the action of tyrosinase
Mushroom tyrosinase (o-diphenol: O2 oxidoreductase, EC
1.14.18.1) (8300 units/mg) was supplied by Sigma (Madrid,
Spain). The enzyme was purified as described by Rodriguez-
D
Eox
on o-diphenol fit Equation (2). From this equation, ½Qꢁ and ꢀ
1
3
Lopez et al. . Protein concentration was determined by the
6
can be determined.
4
1
Lowry method . The substrates TBC and TBF were purchased
from Sigma (Madrid, Spain). All other chemicals were of
Simulation assays
analytical grade. Stock solutions of the diphenolic substrates The simulation shows how the concentrations of the ligand and
were prepared in 0.15 mM of phosphoric acid to prevent auto- enzymatic species involved in the reaction mechanism here
oxidation. Milli-Q system (Millipore Corp., Billerica, MA) proposed for tyrosinase evolve. The respective systems of
ultrapure water was used throughout the experiment.
differential equations have been solved numerically for particular
sets of values of the rate constants and of initial concentrations of
the species of the reaction mechanism. The numerical integration
is based on the Runge–Kuta–Fehlberg algorithm, implemented on
a PC-compatible computer program (WES) .
Simulated data of time-based assays for the accumulation of Q
were fitted to Equation (2) (see Supplementary material).
Spectrophotometric assays
Diphenolase activity
4
4
These assays were carried out with a Perkin-Elmer Lambda-35
spectrophotometer (Perkin-Elmer Corp., Waltham, MA), on line
interfaced to a PC-computer, where the kinetic data were recorded,
stored and later analyzed. The product of the enzyme reaction 4-
tert-o-benzoquinone is stable at long assay times, for which reason,
the reaction was followed by measuring the appearance of Q at
R
d
T
d
Generation of Eox, E , E and Em
The enzymatic forms were generated and their inactivation was
4
studied following Mu n˜ oz-Mu n˜ oz et al. .
0
7
,10
ꢀ1
ꢀ1
4
studied in 30 mM sodium phosphate buffer (pH 7.0).
10 nm , with " ¼ 1200 M cm . The inactivation kinetics was
R
d
Evaluation of enzymatic species Em, E and Eox in an
enzymatic preparation of tyrosinase
Monophenolase activity
These assays were carried out with the same apparatus as in the The evaluation of these enzymatic species in an enzymatic
diphenolase activity assays. The reaction was followed by preparation of tyrosinase was carried out as described by Mu n˜ oz-
4
measuring the Q accumulated in the reaction medium at Mu n˜ oz et al. .
0
ꢀ
1
ꢀ1 7,10
) . The inactivation kinetics
ꢀ
¼ 410 nm (" ¼ 1200 M cm
1
3
was studied in 30 mM sodium phosphate buffer (pH 7.0), adding
the amount of o-diphenol (TBC) necessary for the system to reach
the steady state when t ! 0, that is an initial steady state.
C-NMR assays
1
3
C-NMR spectra of TBC and TBF were obtained as described by
4
Mu n˜ oz-Mu n˜ oz et al. .
0
Oxymetric assays
Results and discussion
Measurements of dissolved oxygen concentration were made with
a Hansatech (Kings Lynn, Cambs, UK) oxygraph unit controlled In this work, we study the reaction of different enzymatic forms of
by a PC. The oxygraph used a Clark-type silver/platinum tyrosinase in its catalytic cycle with TBF/TBC both aerobic (Eox)

4
J. L. Mu n˜ oz-Mu n˜ oz et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Scheme 1. Kinetic mechanism proposed to
explain the monophenolase together with
diphenolase and suicide inactivation path-
ways of tyrosinase acting on TBF/TBC
k2
k1
E D
E M
m
M + E + D
m
m
k-2
k
-1
k54
k3
k−3
1
(
see Scheme 1SM for further details).
1
k73
k
−54
(
Em-D)
4
(Em-D)1
k
55
(
E -D)
ox
2
k3
k−32
2
k
−53
k53
Q
k−55
(
Em-D)2
k72
(E -D)3
m
0
Q + H O + Cu
2
2
k33
i
73
i
7
k52
k
k
2
(
E -D)
ox 1
Ei
(E -D)
ox
3
E
d
+
O2
(Eox-M)1
k71
k−51 k51
k8 k-8
k6
k4
E D
D + Eox + M
E M
ox
ox
k-6
k-4
k9
D + DC + H+
2Q
k10
Q + NADH + H+
D + NAD+
R
d
T
d
and anaerobic (Em, E and E ) conditions (Scheme 1 in its kinetic
form and Scheme 1SM in its structural form).
0.12
0.10
0.08
Calculation of the o-diphenol concentration necessary for
the system to reach its steady state
(a)
First, to eliminate the lag period characteristics of the mono-
phenolase activity when TYR acts on L-tyrosine, the value of the
dimensionless parameter C, which represents the ratio between
½
Dꢁ and ½Mꢁ necessary for the system to reach the steady state,
ss
ss
was calculated . If the substrate varies very little in the assay,
(g)
1
0
A400
0.06
then ½Mꢁ ꢃ½Mꢁ and C can be expressed as C ¼ ½Dꢁ =½Mꢁ [see
ss
0
ss
0
1
0
the work by Garcia-Molina et al. ]. If the concentration of
o-diphenol added exceeds the value predicted by C, a burst of
o-quinone accumulation results. However, when the o-quinone
does not generate o-diphenol in the medium, as is the case of
TBF, C was calculated in an opposite sense. That is, first, a burst
was generated by adding a concentration of o-diphenol above the
steady state and, then, the o-diphenol concentration added was
0.04
0.02
0.00
8
,10
.
gradually reduced until the burst was eliminated (Figure 1)
Figure 1 shows the C obtained by adding different concentra-
tions of TBC while maintaining the initial concentration of TBF
constant. As can be seen from Figure 1 curve (a), a burst appears
in the accumulation of o-quinone as a consequence of the
0
50
100
150
t (s)
200
250
300
Figure 1. Spectrophotometric recordings to calculate the constant
excessive concentration of TBC in the reaction medium. As this [TBC] /[TBF] in the steady state, C. The experimental conditions are
ss
ss
concentration diminishes [Figure 1, curves (b–g)], the burst is sodium phosphate buffer (pH 7.0) 30 mM, [E]
0
¼ 36 nM,
(mM) concentrations are:
a) 35, (b) 30, (c) 28, (d) 25, (e) 20, (f) 18 and (g) 15.
[
TBF]
0
¼ 0.3 mM and the initial [TBC]
0
eliminated and the steady state is reached at t ! 0 [curve (g)].
(
The value obtained of C for the pair TBC/TBF is 0.05.
Inactivation of tyrosinase in aerobic conditions: suicide
inactivation
suicide inactivation of the enzyme. The structural mechanism is
depicted in Scheme 1SM (see Supplementary material).
In the mechanism proposed in Scheme 1, it can be seen how
The kinetic mechanism proposed in Scheme 1 depicts the action
of tyrosinase on monophenols and o-diphenols. It also depicts the the enzyme is inactivated when its acts on the o-diphenol and, as

DOI: 10.3109/14756366.2013.782298
Indirect inactivation of tyrosinase
5
the number of turnovers that inactivates one mole of enzyme is a
constant for a given enzyme, an analytical expression can be
established that relates the number of turnovers in the
diphenolase cycle (Scheme 1 in the absence of M) with the number
of inactivating turnovers in the mechanism of Scheme 1.
270
2
2
60
50
The diphenolase activity (action of tyrosinase on TBC) can be
monitored spectrophotometrically by measuring the formation of
o-quinone, since, in each turnover (Scheme 1 in the absence of M)
two molecules of o-quinone are formed at t ! 1, the quantity of
(
a)
240
o-quinone is ½Qꢁ , and the number of turnovers is calculated by
1
measuring the formation of o-quinone (Q) is:
2
2
2
30
20
10
(g)
½
Qꢁ =2 ꢀ 2½Eꢁ =2 ½Qꢁ =2
½Qꢁ =2
Q
D
1
0
1
0
1
0
r ¼
¼
ꢀ 1ꢃ
ð3Þ
½Eꢁ
½Eꢁ
½Eꢁ
0
In the mechanism of Scheme 1, the number of turnovers made
by the enzyme acting on M and measured by the consumption of
oxygen is:
½
O2ꢁ
O2
M
1
0
r
¼
ð4Þ
½
Eꢁ
200
0
40
80
120
160
These turnovers are of two types, one for the hydroxylase cycle
O2
t (min)
O2
MðHÞ
(r
)
and the other for the oxidase cycle (r
).
MðOÞ
The abovementioned turnovers can produce inactivation, and, as Figure 2. Oxymetric recordings of the suicide inactivation of tyrosinase
two turnovers are produced in the hydroxylase cycle for every one in its action on TBF, varying the initial enzyme concentration.
The experimental conditions are sodium phosphate buffer (pH 7.0)
30 mM, [TBF] ¼ 0.5 mM, [TBC] ¼ 25 mM and the initial enzyme (nM)
in the oxidase cycle, the turnovers realized in the oxidase cycle as
4
5
a function of the oxygen consumed will be :
0
0
concentrations are: (a) 0.008, (b) 0.017, (c) 0.026, (d) 0.034, (e) 0.044,
M
½
O2ꢁ =3
(f) 0.052 and (g) 0.06. Inset. Representation of ½O ꢁ (ꢂ) and ꢀ (ꢅ)
O2
MðOÞ
1
0
2
1
Eox
r
¼
ð5Þ
versus ½Eꢁ .
½
Eꢁ
0
The relation between Equations (3) and (5) is:
TBF can be carried out by measuring the consumption of oxygen
or spectrophotometrically measuring the formation of o-quinone.
Figure 2 depicts the oxygen consumption measured during
the tyrosinase action on TBF, while the figure legend details
the conditions. Analysis of the recordings depicted in
½
Qꢁ =2 ꢀ 2½Eꢁ =2 ½Qꢁ =2
½Qꢁ =2
1
0
1
0
1
0
ꢀ
1
Q
D
O2
MðOÞ
r
½Eꢁ
½Eꢁ
½Eꢁ
0
¼
¼
ꢃ
¼ 1 ð6Þ
½
O2ꢁ =3
½O2ꢁ =3
½O2ꢁ =3
r
1
1
1
½Eꢁ
½Eꢁ
½Eꢁ
0
0
0
Figure 2, according to Equation (1), enables ½O2ꢁ
and
to be obtained. The inset in Figure 2 shows these parameters
1
M
Eox
ꢀ
as a function of the initial enzyme concentration, and from the
If the activity on monophenols is followed by measuring the
o-quinone formation, which, in this case, is stable, bearing in
mind the stoichiometry 3O2 :4Q (in the oxidase cycle 1O2 :2Q
and in the hydroxylase cycle 2O2 :2Q), Equation (6) can be
expressed as:
O2
slope of ½O2ꢁ1 versus ½Eꢁ the value of r can be obtained –
0
M
one-third of these turnovers take place in the oxidase cycle. The
M
inset also shows how the apparent inactivation constant (ꢀ
does not depend on the initial enzyme concentration.
)
Eox
½
Qꢁ =2 ꢀ 2½Eꢁ =2 ½Qꢁ =2
½Qꢁ =2
1
0
1
0
1
0
Figure 3 shows the spectrophotometric recordings of
the accumulation of o-quinone in the action of tyrosinase on
TBF. Analysis of the data by Equation (2) gives the values
ꢀ
ꢀ
1
1
Q
D
r
Q
½Eꢁ
½Eꢁ
½Eꢁ
0
¼
¼
ꢃ
¼ 1 ð7Þ
½Qꢁ =2 ꢀ 2½Eꢁ =2 ½Qꢁ =2
½Qꢁ =2
r
1
0
1
0
1
MðOÞ
M
Eox
of ½Qꢁ and ꢀ . The inset in Figure 3(a) shows the values of
½Eꢁ
½Eꢁ
½Eꢁ
1
0
0
½
Qꢁ versus ½Eꢁ which, from the slope of the straight line, give
1
0
Q
Experimental demonstration of the validity of the analytical
expressions shown in Equations (6) and (7) would confirm the
hypothesis that tyrosinase is not inactivated in the hydroxylation
the value of r . Taking Scheme 1 into account, half of ½Qꢁ
M
1
is
Q
comes from the oxidase pathway and so the value of r
MðOÞ
Q
M
of monophenols, as has been demonstrated in the case of r =2. Note how ꢀ is independent of the enzyme concentra-
M
Eox
4
6,47
L-tyrosine and L-dopa, compounds that generate very unstable
o-quinones (o-dopaquinone) that lead to the accumulation of
4
L-dopa in the medium .
In the present case, the monophenol used (TBF) originates a
very stable o-quinone and the only way to study the process is to
add a quantity of o-diphenol (TBC) to the medium to prevent the
burst while ensuring that the enzyme remains in the steady
state (Figure 1). Once [D]ss has been established, the ratio
tion
described Mu n˜ oz-Mu n˜ oz et al. and the values of the kinetic
. The suicide inactivation of tyrosinase on TBC is
2
5
0
analysis are detailed in Table 1. The inset of Figure 3(b) shows the
Q
½
Qꢁ versus ½Eꢁ and from the slope the value of rD can be
1
0
obtained (Table 1).
The experimental results shown in Figures 2–3 and their
kinetic analysis according to Equations (1) and (2), give the values
O2
MðOÞ
Q
MðOÞ
of r
and r
of enzyme acting on TBF/TBC, measuring the consumption of
, and the number of turnovers made by one mole
[
D] / [M] ¼ C can be established and maintained in all the
ss
ss
experiments.
oxygen or formation of o-quinone, respectively. From the data
25
obtained for the inactivation of the enzyme acting on TBC ,
NADH
we obtain the value of r
Q
¼ ½NADHꢁ =ð2½Eꢁ Þ, which is
D
0
0
Experimental approach
equivalent to r ¼ ½Qꢁ =ð2½Eꢁ Þ. From these results and using
D
1
0
Q
O2
Since the substrate originates a stable quinone, a kinetic study of Equations (6) and (7), we see that rD=r
the suicide inactivation that occurs when tyrosinase acts on rD=r
¼ 1:07 ꢄ 0:08 and
MðOÞ
Q
Q
MðOÞ
¼ 1:01 ꢄ 0:10.

6
J. L. Mu n˜ oz-Mu n˜ oz et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
These results are resumed in Table 2. Note that in the case Hence, in the ‘‘Supplementary material’’, we show the set of
of the pair TBF/TBC, the great stability of the o-quinones differential equations that express the monophenolase and
jeans that record the formation of o-quinone can be obtained both diphenolase mechanism of tyrosinase.
with the monophenol and o-diphenol, which cannot be done with Figure 1SM shows the recordings obtained for the formation of
the pair L-tyrosine/L-dopa due to the instability of the o-quinone (Q) in the diphenolase activity of the enzyme by
4
dopachrome .
0
varying the initial concentration. Analysis of the curves by means
D
of Equation (2) gives the values of ½Qꢁ and ꢀ
, while their
dependence on ½Eꢁ is shown in Figure 1SM (inset). From this
1
EoxðmaxÞ
Numerical integration of the mechanisms proposed
0
Q
dependence of ½Qꢁ versus ½Eꢁ we obtain the value r ¼ 89040.
1
0
D
Figure 2SM shows the recordings obtained for the formation of
The numerical integration of the mechanisms proposed in
Scheme 1 in the presence and the absence ([M] ¼ 0) of
0
o-quinone (Q) in the monophenolase and diphenolase activity
according to Scheme 1. Analysis of the curves by means of
monophenol confirms the predictions indicated in Equation (7).
M
Equation (2) gives the values of ½Qꢁ and ꢀ
, while their
dependence on ½Eꢁ is shown in Figure 2SM (inset). From the
1
EoxðmaxÞ
t (min)
0
Q
MðOÞ
0
5
10
15
20
25
30
values of ½Qꢁ
we obtain the value of r
As expected, these values comply with Equation (7) (Table 2).
¼ 89040.
1
A
B
0.16
0.12
0.08
0.04
0.00
R
d
T
d
Inactivation of E , E and Em under anaerobic conditions
by TBC and its protection by TBF
Figure 4 shows the residual activity of tyrosinase when ER is
preincubated with TBC. Substituting these data into Equation (8)
d
(
h)
D
ꢀ
ꢀ
t
R
R
d
R
d 0
E
½
E ꢁ ¼ ½E ꢁ e
d
ð8Þ
, and the analysis of these data according
A400
D
R
gives the value of ꢀEd
to Equation (9) (Figure 4, Inset ꢅ), gives the values of k and
(
e)
ꢆ
iD
D
K
R
(Table 3).
ꢆ
E
(
d)
d
k ½Dꢁ
D
iD
0
ꢀ
R
¼ K
ð9Þ
E
D
d
þ ½Dꢁ0
R
d
E
(
a)
In the presence of TBC and the monophenol (TBF) (Figure 4,
Inset ꢂ), the residual activity data fit Equation (10) and the values
DðMÞ
ꢆ
DðMÞ
R
. Analysis of
ofꢀ
R
E
fit Equation (11), giving k
and KE
iDðMÞ
d
d
these data by means of Equation (12) gives the values of
M
0
50
100
150
200
KEd (Table 3).
R
DðMÞ
t
d
t (min)
ꢀꢀ
R
E
R
d
R
d 0
½
E ꢁ ¼ ½E ꢁ e
ð10Þ
(ꢂ)
Figure 3. Spectrophotometric recordings of the suicide inactivation of
tyrosinase in its action on TBF, varying the initial enzyme concentration.
The experimental conditions are sodium phosphate buffer (pH 7.0) versus [TBC] , which demonstrate how the monophenol protects
DðMÞ
D
Figure 4 (inset) shows the values of ꢀEd
R
(ꢅ) and ꢀEd
R
0
3
0 mM, [TBF]
concentrations are: (a) 0.045, (b) 0.05, (c) 0.067 and (d) 0.075. The curves
e)–(h) represent the action of the enzyme on TBC. The experimental
conditions are the same as curves (a)–(d), but [TBF]
¼ 0 and
0
¼ 0.1 mM, [TBC]
0
¼ 5 mM and the initial enzyme (nM) the enzyme against inactivation by o-diphenol. Similar results are
T
shown in Figure 5 and its inset for E . The results obtained are
d
(
shown in Table 3. The protection presented by the monophenol
against the irreversible inactivation caused by the o-diphenol is
depicted in Schemes 2(A), 2(B) and 3. Possible molecular
0
[
TBC]
0
¼ 9 mM. The initial enzyme concentrations (nM) are: 0.30
(
e), 0.32 (f), 0.34 (g) and 0.37 (h). Inset A. Representation of ½Qꢁ (ꢂ)
1
M
Eox
D
Eox
and ꢀ (ꢅ) versus ½Eꢁ . Inset B. Representation of ½Qꢁ (ꢂ) and ꢀ (ꢅ) mechanisms of these inactivations are shown in the
0
1
versus ½Eꢁ0.
‘‘Supplementary material’’. Analysis of the residual activity
Table 1. Kinetic constants which characterize the suicide inactivation of tyrosinase by TBC and values of the chemical shifts of this compound
obtained by C-NMR for the C-2 and C-1 carbons at pH 7.0*.
1
3
D
ox
3
ꢀ1
D
D
ox
k cD at (s
ꢀ1
D
O2
o-Diphenol ꢀE ðmaxÞ ꢇ 10 (s
)
r ¼ kcat=ꢀE ðmaxÞ
)
Vmax (mM/s)
Km (mM)
Km (mM)
ꢁ2 (p.p.m.) ꢁ1 (p.p.m.)
147.75 146.24
TBC
7.28 ꢄ 0.28
88 788 ꢄ 1864
640.1 ꢄ 28.10 0.12 ꢄ 0.01
1.45 ꢄ 0.12 27.82 ꢄ 2.52
2
Data taken from the work by Mu n˜ oz-Mu n˜ oz et al. .
5
*
Table 2. Experimental confirmation of the relations described in Equations (6) and (7), according to which the theoretical value is 1.
Equation (6)
Qꢁ =ð2½Eꢁ Þ
Equation (7)
½Qꢁ =ð2½Eꢁ Þ
½
Q
D
O2
MðOÞ
Q
MðOÞ
Q
D
O2
MðOÞ
1
1
0
Q
D
Q
MðOÞ
1
1
0
r
r
r
r =r
¼
¼ 1 r =r
¼
¼ 1
½
O2ꢁ =ð3½Eꢁ Þ
½Qꢁ =ð2½Eꢁ Þ
Results
0
0
[
TBF]
TBC]
0
(0.5 mM)
(25 mM)
–
82 891 ꢄ 8102 87 099 ꢄ 7566
–
–
–
–
[
Applying Equations (6) and (7)
0
88 788 ꢄ 1864
–
–
1.07 ꢄ 0.08
1.01 ꢄ 0.10

DOI: 10.3109/14756366.2013.782298
Indirect inactivation of tyrosinase
7
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
1.2
1.0
0.8
0.6
A/A0
A/A0
0
0
0
.4
.2
.0
0
5
10
15
20
0
10
20
30
40
50
t (min)
t (min)
0
Figure 4. Residual activity, A/A , corresponding to the irreversible
0
Figure 5. Residual activity, A/A , corresponding to the irreversible
inactivation of Ed by TBC and its protection by TBF. The form E dR was
obtained as described in the section ‘‘Material and methods’’ and
immediately incubated with o-diphenol (TBC) in the presence of
monophenol (TBF) or in its absence, taking aliquots at different times
R
inactivation of E dT by TBC and its protection by TBF. The form ET was
d
obtained as described in the section ‘‘Material and methods’’ and
immediately incubated with o-diphenol (TBC) in the presence of
monophenol (TBF) or in its absence, taking aliquots at different times
to measure the residual activity with 9 mM TBC (ꢀ ¼ 400 nm). The
experimental conditions were: 30 mM sodium phosphate buffer (pH 6.0),
to measure the residual activity with [TBC]
The experimental conditions were: 30 mM sodium phosphate buffer
0
¼ 9 mM (ꢀ ¼ 400 nm).
ꢂ
(
The [TBC]
pH 6.0), 25 C, ½Eꢁ ¼ 0.1 mM (see ‘‘Material and methods’’ section).
ꢂ
0
25 C, ½Eꢁ ¼ 0.1 mM (see ‘‘Material and methods’’ section). The [TBC]
0
0
0
concentrations were (mM): (a) 10 ꢅ, (b) 20 ꢂ, (c) 35 g,
concentrations were (mM): (a) 10 ꢅ, (b) 20 ꢂ, (c) 50 g, (d) 100 œ,
(
results follow Equation (8) and the apparent inactivation constants follow
d) 50 œ, (e) 60 m, (f) 80 i, (g) 100 H and (h) 120 O. The experimental
(
e) 150 m and (f) 200 i. The experimental results follow Equation (8)
and the apparent inactivation constants follow Equation (9). Inset.
D
Equation (9). Inset. Representation of the values of ꢀE
R
d
versus [TBC]
0
in
D
T
Representation of the values of ꢀEd
versus [TBC]
0
in the absence (ꢅ)
the absence (ꢅ) of TBF. In the presence of TBF (ꢂ) ([TBF]
0
¼ 40 mM), the of TBF. In the presence of TBF (ꢂ) ([TBF]
0
¼ 40 mM), the experimental
DðMÞ
DðMÞ
experimental results follow Equation (10) to calculate ꢀ
DðMÞ
R
E
. These results were substituted into Equation (10) to calculate ꢀE
. These
DðMÞ
T
d
d
results were substituted into Equation (11) to obtain KEd
R
and its analysis results were again substituted into Equation (11) to obtain KEd
T
M
M
by Equation (12) gives KEd
R
.
and its analysis by Equation (12) gives KEd .
T
R
d
T
d
Table 3. Kinetic constants which characterize the inactivation of E , Em and E by TBC and their protection by TBF under
anaerobic conditions. The constants were obtained using the equations indicated.
D
E
(a)
ꢆ
3
ꢀ1
ꢆ
3
ꢀ1
M
y
K
E
[Equation (12)]
Enzymatic
form
K
(mM)
[Equation (9)]
k
ꢇ 10 (min
)
k
ꢇ 10 (min
)
(mM)
iD
iD
[Equation (11)]
Substrate
[Equation (9)]
T
E
d
TBC
TBF
TBC
TBF
TBC
TBF
55.46 ꢄ 4.68
0.32 ꢄ 0.01
–
–
–
–
0.32 ꢄ 0.03
110.27 ꢄ 17.13
R
E
52.91 ꢄ 5.90
0.47 ꢄ 0.04
–
–
d
–
–
0.47 ꢄ 0.06
–
0.47 ꢄ 0.05
125.61 ꢄ 17.51
Em
52.91 ꢄ 5.90
0.47 ꢄ 0.04
–
–
–
125.61 ꢄ 17.51
y
In K ED and K , E corresponds to the enzymatic forms E or E . When the reaction is started with Em, the data obtained
M
T
d
R
E
d
correspond to E , because of the rapid transformation of Em into E (Scheme 3).
R
R
d
d
data (for ER and E ) in the presence of monophenol (TBF) fits
T
d
M
KE
R
d
d
(
A)
M
(B)
KEd
T
Equation (10) but the apparent inactivation constant is now given
by Equation (11):
R
d
T
R
E dT + M
+
D
E M
E M
Ed + M
+
d
ꢆ
k
½Dꢁ0
D
DðMÞ
iDðMÞ
ꢀ
¼
DðMÞ
K
Ed
ð11Þ
Ed
D
D
R
Q
K
Q
þ ½Dꢁ0
K
*
d
Ed
E
T
ki
k
iD
DðMÞ
M
D
T
d
R
where K
calculate K .
is expressed by Equation (12), which is used to
E D
Ei
E D
Ei
Ed
d
!
E
d
½
Mꢁ
T
DðMÞ
D
Ed
0
M
Scheme 2. (A) Effect of monophenols on E , inactivation by o-diphenol.
R
K
¼ K
1 þ
K
Ed
ð12Þ
d
B) Effect of monophenols on E , inactivation by o-diphenol.
Ed
(
d
This analysis was also carried out for the Em form (Table 3).

8
J. L. Mu n˜ oz-Mu n˜ oz et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Q
diphenolase activities of tyrosinase. J Agric Food Chem 2007;55:
9739–49.
11. Esp ´ı n JC, Var o´ n R, Tudela J, Garc ´ı a-C a´ novas F. Kinetic study of the
oxidation of 4-hydroxyanisole catalyzed by tyrosinase. Biochem
Mol Biol Int 1997;41:1265–76.
2. Esp ´ı n JC, Var o´ n R, Fenoll LG, et al. Kinetic characterization of
the substrate specificity and mechanism of mushroom tyrosinase.
Eur J Biochem 2000;267:1270–9.
3. Esp ´ı n JC, Garc ´ı a-Ruiz PA, Tudela J, Garc ´ı a-C a´ novas F. Study of the
stereospecificity of pear and strawberry polyphenol oxidases. J Agric
Food Chem 1998;46:2469–73.
M
KE
k2
k3
d
R
d
+
D
R
Em + D
E D
E + M
E M
d
m
k-2
+
M
1
1
1
D
KE
R
d
K1
E M
m
R
E D
d
4. Fenoll L, Rodriguez-Lopez JN, Garcia-Sevilla F, et al. Analysis and
interpretation of the action mechanism of mushroom tyrosinase on
monophenols and diphenols generating highly unstable o-quinones.
Biochim Biophys Acta 2001;1548:1–22.
kiD
Q
15. Fenoll LG, Rodriguez-Lopez JN, Garc ´ı a-Sevilla F, et al. Oxidation
by mushroom tyrosinase of monophenols generating slightly
unstable o-quinones. Eur J Biochem 2000;267:5865–78.
Ei
Scheme 3. Effect of monophenols on Em, inactivation by o-diphenol.
1
6. Gupta AK, Gover MD, Nouri KN, Taylor SJ. The treatment of
melasma: a review of clinical trials. J Am Acad Dermatol 2006;55:
Conclusions
In anaerobic conditions, monophenols protect the enzyme (E , E
and Em forms) against inactivation by o-diphenol. In aerobic
1
048–65.
R
d
T
d
17. Ashida M, Brey P. Role of the integument in insect defense: pro-
phenol oxidase cascade in the cuticular matrix. Proc Natl Acad Sci
1
995;92:10698–702.
conditions, the monophenols do not inactivate the enzyme
(Eox form) in the hydroxylase cycle directly, but, as this activity
1
1
2
8. Sonmez F, Sevmezler S, Atahan A, et al. Evaluation of new chalcone
derivatives as polyphenol oxidase inhibitors. Bioorg Med Chem Lett
2011;21:7479–82.
9. Demir D, Gencer N, Arslan O, et al. In vitro inhibition of polyphenol
oxidase by some new diarylureas. J Enzyme Inhib Med Chem 2012;
does not occur without the oxidase cycle acting on o-diphenol, it
may be claimed that the monophenols inactivate tyrosinase
indirectly. This implies that monophenols such as TBF, which,
after oxidation, produce stable o-quinones that do not give rise to
o-diphenol in the medium, do not bring about the suicide
inactivation of the enzyme, but neither would the enzyme act on
them due to the lack of o-diphenol in the medium.
2
7:125–31.
0. Gencer N, Demir D, Sonmez F, Kucukislamoglu M. New saccharin
derivatives as tyrosinase inhibitors. Bioorg Med Chem 2012;20:
2
811–21.
21. Mu n˜ oz-Mu n˜ oz JL, Garcia-Molina F, Varon R, et al. Suicide
inactivation of the diphenolase and monophenolase activities of
tyrosinase. IUBMB Life 2010;62:539–47.
Declaration of interest
2
2. Thorneby-Anderson K, Sterner O, Hansson C. Tyrosinase-mediated
formation of a reactive quinone from the depigmenting agents,
The authors report no declarations of interest. This paper was partially
supported by grants from: Ministerio de Educaci o´ n y Ciencia (Madrid,
Spain), project BIO2009-12956; Fundaci o´ n S e´ neca (CARM, Murcia,
Spain), projects 08856/PI/08 and 08595/PI/08, and Consejer ´ı a de
Educaci o´ n (CARM, Murcia, Spain), BIO-BMC 06/01-0004. JLMM
hold fellowships from Fundaci o´ n Caja Murcia (Murcia, Spain).
4
1
-tert-butylphenol and 4-tert-butylcatechol. Pigment Cell Res 2000;
3:33–8.
2
2
3. Yang F, Boissi RE. Effects of 4-tertiary butylphenol on the
tyrosinase activity in human melanocytes. Pigment Cell Res 1999;
1
2:237–45.
4. Yang F, Sarangarajan R, Le Poole IC, et al. The cytotoxicity and
apoptosis induced by 4-tertiary butylphenol in human melanocytes
are independent of tyrosinase activity. Inves Dermatol 2000;114:
References
1
.
Prota G, d‘Ischia M, Napolitano A. The chemistry of melanins and
related metabolites. In: Nordlund JJ, Boissy RE, Hearing VJ, et al.,
eds. The pigmentary system, physiology and pathology. Oxford:
Oxford University Press; 1998:307–32.
1
57–64.
2
5. Mu n˜ oz-Mu n˜ oz JL, Garcia-Molina F, Garcia-Ruiz PA, et al. Phenolic
substrates and suicide inactivation of tyrosinase: kinetics and
mechanism. Biochem J 2008;416:413–40.
2
3
.
.
Solomon EI, Sundaran UM, Machonkin TE. Multicopper oxidases
and oxygenases. Chem Rev 1996;96:2563–606.
Dietler C, Lerch K. Reaction inactivation of tyrosinase. In: King TE,
Mason HS, Morrison M, eds. Oxidases and related redox systems.
New York: Pergamon Press; 1982:305–17.
2
2
6. Silverman
RB. Mechanism-based
Meth Enzymol 1995;249:240–83.
enzyme
inactivators.
7. Zhong JY, Groutas WC. Recent developments in the design of
mechanism-based and alternate substrate inhibitors of serine
proteases. Curr Top Med Chem 2004;4:1203–16.
4
5
.
.
Martinez MV, Whitaker JR. The biochemistry and control of
enzymatic browning. Trends Food Sci Technol 1995;6:195–200.
Kondo T, Hearing VJ. Update on the regulation of mammalian
melanocyte function and skin pigmentation. Expert Rev Dermatol
2
2
3
8. Tudela J, Garcia-Canovas F, Varon R, et al. Kinetic characterization
of dopamine as a suicide substrate of tyrosinase. J Enzyme
Inhibition 1987;2:47–56.
9. Arnao MB, Acosta M, del Rio JA, et al. A kinetic-study on the
suicide inactivation of peroxidase by hydrogen-peroxide. Biochim
Biophys Acta 1990;1041:43–7.
0. Hiner A, Hernandez-Ruiz J, Arnao MB, et al. A comparative study
of the purity, enzyme activity, and inactivation by hydrogen peroxide
of commercially available horseradish peroxidase isoenzymes A and
C. Biotechnol Bioeng 1996;50:655–62.
2
011;6:97–108.
6
.
Smith NPM, Pavel S, Riley PA. Mechanisms of control of the
cytotoxicity of orthoquinone intermediates of melanog e´ nesis.
In: Creveling CR, ed. Role of catechol quinone species in cellular
toxicity. Tenesee: F.P. Graham Publishing Co; 2000;191–246.
Mu n˜ oz JL, Garcia-Molina F, Varon R, et al. Calculating molar
absortivities for quinones: application to the measurements of
tyrosinase activity. Anal Biochem 2006;351:128–38.
Ros JR, Rodriguez-Lopez JN, Varon R, Garc ´ı a-C a´ novas F. Kinetic
study of the oxidation of 4-tert-butylphenol by tyrosinase. Eur J
Biochem 1994;222:449–52.
7
8
9
.
.
.
3
3
1. Escribano J, Tudela J, Garcia-Carmona F, Garcia-Canovas F.
A kinetic-study of the suicide inactivation of an enzyme measured
through coupling reactions: application to the suicide inactivation of
tyrosinase. Biochem J 1989;262:597–603.
2. Garc ´ı a-Canovas F, Tudela J, Varon R, V a´ zquez AM. Experimental
methods for kinetic study of suicide substrates. J Enzyme Inhibition
1989;3:81–90.
Rodr ´ı guez-Lopez JN, Serna Rodr ´ı guez P, Tudela J, et al. A
continuous spectrophotometric method for the determination of
diphenolase activity of tyrosinase using 3,4-dihydroxymandelic
acid. Anal Biochem 1991;195:369–74.
33. Mu n˜ oz-Mu n˜ oz JL, Acosta-Motos JR, Garcia-Molina F, et al.
Tyrosinase inactivaci o´ n in its action on L-dopa. Biochim Biophys
Acta 2009;1804:1467–75.
1
0. Garcia-Molina F, Munoz JL, Varon R, et al. A review of
spectrophotometric methods for measuring the monophenolase and

DOI: 10.3109/14756366.2013.782298
Indirect inactivation of tyrosinase
9
3
4. Land E, Ramsden C, Riley PA. The mechanism of suicide- 41. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measure-
inactivation of tyrosinase: a substrate structure investigation.
Tohoku J Exp Med 2007;212:341–8.
ment with the Folin phenol reagent. J Biol Chem 1951;193:265–75.
42. Rodr ´ı guez-Lopez JN, Ros-Martinez JR, Varon R, Garcia-Canovas F.
Calibration of a Clark-type oxygen electrode by tyrosinase
3
3
5. Ramsden C, Riley PA. Mechanistic studies of tyrosinase suicide
inactivation. ARKIVOC 2010;i:260–74.
6. Rodriguez-Lopez JN, Fenoll LG, Garcia-Ruiz PA, et al. Stopped-
catalyzed-oxidation of 4-tert-butylcatechol. Anal Biochem 1992;
202:356–60.
TM
flow and steady-state study of the diphenolase activity of mushroom 43. Jandel Scientific. Sigma Plot 9.0 for Windows . Corte Madera:
tyrosinase. Biochemistry 2000;39:10497–506.
7. Koval IA, Gamez P, Belle C, et al. Synthetic models of the active 44. Garcia-Sevilla F, Garrido del Solo C, Duggleby RG, et al. Use of a
Jandel Scientific; 2006.
3
3
site of catechol oxidase: mechanistic studies. Chem Soc Rev 2006;
5:814–40.
8. Palavicini S, Granata A, Monzani E, Casella L. Hydroxylation of
window program for simulation of the progress curves of reactants
and intermediates involved in enzyme-catalyzed reactions.
Biosystems 2000;54:151–64.
3
phenolics compounds by a peroxodicopper (II) complex: further 45. Rodriguez-Lopez JN, Tudela J, Varon R, et al. Analysis of a kinetic
insight into the mechanism of tyrosinse. J Am Chem Soc 2005;127:
8031–6.
9. Rolff M, Schottenheim J, Decker H, Tuczek F. Copper-O
model for melanin biosynthesis pathway. J Biol Chem 1992;267:
3801–10.
reactivity 46. Mu n˜ oz-Mu n˜ oz JL, Garcia-Molina F, Berna J, et al. Kinetic
1
3
2
of tyrosinase models towards external monophenolic substrates:
molecular mechanism and comparison with the enzyme. Chem Soc
Rev 2011;40:4077–98.
characterisation of o-aminophenols and aromatic o-diamines as
suicide substrates of tyrosinase. Biochim Biophys Acta 2012;1824:
647–55.
4
0. Mu n˜ oz-Mu n˜ oz JL, Garcia-Molina F, Acosta-Motos JR, et al. 47. Munoz-Munoz JL, Berna J, Garcia-Molina F, et al. Unravelling the
Indirect inactivation of tyrosinase in its action on L-tyrosine.
Acta Biochim Pol 2011;58:477–88.
suicide inactivation of tyrosinase: a discrimination between mech-
anisms. J Mol Catal B-Enzymatic 2012;75:11–19.