Action of tyrosinase on caffeic acid and its n-nonyl ester. Catalysis and suicide inactivation

Article abstract of DOI:10.1016/j.ijbiomac.2017.10.151

Different mechanisms for inhibiting tyrosinase can be designed to avoid postharvest quality losses of fruits and vegetables. The action of tyrosinase on caffeic acid and its n-nonyl ester (n-nonyl caffeate) was characterized kinetically in this work. The

Full text of DOI:10.1016/j.ijbiomac.2017.10.151

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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: www.elsevier.com/locate/ijbiomac
Action of tyrosinase on caffeic acid and its n-nonyl ester. Catalysis and
suicide inactivation
Antonio Garcia-Jimeneza, Jose Antonio Teruel-Pucheb, Pedro Antonio Garcia-Ruizc,
Adrian Saura-Sanmartind, Jose Bernad, Jose Neptuno Rodríguez-Lópeza,
Francisco Garcia-Canovasa,∗
a GENZ-Group of research on Enzymology, Department of Biochemistry and Molecular Biology-A, Regional Campus of International Excellence “Campus
Mare Nostrum”, University of Murcia, E-30100, Espinardo, Murcia, Spain1
b Group of Molecular Interactions in Membranes, Department of Biochemistry and Molecular Biology-A, University of Murcia, E-30100, Espinardo, Murcia,
Spain
c University of Murcia, Faculty of Veterinary, Group of Chemistry of Carbohydrates, Industrial Polymers and Additives, Department of Organic Chemistry,
E-30100 Murcia, Spain
d Group of Synthetic Organic Chemistry, Department of Organic Chemistry, Faculty of Chemistry, University of Murcia, E-30100 Espinardo, Murcia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2017
Received in revised form 24 October 2017
Accepted 24 October 2017
Available online xxx
Different mechanisms for inhibiting tyrosinase can be designed to avoid postharvest quality losses of
fruits and vegetables. The action of tyrosinase on caffeic acid and its n-nonyl ester (n-nonyl caffeate) was
characterized kinetically in this work. The results lead us to propose that both compounds are suicide
substrates of tyrosinase, for which we establish the catalytic and inactivation efficiencies. The ester is
more potent as inactivator than the caffeic acid and the number of turnovers made by one molecule of
the enzyme before its inactivation (r) is lower for the ester. We proposed that the anti-browning and
antibacterial properties may be due to suicide inactivation processes.
Keywords:
Tyrosinase
Caffeic acid
n-Nonyl caffeate
© 2017 Published by Elsevier B.V.
1. Introduction
and, so, have important applications in this respect [10–13]. In the
case of Chinese olive, it was demonstrated that n-nonyl caffeate
The process of food browning due to the oxidation of polyphe-
nols is notorius since it leads to a loss of nutritional, aesthetic and
organoleptic properties, and, so, of the food’s commercial value
[1–4]. Tyrosinase (EC 1.14.18.1), a copper-containing enzyme, is
one of the main agents responsible for this process. Its catalytic
centre has two copper atoms, each coordinated with three his-
tidine residues [5,6]. This enzyme catalyzes the hydroxylation of
monophenols to o-diphenols (monophenolase activity) and the
subsequent oxidation of o-diphenols to o-quinones (diphenolase
activity) [7].
can prevent postharvest putrefaction because of its anti-browning
and anti-bacterial effect [8]. Indeed, it was seen to attack Bacil-
lus subtilis, Escherichia coli, Staphylococcus aureus, Salmonella and
Klebsiella pneumoniae, but not Pseudomonas aeruginosa or Agrobac-
terium tumefaciens. A kinetic analysis of this compound led the same
authors to propose that n-nonyl caffeate is a reversible inhibitor of
tyrosinase at low concentrations and an irreversible inhibitor at
high concentrations [8]. It was this atypical behaviour of n-nonyl
caffeate that led us to study the action of the enzyme on this com-
pound based in the knowledge gained in our previous studies about
the action mechanism of tyrosinase [7].
Recently, a study was published on the protective effect of
n-nonyl caffeate on the Chinese olive (Canarium album) [8], the
quality of which during postharvest is affected by browning and
bacterial putrefaction [9]. Tyrosinase inhibitors reduce these effects
Therefore, n-nonyl caffeate and caffeic acid (CAFA), from which
it is derived, were studied to understand the mechanism involved,
while considering the possibility that such compounds, being sub-
strates of tyrosinase, may give rise to unstable o-quinones.
∗
Corresponding author at: Departamento Bioquímica y Biología Molecular A,
Facultad de Veterinaria, Campus de Espinardo, E-30100, Spain.
E-mail address: canovasf@um.es (F. Garcia-Canovas).
www.um.es/genz.
1
https://doi.org/10.1016/j.ijbiomac.2017.10.151
0141-8130/© 2017 Published by Elsevier B.V.
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Fig. 1. Chemical structures of (a) CAFA, (b) n-nonyl caffeate, (c) DHPAA, (d) PCA and (e) DHPPA.
2. Materials and methods
ratio [21–24]. The corresponding analytic expression and data anal-
ysis provided r.
2.1. Materials
2.2.3. 13C NMR assays
Mushroom
namide adenine dinucleotide (NADH), ascorbic acid (AH2),
3-(3,4-dihydroxyphenyl)propionic acid (DHPPA), 3,4-
tyrosinase
(3130 U/mg),
reduced
nicoti-
13C NMR spectra of n-nonyl caffeate, CAFA and DHPPA were
obtained on a Bruker Avance 300 MHz instrument. In the case
of DHPAA and PCA, the instrument used was a Bruker Avance
400 MHz. In all the cases DMSO was used as solvent. The ␦ values
were measured relative to those for tetramethylsilane using the
carbon signals of the deuterated solvent. The maximum line width
accepted in the NMR spectra was 0.06 Hz, so that the maximum
accepted error for each peak was 0.03 ppm.
dihydroxyphenylacetic acid (DHPAA), protocatechuic acid (PCA)
and CAFA were purchased from Sigma (Madrid, Spain). The
enzyme was purified as previously described [14] and the protein
concentration was determined by Bradford’s method [15], using
bovine serum albumin as the standard. Stock solutions of CAFA
and n-nonyl caffeate were prepared in 0.15 mM phosphoric acid
(aqueous solution) to prevent auto-oxidation and, this latter
compound was also solubilised in DMSO (4% (v/v)) as cosolvent.
Milli-Q system ultrapure was used throughout.
2.2.4. Computational docking
The chemical structure of n-nonyl caffeate was constructed
with PyMOL 1.8.4.0 [25] and its geometry was optimized with
MOPAC2012 software [26] and PM7 semiempirical Hamiltonian.
Rotatable bonds in the ligand were assigned by AutoDock-
Tools4 program [27,28]. Oxytyrosinase was prepared as previously
described [29]. Ligand was docked into the catalytic site of mush-
room tyrosinase from Agaricus bisporus (PDB code: 2Y9W) using
AutoDock Vina [30]. AutoDock Vina parameters were as follows:
receptor file; ligand file; xyz centre coordinate of the pocket residue
centred in the two copper ions; search space in each dimen-
sion, 11.3 Å; exhaustiveness, 24; and generation number of binding
modes, 10.
2.2. Methods
2.2.1. Synthesis method of n-nonyl caffeate
Catalytic amounts of sulfuric acid were added to a solution of
CAFA (100 mg, 0.6 mM) in nonanol (10 mL) and the reaction mixture
was stirred at 100 ◦C for 6 h. After this time, nonanol was removed
by vacuum distillation, and the resulting solid was crystallized from
ether, yielding the product of interest as a white solid.
2.2.5. Data analysis
2.2.2. Tyrosinase activity
The experimental disappearance of NADH with time follows the
equation:
Spectrophotometric assays were carried out with a PerkinElmer
Lambda-35 spectrophotometer (using water in the reference
cuvette), online interfaced with a compatible PC 486DX microcom-
puter controlled by UV-Winlab software, where the kinetic data
were recorded, stored, and analyzed. The diphenolase activity on L-
dopa and the monophenolase activity on L-tyrosine were followed
by measuring the increase of absorbance at ␭ = 475 nm led by the
formation of dopachrome. For its part, the diphenolase activity on
TBC (4-tert-butylcatechol) was measured at ␭ = 410 nm [16]. The
suicide inactivation of tyrosinase acting on CAFA was followed by
measuring the disappearance of NADH [17–20], originated by its
oxidation by the o-quinone, at 380 nm. Finally, the suicide inac-
tivation of tyrosinase acting on n-nonyl caffeate was followed by
reacting the enzyme with this compound in the presence of AH2
and measuring the on-going residual activity of tyrosinase in a high
concentration of TBC [17–20]. All the experiments were made in
30 mM phosphate buffer pH 7.0.
ꢀ
ꢁ
ꢂ
ꢃ
t
2
NADH = NADH − Q = NADH
−
c1 1 − e−c + c3 + ct
[
]
[
]
[
]
[
]
0
0
4
(1)
where
Q is the concentration of the product of the enzymatic
]
[
reaction, t is time and in which ci (i = 1–4) can be obtained by non-
linear regression [31].
The parameter c1 is equal to
Q
= NADH − NADH (the
∞
[
]
[
]
[
]
0
f
concentration of product obtained at the end of the reaction) and
c2 is equal to the corresponding ␭. The coefficient c3 represents the
uncertainty at zero time absorbance caused by the addition of the
enzyme at the start of the reaction, while c4 corresponds to the
slow spontaneous oxidation of o-diphenol and NADH. The effects
of both experimental artefacts should be computer subtracted in
further NADH against time.
In order to calculate the parameter r (the partition ratio or the
turnover numbers made by one mol of the enzyme before its inac-
tivation) when tyrosinase acts on n-nonyl caffeate, the enzyme was
incubated with this compound while bubbling with air. After that,
aliquots were taken at t → ∞ and the activity was assayed with TBC,
providing the values of residual activity vs the substrate/enzyme
3. Results and discussion
CAFA and n-nonyl caffeate are ortho-diphenolic compounds
(Fig. 1), and so can be chemically (by sodium periodate) [32] and
enzymatically (by tyrosinase) oxidized [7].
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Fig. 2. A. Scan spectra of the evolution of CAFQ obtained by the oxidation of CAFA by NaIO4 in default. The experimental conditions were [CAFA]0 = 150 ␮M and
[NaIO4]0 = 50 ␮M. Recordings were made every 2 min. Inset. Scan spectra (recordings “a–i” and spectrophotometric recording (“g”) at 550 nm of the evolution of the quinone
of n-nonyl caffeate obtained by the oxidation of this compound by NaIO4 in default. The experimental conditions were [n-nonyl caffeate]0 = 200 ␮M and [NaIO4]0 = 60 ␮M.
Recordings were made every 2 min. B. Scan spectra of the evolution of CAFQ obtained by the oxidation of CAFA by NaIO4 in excess. The experimental conditions were
[CAFA]0 = 50 ␮M and [NaIO4]0 = 400 ␮M. Recordings were made every 2 min. Inset. Scan spectra of the evolution of the quinone of n-nonyl caffeate obtained by the oxidation
of this compound by NaIO4 in excess. The experimental conditions were [n-nonyl caffeate]0 = 200 ␮M and [NaIO4]0 = 600 ␮M. Recordings were made every 2 min.
3.1. Characteristics of the o-quinones of the CAFA and n-nonyl
caffeate
Sodium periodate (NaIO4) is known to oxidize o-diphenols to o-
quinones [32]. Fig. 2A shows the spectrophotometric recordings of
the oxidation of CAFA by NaIO4 in default. Note the presence of two
imperfect isosbestic points due to the evolution of the o-quinone
in the presence of o-diphenol and the accumulation of interme-
diate compounds as is represented in Fig. 1SM, which shows the
possible set of reactions until the formation of a practically stable
product [33]. The same experiment in the case of n-nonyl caffeate
is shown in Fig. 2A Inset, where an increase of absorbance after
an initial decay can be seen in the form of scans (recordings “a–i”)
and as a spectrophotometric recording at 550 nm (recording “j”).
When CAFA is oxidized by NaIO4 in excess, the isosbestic points are
more clearly defined (Fig. 2B), since no intermediate products are
accumulated in the medium (Fig. 2SM). The oxidation of n-nonyl
caffeate by NaIO4 in excess generates an o-quinone, which evolves
in the same way as that of CAFA (Fig. 2B Inset).
3.2. Action of tyrosinase on CAFA and n-nonyl caffeate
Fig. 3. A. Scan spectra of the action of tyrosinase on CAFA. The experimental condi-
tions were [E]0 = 30 nM and [CAFA]0 = 50 ␮M. Recordings were made every 2 min. B.
Scan spectra of the action of tyrosinase on n-nonyl caffeate. The experimental con-
ditions were [E]0 = 30 nM and [n-nonyl caffeate]0 = 50 ␮M. Recordings were made
every 2 min.
The action of tyrosinase on CAFA and n-nonyl caffeate are rep-
resented in Fig. 3 Inset, respectively, the spectra being similar to
those obtained with NaIO4 in default (Fig. 2A Inset). Thus, CAFA and
n-nonyl caffeate are oxidized by tyrosinase, generating o-quinones.
feate, DHPPA, DHPAA and PCA (the last three compounds shown
for comparison). The ␦3 and ␦4 values are shown in Table 1.
3.3. 13C NMR
3.4. Effect of CAFA and n-nonyl caffeate on the monophenolase
and diphenolase activities of tyrosinase on L-tyrosine and L-dopa
The NMR spectra of CAFA and n-nonyl caffeate showed the
chemical shifts of the carbon with the hydroxyl groups (C-3 and
C-4), which indicated the potency of the nucleophilic attack on the
copper atom of the active site by the oxygen of the hydroxyl group
[34]. Hence, Fig. 3SM shows the NMR spectra of CAFA, n-nonyl caf-
When the action of tyrosinase on L-tyrosine is assayed (Fig. 4A),
the formation of dopachrome (recording “a”) shows a lag period,
which corresponds to the time needed for the system to accumulate
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Fig. 4. A. Spectrophotometric recordings at 475 nm of the monophenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of CAFA (␮M):
b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 100 nM and [L-tyrosine]0 = 0.5 mM. Inset. Spectrophotometric recordings at 475 nm of
the monophenolase activity of tyrosinase in the (a) absence and the presence of different concentrations of n-nonyl caffeate (␮M): b) 20, c) 50, d) 120, e) 250, f) 400. The
rest of the experimental conditions were [E]0 = 100 nM and [L-tyrosine]0 = 0.5 mM. B. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in
the (a) absence and the presence of different concentrations of CAFA (␮M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 40 nM and
[L-dopa]0 = 0.5 mM. Inset. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the (a) absence and the presence of different concentrations
of n-nonyl caffeate (␮M): b) 20, c) 50, d) 120, e) 250, f) 400. The rest of the experimental conditions were [E]0 = 40 nM and [L-dopa]0 = 0.5 mM.
Table 1
ever, this set of data can also be explained by a suicide inactivation
process, as is detailed in the following mechanism:
Values of the chemical shifts of the different suicide substrates obtained by 13C NMR
for the C-3 and C-4.
The kinetic mechanism proposed to explain the suicide inacti-
Compound
␦
4
␦
3
vation of tyrosinase is described in Fig. 7. The inactivation constant
of tyrosinase ki7 is the lowest, so the enzymatic forms of the mech-
DHPAA
DHPPA
CAFA
n-nonyl caffeate
PCA
145.7
145.9
149.0
149.2
150.8
144.8
144.3
146.5
146.4
145.6
2
anism of Fig. 7 are in the steady state (ES), which evolves through
a transition phase towards the inactivation of the enzyme [17].
The variation of [ES] and [Q] with time is given by:
d E
[
]
S
= −ki7 f(Eox − S)1[ES]
(2)
2
dt
a certain amount of o-diphenol in the steady state [35]. The addition
of CAFA (Fig. 4A) or n-nonyl caffeate (Fig. 4A Inset) reduces the
lag period and apparently activates the enzyme, since the rate is
increased (recordings “b-f”).
and
d[Q]
= 2k7 f(Eox − S)1[ES]
(3)
2
dt
In the case of the diphenolase assay, the addition of CAFA
(Fig. 4B) or n-nonyl caffeate (Fig. 4B Inset) apparently activates the
enzyme when the formation of dopachrome is measured (record-
ings “b–f”). This happens because these compounds act as electron
donors, converting Em to Ed, and the product formed in each case
absorbs in the visible spectrum, the catalytic constant for these
compounds being higher than for L-dopa (107.4 3.1 s−1) and L-
tyrosine (7.9 0.3 s−1) [34].
where f (Eox − S)1 represents the fraction of the species (Eox − S)1
in the steady state (Es). The integration of Eq. (2) and Eq. (3), con-
sidering [ES] = [E]0 and [Q] = 0 at t = 0, yields:
[Q] = [Q]∞(1 − e−ꢀt
)
(4)
with
2k7
2kcat
2
Q
=
E
[ ]0
=
E
[ ]0
= 2r E
[ ]0
(5)
[
]
∞
max
k7i
␭
2
3.5. Inactivation of tyrosinase by CAFA and n-nonyl caffeate
where the partition ratio (r) is
Fig. 5A and B show the spectrophotometric recordings of the
action of the enzyme on L-dopa in the presence of CAFA and n-nonyl
caffeate, but starting the reaction with the addition of L-dopa. In
this case, the previous contact of the enzyme with CAFA or n-nonyl
caffeate inactivates it, which is particularly evident when the con-
centration of n-nonyl caffeate is increased (Fig. 6). For this reason, it
was proposed that n-nonyl caffeate is a reversible inhibitor at low
concentrations and irreversible at high concentrations [8]. How-
kcat
r =
(6)
(7)
ꢀmax
the apparent inactivation constant (␭)
␭
S
max[ ]0
␭ =
KMS + S
[ ]0
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Fig. 5. A. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the absence of (a) and the presence of different concentrations of CAFA: b) 40,
c) 120, d) 250 and e) 400; pre-incubating the assay with this compound and starting the reaction with L-dopa. The rest of the experimental conditions were [E]0 = 10 nM and
[L-dopa]0 = 0.5 mM. B. Spectrophotometric recordings at 475 nm of the diphenolase activity of tyrosinase in the absence of (a) and the presence of different concentrations
of n-nonyl caffeate: b) 40, c) 120, d) 250 and e) 400; pre-incubating the enzyme with this compound and starting the reaction with L-dopa. The rest of the experimental
conditions were [E]0 = 10 nM and [L-dopa]0 = 0.5 mM.
and the initial rate in the steady state gives
2kcat
KMS + S
S
E
[ ]0[ ]0
V0
=
(8)
(9)
[ ]0
with
k7i k3k7 k7
k7 k7 k7 + k3k7 k7 + 1k3k7 k7 + k3k7 k7
2
3
␭
max
=
1
2
3
2
3
1
3
1
2
and
k7 k3k7 k7
2
3
k7 k7 k7 + k3k7 k7 + 1k3k7 k7 + k3k7 k7
kcat =
(10)
1
2
3
2
3
1
3
1
2
The stoichiometry of Q when it is reduced by NADH is:
NADH = NADH 0 − Q
(11)
(12)
[
]
[
]
giving
NADH = NADH + NADH ∞e−ꢀt
[
]
[
]
[
]
f
with
NADH ( NADH f value at t → ∞,
NADH = NADH
−
[
]
[
[ ]
]
[
]
[
]
Fig. 6. Effect of the concentration of tyrosinase in its action on L-dopa (0.5 mM) in the
absence (᭹) and the presence of different concentrations of n-nonyl caffeate (␮M):
(ꢀ) 20 and (᭿) 60; pre-incubating the enzyme with this compound and starting the
reaction with L-dopa.
∞
0
NADH =f Q ∞), being
␭
(the apparent constant of suicide
[
]
f
inactivation of tyrosinase in the presence of CAFA).
The suicide inactivation can also be studied by measuring the
residual activity of the enzyme, maintaining the concentration of
substrate constant by means of a reductant, such as AH2, which
transforms the o-quinone into o-diphenol, and measuring the
residual activity with a high concentration of TBC. Therefore, the
variation of the active enzyme (Es) corresponds to Eq. (2) and its
expression with time is:
where A is the residual activity corresponding to E − E at time
[
]
[ ]
r
0
i
t and A0 is the initial activity. The apparent inactivation constant
(␭) depends on the concentration of substrate according to Eq. (7).
3.5.1. Suicide inactivation of tyrosinase acting on caffeic acid
When tyrosinase acts on CAFA, an unstable o-caffeoquinone
(CAFQ) is originated (Fig. 3). The spectrophotometric recordings
made at short times allows the characterization of CAFA as sub-
strate of the enzyme, as previously described [36]. In this way, the
kinetic constants of the initial steady state are obtained: Michaelis
constant (KCMAFA), the catalytic constant (kCcaAtFA) and the catalytic
efficiency (kcat/KM) (Table 2).
i
7
f (E −S)
ox
t
= [E ]e−k
(13)
1
2
E
[
]
S
0
and the matter balance:
E
[
= E + E
(14)
]
[
]
[ ]
s
0
i
so
i
7
f (E −S)
ox
t
= E − E = E e−k
(15)
(16)
1
2
E
[
]
[
]
[
]
[
]
0
s
0
i
At long times, the initial steady state evolves through a tran-
sition phase towards the inactivation of the enzyme, in a process
that allows new parameters to be determined: maximum apparent
and
Ar = A0e−␭t
inactivation constant (␭max), the inactivation efficiency (␭max/KM
)
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Fig. 7. Kinetic mechanism proposed to explain the suicide inactivation of tyrosinase acting on o-diphenols. Em, metatyrosinase; EmS complex between Em and S; Ed, deoxy-
tyrosinase; Eox, oxytyrosinase; EoxS, complex between Eox and S; (Eox-S)1, axial binding complex of protonated S and Eox; (Eox-S)2, diaxial binding complex of deprotonated
S and Eox; (Eox-S)3, axial binding complex of deprotonated S and Eox; Ei, inactive enzyme.
Table 2
Kinetic constants which characterize the suicide inactivaction of tyrosinase by DHPPA, DHPAA, CAFA, n-nonyl caffeate and PCA.
kcat
KM
(mM)
kcat/KM
␭
max x 103
(s−1
␭
max/KM x 103
r
Ref.
(s−1
)
(mM−1s−1
)
)
(mM−1s−1
)
DHPPA
DHPAA
CAFA
n-nonyl caffeate
PCA
607.2 25.1
433.1 25.9
403 8.30
0.70 0.09
1.30 0.10
0.70 0.05
867 117
333 34
576 43
870 110
116 17
6.93 0.27
6.89 0.37
12.7 0.8
9.9 1.3
5.3 0.5
18.1 1.7
32.0 4.0
12.1 1.8
87646 1315
62838 1187
34300 645
27200 536
10186 224
[17]
[17]
This paper
This paper
[17]
8.1 0.3
0.07 0.01
0.85 0.03
and the partition ratio r. In this way, the study of CAFA as substrate
of tyrosinase is complete.
to Eq. (16), providing the apparent inactivation constant. More-
over Fig. 9A Inset show the dependence of ␭ vs [S]0, whose slope
corresponds to ␭max/KM (inactivation efficiency), according to Eq.
(7).
3.5.1.1. Effect of the concentration of substrate. The inactivation of
tyrosinase at different concentrations of substrate (CAFA) was mea-
sured by following the disappearance of NADH (Fig. 8A) at 380 nm.
The data analysis provides the apparent inactivation constant (␭)
if the spontaneous oxidation of CAFA and NADH are taken into
account (see data analysis) and the curves are fitted to Eq. (12).
3.5.2.2. Effect of the concentration of enzyme. The effect of the con-
centration of tyrosinase is shown in Fig. 9B, while the data analysis
according to Eq. (16) allows us to obtain the value of the apparent
inactivation constant, whose independence with respect to the con-
centration of the enzyme is depicted in Fig. 9B Inset, which agrees
with Eq. (7).
The value of [NADH] does not vary as expected in a suicide inac-
∞
tivation, while ␭
and KM are obtained by means of an analysis
max
of ␭ vs [S]0 (Fig. (8)A Inset) according to Eq. (7). This value of KM
agrees with that obtained with the data for the steady state [36].
The parameter r can be derived from ␭max and kcat, according to Eq.
(6).
3.5.2.3. Deduction and calculation of parameter r. The determina-
tion of r for CAFA was made applying Eq. (6). In the case of n-nonyl
caffeate, it was obtained measuring the residual activity at a con-
stant concentration of enzyme after consumption of the different
concentrations of substrate:
Step 1: the enzyme is pre-incubated with increasing concentra-
tions of substrate and bubbling with air in the medium to maintain
constant the concentration of oxygen.
3.5.1.2. Effect of the concentration of enzyme. The concentration of
tyrosinase was varied with a fixed concentration of substrate and
adding AH2 to maintain the concentration of the substrate constant
during each assay, since this compound reduces the o-quinone.
The residual activity was measured with high concentrations of
TBC (Fig. 8B). For its part, ␭, which is obtained by analyzing these
data according to Eq. (16) does not vary when the concentration
of enzyme changes, as Eq. (7) predicted (Fig. 8B Inset). Therefore,
the characterization of CAFA as suicide substrate is complete and
all the parameters can be gathered, as in Table 2: kcat, KM, kcat/KM
(catalytic efficiency), ␭max, ␭max/KM (inactivation efficiency) and r.
Step 2: the residual activity (Ar) at t → ∞ is determined with a
high concentration of TBC.
Step 3: the Ar/A0 is represented vs [S]0/[E]0, which give rises to
a straight line (Fig. 10). The points at which the straight line cuts
the axis, provide r.
Step 4: deduction of the analytic expression of r and calculation
of its value.
Fig. 7 indicates that the action of tyrosinase on o-diphenols
causes the suicide inactivation of the enzyme, but releases the prod-
uct o-quinone (Q), both in the catalytic and the suicide pathways.
In this way, matter balance:
3.5.2. Suicide inactivation of tyrosinase acting on n-nonyl caffeate
The low solubility and the broad spectra make a kinetic study of
the suicide inactivation of tyrosinase in its action on n-nonyl caf-
feate difficult. To make the characterization, the residual activity of
the enzyme was measured with high concentrations of TBC, main-
taining the concentration of substrate constant in each experiment
by the presence of AH2, which reduces de o-quinone to n-nonyl
caffeate.
S0 = S + Q
(17)
where S0 is the initial concentration of o-diphenol (n-nonyl caffeate
in this case), S is the concentration of substrate after the inactivation
and Q is the product.
3.5.2.1. Effect of the concentration of substrate. The effect of the con-
centration of substrate is shown in Fig. 9A and the data are fitted
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Fig. 8. A. Effect of the concentration of substrate on the suicide inactivation during the action of tyrosinase on caffeic acid using NADH as reductant. The disappearance of
NADH was measured at 380 nm. The experimental conditions were [E]0 = 20 nM, [NADH]0 = 2 mM and [CAFA]0 (mM): a) 0.1, b) 0.2, c) 0.25, d) 0.35, e) 0.4, f) 0.6, g) 0.8 and
h) 1. Inset. Variation of the apparent inactivation constant (␭) with the concentration of CAFA. The experimental conditions were the same as in the main figure. B. Effect
of the concentration of the enzyme on the suicide inactivation caused by the action of tyrosinase on CAFA using AH2 as reductant. Different concentrations of tyrosinase
(nM): a) 10, b) 20, c) 30 and d) 50; were pre-incubated in the presence of AH2 (0.7 mM) and CAFA (0.5 mM). Subsequently, various aliquots (10% of the total on the medium
assay) were taken to test the activity of tyrosinase on TBC (3 mM) after different times of pre-incubation. Inset. Variation of the apparent inactivation constant (␭) with the
concentration of enzyme. The experimental conditions were the same as in the main figure.
Fig. 9. A. Effect of the concentration of substrate on the suicide inactivation caused by the action of tyrosinase on n-nonyl caffeate using AH2 as reductant. Tyrosinase (30 nM)
was pre-incubated in the presence of ascorbic acid (0.7 mM) and different concentrations of n-nonyl caffeate (␮M): a) 10, b) 20, c) 30 and d) 50. Subsequently, various aliquots
(10% of the total on the medium assay) were taken to test the activity of tyrosinase on TBC (3 mM) after different times of pre-incubation. Inset. Variation of the apparent
inactivation constant (␭) with the concentration of n-nonyl caffeate. The experimental conditions were the same as in the main figure. B. Effect of the concentration of the
enzyme on the suicide inactivation caused by the action of tyrosinase on n-nonyl caffeate using AH2 as reductant. Different concentrations of tyrosinase (nM): a) 10, b) 20,
c) 30 and d) 50; were pre-incubated in the presence of ascorbic acid (0.7 mM) and n-nonyl caffeate (30 ␮M). Subsequently, various aliquots (10% of the medium assay) were
taken to test the activity of tyrosinase on TBC (3 mM) after different times of pre-incubation. Inset. Variation of the apparent inactivation constant (␭) with the concentration
of enzyme. The experimental conditions were the same as in the main figure.
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turnovers of the enzyme, giving rise to o-quinones. Hence Eq. (24)
becomes:
E
2 S
]
0
Ar
[
[
]
]
[
S
= 1 −
=
]
0
(25)
E
0
2(r + 1) E
A0
[
and the cut point on the X axis (Ar/A0 = 0) is:
S
E
[
[
]
]
0
0
= r + 1
(26)
(27)
or
S
[
[
]
]
0
0
r =
− 1
E
In the case of n-nonyl caffeate, the catalytic efficiency (kcat/KM
)
(Table 2) can be obtained from r (Fig. 10) and the inactivation effi-
ciency (␭max/KM) (Fig. 9A), resulting:
kcat/KM
kcat
r =
=
(28)
(29)
␭
␭
max/KM
max
Hence, the catalytic efficiency is:
kcat
KM
␭
max
KM
= r
Fig. 10. Determination of parameter r by means of the sensivity of tyrosinase to
inactivation at different molar ratios of n-nonyl caffeate/enzyme. Plot of residual
activity against the [n-nonyl caffeate]/[tyrosinase] ratio. Residual activity was mea-
sured with TBC as substrate after incubations. During the assay, air was bubbled
through to maintain the concentration of oxygen saturating. The line was fitted and
the value of the intercept at the X-axis was used to calculate r.
as is shown in Table 2.
In this way, the kinetic characterizations of CAFA and n-nonyl
caffeate as suicide substrates of tyrosinase is complete.
3.6. Molecular docking
The number of turnovers realized by the enzyme before its inac-
tivation (r) is:
The docking of n-nonyl caffeate at the binuclear copper active
site of oxytyrosinase is shown in Fig. 11 with a dissociation constant
of 0.5 mM, which is lower than that reported for CAFA (1.18 mM)
[36]. The hydrogen atom of the C1-OH phenolic group is 2.7 Å from
the oxygen atom of the amide bond of N260 and 3.9 Å from the oxy-
gen atom of the carboxamide group of N260. The hydrogen atom of
the C2-OH phenolic group is 2.9 Å from an oxygen atom of the per-
oxide ion and 3.8 Å from the oxygen atom of the carboxamide group
of N260. Therefore, hydrogen bonds interactions between the phe-
nolic groups of the ligand and the protein are possible. Besides, the
aliphatic chain of the nonyl group is 3.6 Å from the aliphatic chain of
V283, contributing to the stabilization of the docking configuration
by hydrophobic interactions (Fig. 11).
The disposition of the phenyl ring and the phenolic groups of
n-nonyl caffeate is similar to that reported for CAFA previously
[36]. The presence of the long alkyl chain of the nonyl group makes
the phenyl ring rotates in its plane and only one hydroxyl group
is bonded to the peroxide atom instead of two, as in the case of
CAFA [36]. However, this configuration would allow electrostatic
interactions with N260, hydrophobic interactions with V283 and,
therefore, a lower Kd value than that of CAFA.
Q − 2 E /2
[
]
[ ]
i
r =
(18)
E
[
]
i
since two molecules of Q are released in each turnover, even in
the inactivation pathway.
The expression of Q can be obtained from Eq. (18):
Q = 2(r + 1) E
(19)
[
]
[ ]
i
The expression of E can be obtained according to:
[
]
i
S
[
= S + 2(r + 1) E
[ ]
(20)
(21)
]
[ ]
i
0
S
[
2(r + 1)
− S
[ ]
]
0
E
[
=
]
i
The matter balance of the enzyme is:
E
[
= E − E
(22)
(23)
]
[
]
[ ]
S
0
i
where E is the active enzyme.
[
]
S
E
S
]
0
2(r + 1) E
− S
[ ]
[
[
]
]
[
S
= 1 −
E
0
[
]
0
The docking of n-nonyl caffeate to deoxytyrosinase has already
been reported [8]. This docking proposed interactions with N260
and V283, nevertheless, the oxygen atoms of the phenolic groups
interact with copper ions. In our study, the phenolic groups are
located further from the copper ions because of the presence of
the peroxide ion in the oxytyrosinase. Thus, the phenolic group
would interact with the peroxide ion of oxytyrosinase and with
copper ions of deoxytyrosinase. However, the docking to oxytyrosi-
nase is the most significant, since the oxygen saturates the enzyme
(deoxytyrosinase) [37] and, so, the substrate does not bind when
the studied concentrations are used, as indicated in Fig. 11.
In summary, the results obtained from the docking studies show
that the dissociation constant for n-nonyl caffeate binding to oxy-
tyrosinase (0.5 mM) is lower than that for CAFA (1.18 mM). These
data could explain the fact that the catalytic efficiency and the inac-
tivation efficiency are higher for n-nonyl caffeate, which may be
If the substrate is consumed ( S = 0), Eq. (23) becomes:
[ ]
E
S
]
0
Ar
[
[
]
]
[
S
= 1 −
=
]
0
(24)
E
2(r + 1) E
A0
[
0
Taking into account the evolution of the quinone from n-nonyl
caffeate (see Fig. 1BSM), the oxidation of the compound by the
enzyme is analogous to the oxidation by NaIO4 in default. Assum-
ing that the reaction starts with two molecules of n-nonyl caffeate,
one of them is oxidized to o-quinone, which reacts with the other
molecule of substrate, giving rise to a dimmer, which, in turn,
changes to the leuco form. The leuco form is oxidized by the
enzyme, giving rise to another quinone, which also becomes leuco.
Finally, this latter compound is oxidized by tyrosinase, giving rise
to an o-quinone, which becomes a diquinone by means of another
enzymatic oxidation. Therefore, one initial molecule is oxidized
three times and the other is oxidized only once, which means two
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Fig. 11. Computational docking of n-nonyl caffeate. Lowest energy AutoDock-Vina of n-nonyl caffeate at the tyrosinase active site is shown as green sticks. Distances (Å) are
shown by dotted yellow lines. The atom colors are as follows: red = oxygen, blue = nitrogen, brown = copper, carbon = green and white = hydrogen. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
associated with a lower KM, since the ␦4 values are similar (Table 1)
and, so, the kcat values should be similar too. It is proposed that the
hydrophobic chain of n-nonyl caffeate could lead to a high affinity
for the enzyme, since the hydrophobic repulsion with the medium
pushes the compound into the active site. This indicates that the
n-nonyl caffeate is a more potent suicide substrate for tyrosinase
than CAFA.
Conflict of interest
The authors declare no conflict of interest and funds.
Acknowledgements
This work was supported by the Fundación Seneca (CARM,
Murcia, Spain) under Projects 19545/PI/14, 19304/PI/14 and
19240/PI/14; MINECO under Projects SAF2016-77241-R and
CTQ2014-56887-P (Co-financing with Fondos FEDER); and Univer-
sity of Murcia, Murcia under Projects UMU15452 and UMU17766.
A. Garcia-Jimenez has a FPU fellowship from the University of Mur-
cia.
4. Conclusion
When the inhibition of tyrosinase by CAFA and n-nonyl caf-
feate was studied in this work, both compounds were seen to
act as suicide substrates of the enzyme due to their o-diphenolic
structure. The inhibitory mechanism is the same at low and high
concentrations, but the inactivation rate varies. The irreversible
inactivation (suicide kinetic) is slow at low concentrations, and
fast at high concentrations. n-Nonyl caffeate has a higher cat-
alytic efficiency (870 110 mM−1 s−1) and inactivation efficiency
(32.0 4.0 mM−1 s−1) than the other studied o-diphenols with a
similar structure (DHPPA, DHPAA, CAFA and PCA). Since the ␦4
values for CAFA and n-nonyl caffeate are similar, the catalytic con-
stants might be expected similar too, so the differences in the
catalytic efficiency (1.5 times) and the inactivation efficiency (1.8
times) could be caused by the lower KM value for n-nonyl caf-
feate, which, in turn, is due to the presence of a hydrophobic
chain. This behaviour as suicide substrate could explain its anti-
browning effect and antibacterial activity against Bacillus subtilis,
Escherichia coli, Staphylococcus aureus, Salmonella and Klebsiella
pneumoniae.
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