Kinetic characterisation of o-aminophenols and aromatic o-diamines as suicide substrates of tyrosinase

Article abstract of DOI:10.1016/j.bbapap.2012.02.001

We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow the order aromatic o-diamines < o-aminophenols < o-diphenols, which agrees with the view that the transfer of the proton to the peroxide group of the oxy-tyrosinase form is the slowest step in the catalytic cycle. As regards the apparent inactivation constant, it remains within the same order of magnitude, although slightly lower in the case of the aromatic o-diamines and o-aminophenols than o-diphenols: o-diamines < o-aminophenols < o-diphenols. The efficiency of the second nucleophilic attack of substrate on CuA seems to be the determining factor in the bifurcation of the inactivation and catalytic pathways. This attack is more efficient in o-diamines (where it attacks a nitrogen atom) than in o-aminophenols and o-diphenols (where it attacks an oxygen atom), favouring the catalytic pathway and slowing down the inactivation pathway. The inactivation step is the slowest of the whole process. The values of r, the number of turnovers that 1 mol of enzyme carries out before being inactivated, follows the order aromatic o-diamines < o-aminophenols < o-diphenols. As regards the Michaelis constants, that of the o-diamines is slightly lower than that of the o-diphenols, while that of the o-aminophenols is slightly greater than that observed for the o-diphenols. As a consequence of the above, the inactivation efficiency, λ_max/K_m^S, follows this order: o-diphenols > o-aminophenols > aromatic o-diamines.

Full text of DOI:10.1016/j.bbapap.2012.02.001

Biochimica et Biophysica Acta 1824 (2012) 647–655
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Kinetic characterisation of o-aminophenols and aromatic o-diamines as suicide
substrates of tyrosinase
Jose Luis Muñoz-Muñoz a, Francisco Garcia-Molina a, Jose Berna b, Pedro Antonio Garcia-Ruiz c,
Ramon Varon d, Jose Tudela a, Jose N. Rodriguez-Lopez a, Francisco Garcia-Canovas a,
⁎
a
GENZ: Grupo de Investigación Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100, Espinardo, Murcia, Spain
b
Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100, Espinardo, Murcia, Spain
c
QCPAI: Grupo de Química de Carbohidratos, Polímeros y Aditivos Industriales, Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100, Espinardo,
Murcia, Spain
d
Departamento de Química-Física, Escuela de Ingenieros Industriales de Albacete, Universidad de Castilla la Mancha, Avda. España s/n. Campus Universitario, E-02071, Albacete, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and
compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow
the order aromatic o-diaminesbo-aminophenolsbo-diphenols, which agrees with the view that the trans-
fer of the proton to the peroxide group of the oxy-tyrosinase form is the slowest step in the catalytic cycle.
As regards the apparent inactivation constant, it remains within the same order of magnitude, although
slightly lower in the case of the aromatic o-diamines and o-aminophenols than o-diphenols: o-dia-
minesbo-aminophenolsbo-diphenols. The efficiency of the second nucleophilic attack of substrate on
CuA seems to be the determining factor in the bifurcation of the inactivation and catalytic pathways.
This attack is more efficient in o-diamines (where it attacks a nitrogen atom) than in o-aminophenols
and o-diphenols (where it attacks an oxygen atom), favouring the catalytic pathway and slowing down
the inactivation pathway. The inactivation step is the slowest of the whole process. The values of r, the
number of turnovers that 1 mol of enzyme carries out before being inactivated, follows the order aromatic
o-diaminesbo-aminophenolsbo-diphenols. As regards the Michaelis constants, that of the o-diamines is
slightly lower than that of the o-diphenols, while that of the o-aminophenols is slightly greater than that
observed for the o-diphenols. As a consequence of the above, the inactivation efficiency, λmax/KmS , follows
this order: o-diphenols>o-aminophenols>aromatic o-diamines.
Received 9 November 2011
Received in revised form 31 January 2012
Accepted 1 February 2012
Available online 10 February 2012
Keywords:
Tyrosinase
Aromatic o-diamine
o-Aminophenol
Suicide inactivation
o-Diphenol
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
The crystallisation of tyrosinase from different sources, Strepto-
myces castaneoglobisporus [12], Bacillus megaterium [13,14] and Agar-
Tyrosinase is a cuproprotein that catalyses the hydroxylation of
monophenols to o-diphenols (monophenolase activity) and the oxi-
dation of the latter to o-quinones (diphenolase activity), using molec-
ular oxygen [1–3].
icus bisporus [15], has increased our knowledge of the structure of the
enzyme. In addition, it has been described that mushroom tyrosinase
has an active centre close to the surface [15], which would explain the
low substrate specificity shown by this enzyme [16–20].
Tyrosinase suffers inactivation when it reacts with its o-diphenolic
and triphenolic substrates [4–11]. The catalytic process is related with
the nucleophilic power of the oxygen atom of the hydroxyl group
bound to the carbon atom in para position with respect to the R
group (C-4). However, the inactivation process has no such direct re-
lation, and substrates with low chemical shift (δ) values in C-4 have a
high catalytic constant but low inactivation constant. The relation be-
tween these constants is given by the parameter r, the number of
turnovers that 1 mol of enzyme carries out before being inactivated.
Kinetic studies in the transition phase and the steady state have
shown that the o-diphenols bind to the form met-tyrosinase (Em)
more rapidly than to the form oxy-tyrosinase (Eox) [21]. Furthermore,
studies on the isotopic effect in the presence of deuterated water
have suggested that the transfer of a proton from the hydroxyl
group of C-4 to the peroxide group of Eox is the rate-limiting step in
both diphenolase and monophenolase activities [22,23].
Experiments using aromatic monoamines, aromatic o-diamines
and o-aminophenols as substrates [20,24–27] point to the low values
of the catalytic constant of these processes, lending support to the
suggestion that the transfer of the critical proton to the oxy-
tyrosinase form is the limiting step in the catalytic cycle [20,24,25].
In an earlier publication, we studied the catalytic action of tyrosinase
⁎
Corresponding author. Tel.: +34 868 884764; fax: +34 868 883963.
E-mail address: canovasf@um.es (F. Garcia-Canovas).
1570-9639/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2012.02.001

648
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1824 (2012) 647–655
on aromatic monoamines, aromatic o-diamines and o-aminophenols,
which were found to be substrates of the enzyme [20]. When the
reactions above were followed at long measurement times, the
suicide inactivation of tyrosinase was evident [5–7]. The wide
variety of aromatic o-diamines and o-aminophenols that are avail-
able commercially has enabled us to look more deeply at this sui-
cide inactivation process by studying their corresponding structure/
reactivity relationships.
o-diimines to o-diamines, respectively [20,30,31]. The substrates used
in this method were 2-aminophenol, 3-amino-4-hydroxybenzoic acid,
4-amino-3-hydroxybenzoic acid, 3-amino-L-tyrosine, 1,2-diaminoben-
zene, 3,4-diaminotoluene, 4-methoxy-1,2-phenylenediamine and 2,3-
diaminobenzoic acid.
2.2.2. Oxymetric assays
When the substrate and ascorbic acid spectra overlapped, oxy-
metric assays were carried out [32]. Measurements of dissolved ox-
ygen concentration were made with a Hansatech (Kings Lynn,
Cambs, U.K.) oxygraph unit controlled by a PC. The oxygraph used
a Clark-type silver/platinum electrode with a 12.5 μm Teflon mem-
brane. The sample was continuously stirred during the experiments
and its temperature was maintained at 25 °C. The zero oxygen level
for calibration and the experiments was obtained by bubbling
oxygen-free nitrogen through the sample for at least 10 min. The
substrates 3-amino-4-hydroxytoluene, 4-amino-3-hydroxytoluene,
2-amino-3-hydroxybenzoic acid, 3-amino-2-hydroxybenzoic acid,
3,4-diaminobenzoic acid, 2,3-dihydroxybenzoic acid, 4-fluor-1,2-
phenylenediamine, 4-chloro-1,2-phenylenediamine and 4-bromo-
1,2-phenylenediamine were studied by means of this method.
The effect of various reagents, whose concentrations are given in
detail in the corresponding figures of Section 3, was studied. In all
the assays, the following reagents were maintained constant:
0.26 mM O2 (saturating), 30 mM phosphate buffer pH 7.0 and the con-
centration of AH2 that is indicated in the corresponding figure legend.
2. Material and methods
2.1. Materials
Mushroom tyrosinase or polyphenol oxidase (TYR, o-diphenol: O2
oxidoreductase, EC 1.14.18.1) (8300 units/mg) and L-ascorbic acid
(AH2) were supplied by Sigma (Madrid, Spain). The enzyme was
purified as previously described in Ref. [21]. Protein concentration
was determined by the Lowry method [28]. All the substrates used
in this work were purchased from Sigma (Madrid, Spain) (Table
1SM). All other chemicals were of analytical grade. Stock solutions
of the o-diphenolic substrates, aromatic o-diamines and o-aminophe-
nols were prepared in 0.15 mM phosphoric acid to prevent auto-
oxidation. Milli-Q system (Millipore Corp.) ultrapure water was
used throughout.
2.2. Methods
2.2.3. Data analysis
2.2.1. Spectrophotometric assays
The experimental data of time-based assays for the disappearance
of AH2 follow Eq. (1):
These assays were carried out with a Perkin-Elmer Lambda-35 (Per-
kin-Elmer, Massachusetts, USA) spectrophotometer, on line interfaced
to a PC-computer, where the kinetic data were recorded, stored and
later analysed. The products of the enzyme reaction (o-quinoneimine
or o-diimine) are unstable and evolve towards other products [24,29].
For this reason, the reaction was followed by measuring the disappear-
ance of AH2 at the wavelength and molar absorptivity coefficient
specified for each substrate (Table 2SM). This coupled reagent reduces
o-quinone to o-diphenol, o-quinoneimine to o-aminophenol and
S
E
ox t
½AH2ꢀ ¼ ½AH2ꢀ þ ½AH2ꢀ e−λ
ð1Þ
f
∞
where [AH2]f is the AH2-value at t→∞, [AH2]∞ =[Q]∞ is the AH2 con-
sumed at the end of the reaction and λSE is the apparent constant of
ox
suicide inactivation of tyrosinase in the presence of the corresponding
110
40000
12000
10000
8000
6000
4000
2000
0
1.0
0.8
0.6
0.4
0.2
108
106
104
102
100
98
4
300
30000
(a)
3
20000
8
8
280
2
10000
(f)
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
0
2
4
6
8
260
[E]0 (nM)
[E]
(nM)
0
(a)
(f)
96
240
94
92
0
500
1000
1500
2000
2500
220
0
10
20
30
40
50
t (s)
t (min)
Fig. 1. Corrected recordings of the disappearance of AH2 in the suicide inactivation of ty-
rosinase by the oxidation of 3,4-diaminotoluene for different enzyme concentrations. The
experimental conditions were 30 mM sodium phosphate buffer (pH 7.0), 0.1 mM AH2,
0.26 mM O2, wavelength=265 nm, the initial substrate concentration, [S]0=0.1 mM,
and the initial enzyme concentration, [E]0, (nM): (a) 2, (b) 3.3, (c) 3.8, (d) 4.8, (e) 5.5
Fig. 2. Corrected recordings of the disappearance of oxygen in the suicide inactivation
of tyrosinase by the oxidation of 3-amino-4-hydroxytoluene for different enzyme con-
centrations. The experimental conditions were 30 mM sodium phosphate buffer (pH
7.0), 0.15 mM AH2, 0.26 mM O2, [S]0 =5 mM and [E]0 (nM): (a) 0.5, (b) 1, (c) 1.2,
and (f) 7.5. Inset. Representation of the values of ½AH2ꢀ∞ (○) and λSE (•) vs. enzyme
(d) 2, (e) 2.5 and (f) 3. Inset. Representation of the values of ½O2ꢀ∞ (○) and λES (•)
ox
ox
concentration.
vs. enzyme concentration.

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1824 (2012) 647–655
649
Table 1
Kinetic constants characterising the suicide inactivation of tyrosinase acting on aromatic o-diamines.
Aromatic o-diamine
λE
S x 103
r
kcSat
KmS
k6
δ1
δ2
δ3
δ4
Σδ
ox(max)
(s−1
)
(s−1
)
(mM)
(M−1 s−1
)
(ppm)
(ppm)
(ppm)
(ppm)
(ppm)
4-Fluor-1,2-phenylenediamine
4-Bromo-1,2-phenylenediamine
4-Chloro-1,2-phenylenediamine
4-Methoxy-1,2-phenylenediamine
1,2-Diaminobenzene
3,4-Diaminotoluene
3,4-Diaminobenzoic acid
2,3-Diaminobenzoic acid
3.87 0.32
3.75 0.35
2.57 0.18
1.77 0.12
1.47 0.09
0.85 0.09
0.81 0.07
0.57 0.05
31
16
35
852 89
360 31
599 51
3
2
3
0.12 0.01
0.06 0.01
0.09 0.01
1.48 0.15
0.51 0.06
0.54 0.05
0.07 0.01
0.04 0.01
0.24 0.03
0.73 0.10
0.52 0.07
0.38 0.06
0.44 0.04
0.07 0.01
0.13 0.02
0.30 0.04
500 58
82 11
173 19
3894 416
1159 132
7714 892
538 58
128.9
132.3
131.4
125.6
133.3
–
134.9
135.5
134.7
134.3
133.3
–
–
–
263.8
267.8
266.1
259.9
266.6
263.5
259.6
270.2
–
–
–
–
–
–
–
–
133.2
127.2
135.9
130.3
132.4
–
98
88
9
7
–
–
133 11
–
134.3
Table 2
Kinetic constants characterising the suicide inactivation of tyrosinase acting on o-aminophenols.
× 103
r
kcSat
KmS
k6
δ1
δ2
δ3
δ4
Σδ
S
o-Aminophenol
λE
ox(max)
(s−1
)
(s−1
)
(mM)
(M−1 s−1
)
(ppm)
(ppm)
(ppm)
(ppm)
(ppm)
2-Aminophenol
7.90 0.09
5.34 0.39
4.43 0.41
2.01 0.17
0.62 0.04
0.37 0.02
0.93 0.07
1.38 0.11
7666 815
5503 502
5884 510
701 65
810 76
1230 101
58.12 2.84
25.02 1.58
21.72 2.13
1.21 0.15
0.48 0.01
0.43 0.01
0.15 0.01
0.15 0.01
0.82 0.09
2.77 0.59
2.33 0.41
0.96 0.20
0.14 0.01
0.13 0.01
0.39 0.05
2.20 0.24
70,878 7502
9032 918
9322 950
1260 121
3429 315
3308 295
384 39
143.9
133.9
–
–
–
277.8
274.7
274.7
274.9
283.2
280.5
285.3
282.8
3-Amino-4-hydroxytoluene
4-Amino-3-hydroxytoluene
3-Amino-L-tyrosine
4-Amino-3-hydroxybenzoic acid
3-Amino-4-hydroxybenzoic acid
3-Amino-2-hydroxybenzoic acid
2-Amino-3-hydroxybenzoic acid
–
–
–
–
–
–
–
133.8
143.8
133.8
144.1
132.8
134.7
145.4
140.9
130.9
141.1
139.1
147.7
–
–
–
–
–
140
94
8
7
150.6
137.4
68
7
–
o-diphenol, o-aminophenol or aromatic o-diamine. These parameters
can be obtained by non-linear regression [33]. There is always a slow
spontaneous oxidation of o-diphenol, o-aminophenol, aromatic o-di-
amine and AH2 (result not shown), which should be computer cor-
rected in further [AH2] vs. time plots (in general this treatment is
applied to all the kinetic recordings). Fig. 1 shows the recordings for
the oxidation of 3,4-diaminotoluene. Kinetic analysis of these record-
a Bruker Avance 400 MHz instrument employing buffered solutions
(at pH=7.0) of pure samples in D2O. For those compounds that were
insoluble in this medium (4-fluor-1,2-phenylenediamine, 4-chloro-
1,2-phenylenediamine, 4-bromo-1,2-phenylenediamine, 4-methoxy-
1,2-phenylenediamine, o-phenylenediamine, 3,4-diaminotoluene,
2-aminophenol, 3-amino-4-hydroxytoluene, 4-amino-3-hydroxyto-
luene and 3-amino-L-tyrosine), the respective chemical shifts were esti-
mated with the ChemNMR module included in the ChemDraw v8.0, a
CambridgeSoft Desktop Software and corrected with the chemical
shifts of the corresponding head compound (o-phenylenediamine,
2-aminophenol, o-catechol).
ings according to Eq. (1) provides the values of ½AH2ꢀ∞ and λE S. The
ox
inset of Fig. 1 reveals the dependence of these parameters on the en-
zyme concentration.
When the disappearance of oxygen is measured, the experimental
data of time-based assays for the disappearance follow Eq. (2):
3. Results and discussion
S
Eoxt
½O2ꢀ ¼ ½O2ꢀ þ ½O2ꢀ e−λ
ð2Þ
f
∞
The suicide inactivation kinetic of tyrosinase acting on aromatic o-
diamines and o-aminophenols was studied. From this study, the kinetic
parameters that characterise the suicide inactivation process were
where [O2]f is the O2-value at t→∞, [O2]∞ is the O2 consumed at the
end of the reaction and λES is the apparent constant of suicide inactiva-
ox
obtained: the maximum apparent suicide constant (λE
S), the cat-
ox(max)
tion of tyrosinase in the presence of the corresponding substrate. These
parameters can be obtained by non-linear regression [33]. Note that
[O2]∞ becomes [Q]∞/2, reflecting the stoichiometry of the reaction.
Fig. 2 depicts the recordings for the disappearance of oxygen during
the oxidation of 3-amino-4-hydroxytoluene. Fitting these data to
Eq. (2) gives the values of [O2]∞ and λES . The dependence of these
parameters on the enzyme concentration ocxan be seen from Fig. 2 inset.
alytic constant (kcSat) and the parameter r which represents the number
of turnovers that 1 mol of enzyme makes before it is inactivated.
3.1. Oxidation of aromatic o-diamines
Scheme 1 represents a proposed mechanism to explain the
catalytic oxidation of aromatic o-diamines, which takes into account
(i) the suicide inactivation [6] and (ii) the studies carried out with
3,6-difluorocatechol, in which the existence of two kinetically signif-
icant pK's is evident [34]. Kinetic studies measuring the initial velocity
2.2.4. Determination of 13C NMR chemical shifts
The carbon chemical shifts given in Tables 1–3 were obtained from
the corresponding 13C NMR spectra, which were recorded at 298 K on
Table 3
Kinetic constants characterising the suicide inactivation of tyrosinase acting on o-diphenols.
o-Diphenol
λE
S ×103
r
kcSat
KmS
k6 ×10−3
δ1
δ2
δ3
δ4
Σδ
ox(max)
(s−1
)
(s−1
)
(mM)
(M−1 s−1
)
(ppm)
(ppm)
(ppm)
(ppm)
(ppm)
Catechol
8.92 0.27
8.54 0.25
8.21 0.25
2.21 0.01
0.96 0.08
0.85 0.03
99,994 1604
99,356 1377
98,366 1377
46,133 2306
386 40
874.1 30.2
859.2 28.3
842.1 26.1
102.6 19.2
0.39 0.05
8.11 0.30
0.16 0.01
0.60 0.03
0.10 0.01
0.55 0.08
1.09 0.14
0.07 0.01
5463.13 568.21
1432.00 148.02
8421.00 853.34
186.55 19.36
0.36 0.05
146.59
145.04
144.06
–
–
–
146.59
147.35
146.43
–
–
–
293.18
292.39
290.49
292.98
292.90
296.04
4-Chlorocatechol
4-Methylcatechol
L-Dopa
2,3-Dihydroxybenzoic acid
3,4-Dihydroxybenzoic acid
–
–
–
–
146.92
143.89
146.04
146.06
–
149.01
–
10,186 224
115.86 13.62
150.00

650
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1824 (2012) 647–655
R
R
B1
B2
NH2
B2
B1
k3
1
H2N
NH2
HN
H
N
N
N
2+
Cu
2+
N
k-3
N
N
Cu
2+
Cu
2+
1
Cu
N
O
H
N
O
N
N
N
N
H
(EmS)0
Step 1
Step 2
(
Em-S)1
k2
k-2
R
R
BH
B2
H2N
NH2
B1H
B2
B1
B2
NH
HN
S
H
N
N
N
N
o
N
N
N
N
Cu
Cu
2+
N
2+
Cu
2+
N
N
2+
2+
Cu
Cu
N
Cu
O
H
N
O
H
N
N
N
N
N
Step 3
(Em-S)2
Step 9
H2O
Step 11
Em
Ei
k3
k-3
2
2
k7
R
3
R
H2O2
R
R
k7i
B2H
B1H
B2H
B1H
3
NH
Cu
HN
HN
NH
NH
HN
N
N
N
N
N
N
2+
Cu
N
N
2+
Cu
2+
Suicide
inactivation
pathway
Cu
2+
O
H
HN
NH
O
N
N
N
H
N
O
(Em-S)
Step 4
3
(Eox-S)
Step 8
3k7
k-7
k3
3
2
2
H2O
R
R
B1H
B2
N
Diamine oxidase
activity
k7i
2
B1H
B1H
B2
B2
N
N
+
+
NH
HN
HN
Cu
HN
Cu
pathway
H
N
k-7i
N
N
N
N
N
N
N
N
N
N
N
H
Cu
2+
Cu
2
2+
Cu
2+
Cu
2+
O
O
N
O
O
N
N
H
H
Ed
Step 5
(Eox-S)
Step 10
(Eox-S)2
4
Step 7
O2
O2
k7
k-7
1
k-8
k8
1
R
k-6
k6
R
B2
B1H
B2
B1H
H2N
B2
B1H
R
NH2
NH2
HN
H
N
N
N
N
O
O
2+
Cu
2+
Cu
N
N
N
N
N
O
O
2+
Cu
N
N
O
2+
Cu
2+
Cu
2+
Cu
H2N
NH2
N
O
N
N
N
N
N
N
S
Eox
(Eox-S)1
(EoxS)0
Step 6

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1824 (2012) 647–655
651
gave the catalytic constant (kcSat) and the Michaelis constant (KmS )
102
(Table 1) [20]. Fig. 3 shows the recordings of the disappearance of
AH2 during the oxidation of 3,4-diaminotoluene, at long measuring
0.8
0.6
0.4
0.2
0.0
(a)
(f)
100
98
96
94
92
90
88
86
times. Fig. 3 inset shows the values λES [obtained by means of
ox
Eq. (1)] for different concentrations of 3,4-diaminotoluene. Therefore,
the hyperbolic behaviour observed in this representation allows us
the calculation of λES
and KmS for this compound, according to
ox(max)
Eq. (3) (Table 1) [6].
λS
½Sꢀ
Eoxð maxÞ
λES
¼
ð3Þ
0
KSm þ ½Sꢀ0
ox
0.00
0.05
0.10
0.15
0.20
0.25
[3,4-Diaminotoluene]0 (mM)
In the case of the oxidation of aromatic o-diamines, the velocity of
catalysis reflected in the value of the catalytic constant (kcSat) is very
low compared with those of the corresponding o-diphenols. This is
because Step 7 in Scheme 1 is much slower due to the proton transfer
from an amino group is less unlikely (compared when this transfer is
produced from a hydroxyl group). This slowing down of the catalytic
pathway has no effect on the suicide inactivation pathway because this
0
20
40
60
80
100
t (min)
process is limiting (Tables 1–3). From the values of λSE (max) obtained for
ox
the aromatic o-diamines (Table 1) and bearing in mind the 13C NMR
values of these molecules (Table 1), it is deduced that (a) the substitu-
tion of oxygen by nitrogen slows down both the catalysis and suicide in-
activation; (b) the first nucleophilic attack on CuB in Step 2 and Step 6 is
always carried out by the nitrogen bound to the carbon atom with the
lowest chemical shift (δ) in an attack that is facilitated by base 1 (B1);
(c) the second nitrogen atom can carry out a nucleophilic attack on
the CuA, transferring the proton to the second base (B2) or to the perox-
ide group of the oxy-tyrosinase form; (d) the lower the value of δ of the
carbon atom that carries the NH2 group responsible for the second at-
tack on the CuA, the more nucleophilic the nature of the nitrogen, and
so the catalytic pathway is preferred to the inactivation pathway and
the apparent inactivation constant is lower; and (e) the sum of the
chemical shifts of the carbon atoms that carry the diamine groups,
will be responsible for the values of kcSat; hence, the lower the sum of
the δ values, the greater the kcSat. Based on these predictions, the order
of the aromatic o-diamines is given in Table 1.
Fig. 3. Corrected recordings of the disappearance of AH2 in the suicide inactivation of
tyrosinase by the oxidation of different 3,4-diaminotoluene concentrations. The ex-
perimental conditions were 30 mM sodium phosphate buffer (pH 7.0), 0.26 mM O2,
wavelength=265 nm and the initial concentrations of AH2 and tyrosinase were
0.1 mM and 30 nM, respectively. The substrate concentrations were (mM) (a) 0.02,
(b) 0.05, (c) 0.075, (d) 0.1, (e) 0.15 and (f) 0.2. Inset. Values of λES for different
ox
3,4-diaminotoluene concentrations.
aminophenols (Table 2) are greater, since
kScat
k6
KmS
¼
ð4Þ
The aromatic o-diamines have low kcSat, KmS and k6 values (Table 1).
We have postulated that it can be due that these compounds have an
electron-rich ring and then repulsive π–π interactions with, probably,
a histidine residue of the active site could established [37]. o-diphe-
nols (Table 3) have high kcSat and k6 values [21], and the ring of the
substrate is less electron-rich and so there should be weaker repul-
sion at the active site. In the case of o-aminophenols (Table 2), the
catalytic constant values, kcSat, are higher than those corresponding
to the aromatic o-diamines and so KmS is slightly higher (see
Eq. (4)). Thus, the k6 values are lower in the case of aromatic o-di-
amines than in the case of o-diphenols (Tables 2 and 3).
The compounds 3,4-diaminobenzoic acid and 2,3-diaminoben-
zoic acid deserve additional comment. Taking into account its δ3
and δ4 values, 3,4-diaminobenzoic acid (Table 1) should have a
low λES
and one of the highest kcSat. In the case of 2,3-diamino-
ox(max)
benzoic acid, as the attack on the CuB is carried out by the C-2
group, a possible steric hindrance means that the behaviour of this
compound could well differ from the predicted behaviour (see
Table 1). The carboxylic group of 3,4-diaminobenzoic acid might in-
3.2. Oxidation of o-aminophenols
hibit tyrosinase, diminishing kcSat, λES
and KmS ; in this case, 3,4-
ox(max)
diaminobenzoic would be acting as a stoichiometric inhibitor with
the substrate [35,36].
The mechanism proposed to explain the catalytic oxidation and
the suicide inactivation of o-aminophenols is described in Scheme 2.
Fig. 4 shows the recordings obtained when varying the concentration
of substrate in the oxidation of 3-amino-4-hydroxytoluene. Fig. 4
When the data shown in Table 1 for the aromatic o-diamines are
compared with those obtained for o-diphenols (Table 3) kcSat can be
seen to be smaller by three orders of magnitude, but the apparent
inset depicts the hyperbolic dependence of λES vs. [S]0, while analysis
ox
inactivation constant (λES (max)) remains in the same range, so that
using Eq. (3) permits us to obtain λES
and KmS . When the oxida-
ox(max)
ox
the value of r is much lower (by approximately three orders of mag-
nitude) in the aromatic o-diamines (Tables 1 and 3).
The values of the Michaelis constants for the aromatic o-diamines
are low (Table 1) and of a similar order of magnitude to those of
the o-diphenols (Table 3), while the Michaelis constants of the o-
tion of the o-aminophenols is studied, the catalytic constants are
greater than those obtained for the corresponding aromatic o-di-
amines (Tables 1 and 2) and lower than those of the o-diphenols
(Table 3). The slowest step in the catalysis should be Step 7—the
transfer of the proton to the peroxide group in oxy-tyrosinase. The
Scheme 1. Structural mechanism proposed to explain the catalytic and suicide inactivation pathways of tyrosinase in its action on aromatic o-diamines. Em, met-tyrosinase; (EmS)0,
met-tyrosinase/aromatic o-diamine complex; (Em–S)1, met-tyrosinase/aromatic o-diamine complex axially bound to a CuB atom with the base (B1) forming a hydrogen bridge;
(Em–S)2, met-tyrosinase/aromatic o-diamine complex axially bound to a CuB atom with protonated base (B1H); (Em–S)3, met-tyrosinase/aromatic o-diamine complex axially
bound to the two Cu atoms; Ed, deoxy-tyrosinase; Eox, oxy-tyrosinase; (EoxS)0, oxy-tyrosinase/aromatic o-diamine complex; (Eox–S)1, oxy-tyrosinase/aromatic o-diamine complex
axially bound to a CuB atom; (Eox–S)2, oxy-tyrosinase/aromatic o-diamine complex axially bound to the CuB atom and with the proton transferred to the peroxide group; (Eox–S)3,
oxy-tyrosinase/aromatic o-diamine complex axially bound to the two Cu atoms with one proton transferred to the peroxide group and the other proton transferred to the base B2
(B2H); (Eox–S)4, oxy-tyrosinase/aromatic o-diamine complex axially bound to the CuB atom and with two protons transferred to the peroxide group;Ei, enzyme inactive.

652
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1824 (2012) 647–655
R
R
k3
B2
B1
1
B1
B2
OH
OH
H2N
HN
H
k-3
N
N
N
N
N
N
1
2+
2+
Cu
2+
2+
Cu
Cu
Cu
N
N
O
H
O
N
N
N
N
H
Step 1
(EmS)0
(Em-S)1
Step 2
k-2
k2
R
R
B1
B1H
B2
B2
N
H2N
OH
B1H
B2
O
HN
2+
Cu
N
N
H
2+ 2+
o
N
S
N
Cu
Cu
Cu
N
N
N
N
N
N
O
H
2+
2+
Cu
N
Cu
N
N
N
N
O
N
N
H
Em
Step 3
Step 11
(Em-S)2
Ei
Step 9
H2O
k3
k-3
2
k7
2
3
R
R
H2O2
R
k7i
B2H
R
B1H
3
B2H
O
B1H
HN
HN
O
O
HN
N
N
N
N
Cu
2+
Cu
2+
N
N
2+
N
N
2+
O
Cu
Cu
N
N
O
O
H
N
H
N
HN
O
(Em-S)3
Step 4
(Eox-S)3
Step 8
k3
3
k7
k-7
2
H2O
2
R
R
k7i
B1H
B2
Aminophenol
oxidase
2
B1H
B1H
B2
B2
_
O
HN
N
N
+
+
N
O
HN
Cu
Cu
k-7i
activity
pathway
H
N
N
N
N
N
2
N
N
2+
N
H
2+
N
N
Cu
2+
Cu
Cu
Cu
2+
O
O
N
O
O
N
N
N
H
N
H
Ed
(Eox-S)4
(Eox-S)2
Step 7
Step 10
Step 5
O2
O2
k-8
k8
k7 k-7
1
1
R
R
Suicide
inactivation
pathway
k-6
k6
B2
B1H
B2
B1H
B2
B1H
OH
_
HN
OH
H2N
2+
2+
Cu
O
O
H
R
N
N
N
Cu
N
N
2+
Cu
N
N
N
O
2+
Cu
N
N
N
O
O
2+
Cu
2+
Cu
N
N
O
N
N
N
N
N
H2N
OH
(Eox-S)1
Step 6
(EoxS)0
S
Eox

J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1824 (2012) 647–655
653
220
264
260
256
252
248
244
240
40000
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4
3
2
1
0
(a)
(f)
30000
20000
10000
0
200
180
160
140
120
100
8
0
4
8
12
16
0
2
4
6
[E]
0 (nM)
[3-amino-4-hydroxytoluene]0 (mM)
(a)
(e)
0
50
100
150
200
250
0
10
20
30
40
50
t (min)
t (min)
Fig. 4. Corrected recordings of the disappearance of oxygen in the suicide inactivation of
tyrosinase by the oxidation of different 3-amino-4-hydroxytoluene concentrations. The
experimental conditions were 30 mM sodium phosphate buffer (pH 7.0), 0.26 mM O2
and the initial concentrations of AH2 and tyrosinase were 0.15 mM and 2 nM, respec-
tively. The substrate concentrations were (mM) (a) 1, (b) 2, (c) 3, (d) 4, (e) 5 and (f)
Fig. 5. Corrected recordings of the disappearance of AH2 in the suicide inactivation of
tyrosinase by the oxidation of 3-amino-4-hydroxybenzoic acid for different enzyme
concentrations. The experimental conditions were 30 mM sodium phosphate buffer
(pH 7.0), 0.15 mM AH2, 0.26 mM O2, wavelength=280 nm, [S]0 =0.37 mM and [E]0
(nM): (a) 5, (b) 8, (c) 10, (d) 12 and (e) 15. Inset. Representation of the values of
6. Inset. Values of λES for different 3-amino-4-hydroxytoluene concentrations.
½AH2ꢀ∞ (○) and λES (•) vs. enzyme concentration.
ox
ox
160
speed of the suicide inactivation will depend on the efficiency of
the nucleophilic attack of the oxygen atom of the hydroxyl group. In
principle, the substrate attack will always occur through the amino
group on the CuB and then of the oxygen atom of the hydroxyl
group on CuA. Depending on the δ value of the carbon atom carrying
the hydroxyl group, the catalytic or inactivation pathway will be
favoured. When the δ value is low, the nucleophilic attack of the
oxygen atom of the hydroxyl group on the copper atom (CuA)
would be favoured and the proton transferred to base B2 thereby
favouring the catalytic pathway over the inactivation pathway. In
3
150
2
1
140
0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
this way, the value of λES
would be lower than in the case of o-
ox(max)
130
120
110
[3-Amine-4-hydroxybenzoic acid]0 (mM)
diphenols. The results depicted in Table 2 agree with this reasoning.
The catalytic constants are lower than for the o-diphenols, but, in
this case, only by one order of magnitude (Tables 2 and 3). The appar-
ent inactivation constants remain within the same order of magni-
tude as those corresponding to o-diphenols but are lower (Tables 2
and 3), which agrees with the behaviour of the aromatic o-diamines,
suggesting that the slowest step of the whole process (inactivation
(a)
(h)
0
50
100
150
200
250
pathway) is the transfer of
a proton from the amine group
t (min)
(aromatic o-diamines) or the hydroxyl group (o-aminophenols and o-
diphenols) to the peroxide group in the form Eox (Tables 1–3).
Table 2 shows the compounds ordered by the decreasing value
Fig. 6. Corrected recordings of the disappearance of AH2 in the suicide inactivation of
tyrosinase by the oxidation of different 3-amino-4-hydroxybenzoic acid concentra-
tions. The experimental conditions were 30 mM sodium phosphate buffer (pH 7.0),
0.26 mM O2, wavelength=280 nm and the initial concentrations of AH2 and tyrosi-
nase were 0.15 mM and 15 nM, respectively. The substrate concentrations were
(mM) (a) 0.1, (b) 0.15, (c) 0.22, (d) 0.3, (e) 0.37, (f) 0.45, (g) 0.52 and (h) 0.6. Inset.
of λES (max), where, in addition, it is possible to distinguish between
ox
the compounds bearing -OH (2-aminophenol) or -CH3 (3-amino-4-
hydroxytoluene and 4-amino-3-hydroxytoluene) in C-1, and the
group bearing -CH2-CHNH2-COOH (3-amino-L-tyrosine). In this way,
it is possible to distinguish the derivates of benzoic acid, which, as
mentioned above, may inhibit the enzyme through carboxylic
group. Such compounds would act as stoichiometric inhibitors, and
Values of λES for different 3-amino-4-hydroxybenzoic acid concentrations.
ox
byz varying the concentrations of enzyme and substrate, respectively.
so kcSat, KmS and λES
would decay. Figs. 5 and 6 show the record-
Fig. 5 inset and Fig. 6 inset show the dependence of ½AH2ꢀ∞ and λES on
ox(max)
ox
ings for the oxidation of 3-amino-4-hydroxybenzoic acid obtained
these concentrations. As can be seen, the concentration of ½AH2ꢀ∞ is
Scheme 2. Structural mechanism proposed to explain the catalytic and suicide inactivation pathways of tyrosinase in its action on o-aminophenols. Em, met-tyrosinase; (EmS)0, met-
tyrosinase/o-aminophenol complex; (Em–S)1, met-tyrosinase/o-aminophenol complex axially bound to a CuB atom; (Em–S)2, met-tyrosinase/o-aminophenol complex axially bound
to a CuB atom with the proton transferred to the base B1 (B1H); (Em–S)3, met-tyrosinase/o-aminophenol complex axially bound to the two Cu atoms with the proton of the OH
group transferred to the base B2 (B2H); Ed, deoxy-tyrosinase; Eox, oxy-tyrosinase; (EoxS)0, oxy-tyrosinase/o-aminophenol complex; (Eox–S)1, oxy-tyrosinase/o-aminophenol com-
plex axially bound to a CuB atom; (Eox–S)2, oxy-tyrosinase/o-aminophenol complex axially bound to the CuB atom and with the proton transferred to the peroxide group; (Eox
–
S)3, oxy-tyrosinase/o-aminophenol complex axially bound to the two Cu atoms with one proton transferred to the peroxide group and the other proton transferred to the base
B2 (B2H); (Eox–S)4, oxy-tyrosinase/o-aminophenol complex axially bound to the CuB atom with two protons transferred to the peroxide group; Ei, inactive enzyme.

654
J.L. Muñoz-Muñoz et al. / Biochimica et Biophysica Acta 1824 (2012) 647–655
300
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(a)
280
(h)
260
0
2
4
6
8
240
220
200
[3-hydroxyantrhanilic acid]0 (mM)
0
20
40
60
80
100
120
140
t (min)
Fig. 7. Corrected recordings of the disappearance of oxygen in the suicide inactivation
of tyrosinase by the oxidation of 2-amino-3-hydroxybenzoic acid for different enzyme
concentrations. The experimental conditions were 30 mM sodium phosphate buffer
(pH 7.0), 0.15 mM AH2, 0.26 mM O2, [S]0 =3 mM and [E]0 (nM): (a) 0.5, (b) 0.8,
(c) 0.9, (d) 1.1, (e) 1.2, (f) 1.3, (g) 1.35, (h) 1.4 and (i) 1.5. Inset. Representation of
Fig. 8. Corrected recordings of the disappearance of oxygen in the suicide inactivation
of tyrosinase by the oxidation of different 2-amino-3-hydroxybenzoic acid concentra-
tions. The experimental conditions were 30 mM sodium phosphate buffer (pH 7.0),
0.26 mM O2 and the initial concentrations of AH2 and tyrosinase were 0.15 mM and
0.5 μM, respectively. The substrate concentrations were (mM) (a) 0.5, (b) 1, (c) 2,
S
S
the values of ½O2ꢀ∞ (○) and λE (•) vs. enzyme concentration.
ox
(d) 3, (e) 4, (f) 5, (g) 6 and (h) 7. Inset. Values of λE for different 2-amino-3-hydro-
ox
xybenzoic acid concentrations.
directly proportional to the enzyme concentration, while λES is indepen-
acid and 3-hydroxy-4-aminobenzoic acid, which have similar kcSat, λ-
EoxS(max), KmS and r values. In the case of ortho derivates, the results
are practically identical to those obtained for the dihydroxylated
derivates (Tables 2 and 3) except that the KmS is higher in 2-amino-
3-hydroxybenzoic acid (3-hydroxyanthranilic acid). The attack car-
ried out by C-2 (CuB) points to the steric hindrance involved in the
increase in KmS [38].
ox
dent (Fig. 5 inset). However, λES shows a hyperbolic behaviour vs. the
ox
substrate concentration, while ½AH2ꢀ∞ is independent (Fig. 6 inset). By
means of Eq. (3) it is possible to obtain λSE
and KmS (Table 2).
ox(max)
Figs. 7 and 8 depict the recordings of the disappearance of oxygen dur-
ing the oxidation of 2-amino-3-hydroxybenzoic acid by tyrosinase,
varying the enzyme and substrate concentrations, respectively. Fig. 7
inset depicts the values of ½O2ꢀ∞ and λES vs. enzyme concentration.
The results described above highlight the importance of proton
transfer from the aromatic o-diamines and o-aminophenols to the
peroxide group in the oxy-tyrosinase. During the catalysis, two trans-
fers may occur to the oxy-tyrosinase, one to base B2 and the other to
the peroxide group in what is probably the limiting step (Step 7 in
Schemes 1 and 2). The transfer of two protons probably occurs during
suicide inactivation as well, but, in this case, both are transferred to
the peroxide group of the oxy-tyrosinase. The first nucleophilic at-
tack of the oxygen atom from the hydroxyl group involving the
transfer of the first proton to the peroxide group is the limiting
step of the catalysis, while the deprotonation involving no nucleo-
philic attack of the oxygen atom of the second hydroxyl group is
the limiting step in the inactivation (Step 10 en Schemes 1 and 2).
As regards Schemes 1 and 2, the first proton to the peroxide group
corresponds to Step 7 in both cases (limiting step of the catalysis).
The catalytic pathway continues in both Schemes with Step 8, in
which the proton is transferred to base B2. The inactivation path-
way in both Schemes continues with Step 10, both for aromatic o-
diamines (Scheme 1) and o-aminophenols (Scheme 2). This step
would be limiting for the suicide inactivation.
ox
Fig. 8 inset represents λES vs. the substrate concentration. From
Eq. (3), it is possible to obtoaxin λSE
and KmS (Table 2). The results
ox(max)
are similar to those obtained in the insets of Figs. 5 inset and 6.
Table 2 shows that the compound 2-aminophenol has the highest
kcSat and, as the δ value of the carbon atom carrying the hydroxyl
group is high, its λES
is also high and the r is the highest in
ox(max)
Table 2. Its KmS is higher than that of catechol, possibly because the
binding constant to the enzyme (k6) is hindered by the repulsion in-
volved in the π–π interactions (Table 2). As can be seen, the derivates
of toluene show similar values of kcSat and λES (max) and, therefore, of r.
Note that the Michaelis constants are one oorxder of magnitude higher
than that corresponding to 4-methylcatechol even though the kcSat is
one order of magnitude less, probably because the lower binding con-
stant (k6) resulting from the greater electron density of the aromatic
ring would hinder the binding (see Table 2). In the case of 3-amino-L-
tyrosine, the nitrogen in C-3 would be responsible for the binding in
CuB; the δ4 value is very high and so the catalytic constant falls to
1.41 s−1; furthermore, the first attack on CuB occurs through the
amino group bound to C-3, and the Michaelis constant increases
with respect to L-dopa, pointing to steric hindrances, as has been de-
scribed previously for the compounds 2-amino-3-hydroxybenzene-
sulphonamide and 3-amino-2-hydroxybenzenesulphonamide [38].
In the case of the benzoic acid derivates, as in the case of aromatic
o-diamines, the carboxylic group may react with the copper atoms,
Supplementary materials related to this article can be found on-
line at doi:10.1016/j.bbapap.2012.02.001.
Acknowledgements
inhibiting the enzyme and resulting in an apparent diminution of kcSat
λSE and KmS [35,36]. Note the low values of these parameters in
,
This paper was partially supported by grants from Ministerio de
Educación y Ciencia (Madrid, Spain) Project BIO2009-12956, Fundación
Séneca (CARM, Murcia, Spain) Projects 08856/PI/08 and 08595/PI/08,
and Consejería de Educación (CARM, Murcia, Spain) BIO-BMC 06/01-
0004. JLMM and FGM hold two fellowships from Fundación Caja Murcia
ox(max)
Table 2 and how the carbon atoms that support the NH2 group have a
lower δ value. In the same way, the withdrawing effect of the carbox-
ylic group is stronger if the substituent in C-4 is a hydroxyl group.
This is confirmed by comparing the 3-amino-4-hydroxybenzoic

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[31] J.L. Munoz, F. Garcia-Molina, R. Varon, J.N. Rodriguez-Lopez, F. Garcia-Canovas, J.
Tudela, Calculating molar absorptivities for quinones: application to the measure-
ment of tyrosinase activity, Anal. Biochem. 351 (2006) 128–138.
[32] J.N. Rodriguez-Lopez, J.R. Ros-Martinez, R. Varon, F. Garcia-Canovas, Calibration of
a Clark-type oxygen electrode by tyrosinase-catalyzed oxidation of a 4-tert-butyl-
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(Murcia, Spain). J.B. thanks the MICINN for a Ramon y Cajal Fellowship,
co-financed by the European Social Fund.
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