Structure-activity relationships of various amino-hydroxy-benzenesulfonic acids and sulfonamides as tyrosinase substrates

Article abstract of DOI:10.1016/j.bbagen.2011.05.002

Background: o-Aminophenols have been long recognised as tyrosinase substrates. However their exact mode of interaction with the enzyme's active site is unclear. Properly vic-substituted o-aminophenols could help gain some insight into tyrosinase catalytic

Full text of DOI:10.1016/j.bbagen.2011.05.002

Biochimica et Biophysica Acta 1810 (2011) 799–807
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Structure–activity relationships of various amino-hydroxy-benzenesulfonic acids and
sulfonamides as tyrosinase substrates
c
d
a
b,e
Antonio Rescigno a,b, , Frédéric Bruyneel , Alessandra Padiglia , Francesca Sollai , Andrea Salis
,
⁎
Jaqueline Marchand-Brynaert c, Enrico Sanjust a,b
Dipartimento di Scienze e Tecnologie Biomediche, Università di Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy
Consorzio per lo Sviluppo dei Sistemi a Grande Interfase (CSGI), via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy
Université Catholique de Louvain, Institute of Condensed Matter and Nanosciences, Bâtiment Lavoisier, Place Louis Pasteur 1, 1348 Louvain-la-Neuve, Belgium
a
b
c
d
Dipartimento di Scienze Applicate ai Biosistemi, Università di Cagliari, Monserrato (CA), Italy
Dipartimento di Scienze Chimiche, Università di Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: o-Aminophenols have been long recognised as tyrosinase substrates. However their exact mode
of interaction with the enzyme's active site is unclear. Properly vic-substituted o-aminophenols could help
gain some insight into tyrosinase catalytic mechanism.
Methods: Eight vic-substituted o-aminophenols belonging to two isomeric series were systematically evaluated
as tyrosinase substrates and/or activators and/or inhibitors, by means of spectrophotometric techniques and
HPLC-MS analysis. Some relevant kinetic parameters have also been obtained.
Received 13 January 2011
Received in revised form 3 May 2011
Accepted 4 May 2011
Available online 13 May 2011
Keywords:
Results: Four o-aminophenolic compounds derived from 3-hydroxyorthanilic acid (2-amino-3-hydroxybenze-
nesulfonic acid) and their four counterparts derived from the isomeric 2-hydroxymetanilic acid (3-amino-2-
hydroxybenzenesulfonic acid) were synthesised and tested as putative substrates for mushroom tyrosinase.
While the hydroxyorthanilic derivatives were quite inactive as both substrates and inhibitors, the
hydroxymetanilic compounds on the contrary all acted as substrates for the enzyme, which oxidised them to
the corresponding phenoxazinone derivatives.
Tyrosinase activity
Active site
Benzenesulfonic acid
Benzenesulfonamide
Phenoxazinone
General significance: Based on the available structures of the active sites of tyrosinases, the different affinities of
the four metanilic derivatives for the enzyme, and their oxidation rates, we propose a new hypothesis regarding
the interaction between o-aminophenols and the active site of tyrosinase that is in agreement with the obtained
experimental results.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
enzyme's reduced (deoxy) form the two copper ions are in their cuprous
state; in the oxy form, a peroxo μ–η2:η2 bridge between the two cupric
Tyrosinases (monophenol, o-diphenol:dioxygen oxidoreductases, EC
1.14.18.1) are copper-containing enzymes that catalyse the hydroxyl-
ation of monophenols to the corresponding o-diphenols (catechols) and
the oxidation of o-diphenols to o-quinones [1]. The first activity is often
referred to as cresolase or monophenolase activity and the second as
diphenolase or catecholase activity. In its active site, tyrosinase binds two
non-equivalent cupric ions that form a cluster of spectroscopic type III
(antiferromagnetic coupling that renders the enzyme EPR-silent) and
are joined by a water or hydroxide bridge in the enzyme's resting
state (met form). The two cupric ions forming the dicopper cluster are
coordinated by six conserved histidine residues belonging to the same
polypeptide chain and are named CuA (the cupric ion that is coordinated
by three histidines proximal to the N-terminus) and CuB (that coor-
dinated by three histidines proximal to the C-terminus) [2]. In the
ions is present. The active site of the enzyme is the subject of several past
and ongoing studies aimed at outlining its shape and identifying the
amino acid residues involved [3], studying the modes of substrate
recognition and binding, and elucidating the precise mechanisms of the
reactions throughout the catalytic cycle [4–8]. Mushroom tyrosinase is
undoubtedly the most studied tyrosinase and has been used to generate
much kinetic data. Unfortunately, it has not yet been crystallised, so its
3D structure is unknown. Comparative modelling is currently the most
accurate method to predict the 3D structure of proteins when no
experimental structure is available [9].
With respect to tyrosinase substrates, a wide range of phenols and
catechols have been found to be o-hydroxylated and quinonised,
respectively, by the enzyme [1]. All known tyrosinases (with rare
exception [10]) show a typical lag time when acting on monophenols
[11–13]. This lag time can be defined as the time required to reach
the stationary state speed with respect to the diphenol concentration.
The lag time can be influenced by many factors, including the presence
of minute amounts of diphenols such as L-dopa. Conversely, several
⁎
Corresponding author at: Dipartimento di Scienze e Tecnologie Biomediche, Università
di Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy. Fax: +39 070 675.4527
E-mail address: rescigno@unica.it (A. Rescigno).
0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2011.05.002

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properly substituted phenols have been found to be more or less effec-
tive inhibitors of the enzyme [14].
H-phenoxazin-3-one-1,9-dicarboxylic acid, CA) after the white-rot
fungus Picnoporus cinnabarinus, from which it is isolated [21]. Other
phenoxazinone derivatives closely resembling CA are well known to
exist in nature. These include 2-amino-3-H-4,6-dimethyl-3-phenoxazin-
3-one-1,9-dicarboxylic acid (4,6-dimethylcinnabarinic acid, also known
as actinocin), which is the non-amino acidic component of the
dicyclopeptidic antitumor and antibiotic drug actinomycin D [22]. In
general, phenoxazinone derivatives are expected to arise whenever an o-
aminophenol with free 4- and 5-positions is properly oxidised [23].
Very recently, some studies have explored the synthesis of a
number of phenoxazinone derivatives whose outstanding feature is
the presence of sulfonate or sulfonamide functionalities directly
bound to the tricyclic cores of those molecules [24,25]. In the present
study, some o-aminophenols bearing the sulfonate or sulfonamide
functionalities (Table 1) were synthesised and studied as putative
Tyrosinase substrates are not limited to phenols and catechols; many
o-aminophenols and aromatic o-diamines have been found to be
quinonised by tyrosinase, and even monoamines have been reported to
be o-hydroxylated by the enzyme [15–18]. While aromatic o-diamines
are unnatural compounds, o-aminophenols are commonly found in
living systems; the most studied is perhaps 2-amino-3-hydroxybenzoic
acid (3-HAA, 3-hydroxyanthranilic acid), which arises from tryptophan
metabolism [19]. 3-HAA is not only a good substrate for tyrosinase but
also an activator of its monophenolase activity; treatment of tyrosinase
with 3-HAA substantially shortens the lag time [20]. The oxidation
product of 3-HAA arises from an oxidative coupling of an intermediate
quinoneimine derivative; it has a peculiar structure, based on a tricyclic
phenoxazinone nucleus, and is known as cinnabarinic acid (2-amino-3-
Table 1
The structures of the studied compounds, series 1(a–d) and 2(a–d).
Compound
IUPAC name
Chemical structure
1a
2-Amino-3-hydroxy-benzenesulfonic acid (3-hydroxy-orthanilic acid, 3-HOA)
1b
1c
2-Amino-3-hydroxy-benzenesulfonamide
2-Amino-3-hydroxy-benzene-N-cyclohexyl sulfonamide
1d
2-Amino-3-hydroxy-benzene-N-(3-dimethylamino-propyl)sulfonamide
2a
2b
3-Amino-2-hydroxy-benzenesulfonic acid (2-Hydroxymetanilic acid, 2-HMA)
3-Amino-2-hydroxy-benzenesulfonamide
2c
3-Amino-2-hydroxy -benzene-N-cyclohexyl sulfonamide
2d
3-Amino-2-hydroxy- benzene-N-(3-dimethylamino-propyl)sulfonamide

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Scheme 1. Synthesis of 3-amino-2-hydroxy-benzenesulfonamide (Method A: [24,25]). Reagents and conditions: (i) n-BuLi (2.1 equiv.), −78 °C to −45 °C, 2 h; (ii) SO2Cl2 (5 equiv.),
−78 °C to 25 °C, overnight; (iii) 25% aq. NH3, DCM, 25 °C, overnight; (iv) 5 N HCl, 100 °C, overnight.
tyrosinase substrates with the aims of shedding light on the structural
features required for a good fit in the enzyme active site and of gaining
more information about the mode of action of tyrosinase.
produced a mixture of 2b (quantitatively determined to be minor) and
2a (2-HMA; quantitatively determined to be major). Pure 3-amino-2-
hydroxy-benzenesulfonamide (2b) was obtained by concentration in
vacuo and flash chromatography (ether/ethyl acetate, 7:3) as a brown
powder (yield=20%): 1H NMR (300 MHz, CD3OD) δ=7.08 (d,
J=7.8 Hz, 1 H), 6.93 (d, J=7.2 Hz, 1 H), 6.77 (dd, J=7.8, 7.2 Hz, 1 H)
ppm. 13C NMR (75 MHz, CD3OD) δ=143.7, 138.8, 128.5, 121.5, 120.7,
117.8 ppm. MS (ESI) m/z=187.1 [M–H+] for C6H8N2O3S. The com-
pound synthesised here was identical to a reference sample obtained
according to Method B (Scheme 2) from Binggeli et al. (Hoffman-La
Roche Patent US 2007093521, WO 2007025897) [26], which was
practical on the multigram scale.
2. Experimental
2.1. Preparation of aminophenols
The tyrosinase substrates were prepared using the standard
equipment in the Laboratory of Organic and Medicinal Chemistry at
UCL (Louvain-la-Neuve, Belgium).
The multistep syntheses of compounds 1a (3-HOA) and compounds
1b, 1c, 1d, 2a (2-MA), 2c and 2d fully described in references [24] and
[25] respectively, were performed according to the literature. The novel
compound 2b was similarly prepared, as depicted in Scheme 1 (Method
A). Briefly, 2-tert-butylbenzoxazole-7-sulfonyl chloride (4), prepared in
two steps from N-(3-chlorophenyl)-2,2-dimethylpropionamide (3),
was dissolved in CH2Cl2 (5 mL/mmol) and treated at 0 °C with 25%
NH4OH aqueous solution (5 mL/mmol). The mixture was stirred for
17 h at 20 °C. The organic phase was separated, washed with 1 N HCl
(3×5 mL/mmol), dried (MgSO4), and concentrated. The residue was
purified by flash chromatography (hexane/ethyl acetate, 7:3) to
produce 2-tert-butylbenzoxazole-7-sulfonamide (5) as a white solid
(yield=40%). Hydrolysis with 6 N HCl (10 mL/mmol) at 100 °C for 17 h
All other chemicals used were obtained from Sigma-Fluka-Aldrich
(Milan, Italy) and were of reagent grade and used without further
purification.
2.2. Enzyme and assays
Mushroom tyrosinase was purified as previously described [27].
Enzyme activity was estimated in the presence of a given amount of
enzyme, 100 mM potassium phosphate buffer (pH 6.5), and 0.33 mM
L-tyrosine. We consider the amount of enzyme that increases the
absorbance at 305 nm by 0.001 per min in the assay conditions to be one
enzyme unit [28]. Because tyrosinase catalyses a reaction between two
Scheme 2. Synthesis of 3-amino-2-hydroxy-benzenesulfonamide (Method B: US Patent 2007093521). Reagents and conditions: (i) HO3SCl (7 equiv.), 0 °C to 20 °C, 16 h; (ii) HNO3-
H2SO4 (1:1, 1.5 equiv.), DCM, 5 °C to 20 °C, 2 h; (iii) 25% aq. NH3, (1.5 equiv.), THF, Et3N (1.5 equiv.), 0°C to 20 °C, 16 h; (iv) H2 (1 atm), Pd-C (10%), MeOH, 4 h, 20 °C.

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substrates, molecular oxygen and a mono- or diphenol, the assays were
carried out in air-saturated solutions.
ability to behave as enzyme substrates, and therefore their absorption
spectra remained almost unchanged.
Stock solutions of aminophenolic derivatives to be tested as
putative tyrosinase substrates were prepared at 1 mM in 10% (v/v)
aqueous ethanol. The effect of tyrosinase on each of the aminophe-
nolic compounds was measured in a final volume of 1 mL 0.1 M
potassium phosphate buffer at pH 6.5. The reaction mixture also
included 200 μM aminophenol and 130 tyrosinase units. The reaction
mixtures contained 1% (v/v) ethanol. This concentration of ethanol
had no measurable effect on enzyme activity.
In some experiments monophenolase activity was also measured
in the presence of tert-butylphenol (BP), while diphenolase activity
was tested with 4-tert-butylcatechol (BC) as the substrate. Indeed, the
catalysed oxidation of these substrates gives rise to 4-tert-butyl-o-
benzoquinone (BQ), which is more stable than the product derived
from oxidation of L-tyrosine or L-dopa. Moreover, this o-quinone can
be coupled to an aromatic amine to yield a blue adduct [29,30]. This
adduct exhibits spectral features different from those of the parent
phenol and its o-quinone counterpart and is therefore a better readout
of the effect of some of the experimental compounds on enzyme
activity. Furthermore, the higher sensitivity of this test allowed for the
use of lower enzyme amounts (13 U) when measuring diphenolase
activity. KM values for compounds 2(a–d) were measured using
20 mM stock solutions in 20% (v/v) ethanol. All other conditions were
the same.
On the contrary, compounds 2(a–d) were all enzyme substrates,
although enzymatic oxidation of compound 2a was comparatively
slow. Fig. 1 shows the spectral variations caused by tyrosinase action
towards compounds 2(a–d). Fig. 2 shows a quantitative comparison of
tyrosinase action on the four different substrates.
It is worth noting that sulfonyl moieties in position 1 should render
the corresponding quinoneimines particularly reactive as electrophiles
due to the strong electron-withdrawing effect of these substituents.
Hence, the formation rates for the corresponding phenoxazinone
derivatives should be very high, with absorption maximum centred
around 440 nm (at pH 7) [16,25]. In fact, recent studies demonstrated
that the laccase-mediated oxidation of o-aminophenols brings to the
formation of phenoxazinone derivatives [25,33]. Here, mushroom
tyrosinase allowed to obtain the same kind of oxidised products.
Indeed, Fig. 3 shows the mass spectrum (positive electrospray ionisation
mode) of the main products obtained by the oxidation of compound 2b.
The peaks at 372 and 373 m/z have quite high abundances and are
compatible with phenoxazinone derivatives. This is also confirmed by
the intense peaks at 356 and 357 m/z, due to the formation of stable
ions likely obtained by the loss of a NH2 group (16 m/z), and by those
at 340 and 341 due to the loss of a further NH2 group. As the four
phenoxazinone derivatives have similar structures, the respective ε
values should also be similar, allowing for quantitative comparison. In
All the experiments were performed in triplicate and the given
data are the corresponding mean values.
Characterization of reaction products was carried out using a LC–
MS system composed by a Varian 212-LC HPLC (Varian Inc, CA, USA)
and a Varian 310-MS triple quadrupole mass spectrometer (Varian
Inc, CA, USA) with positive electrospray ionisation mode using full
scan monitoring (m/z: 330.0→380.0). HPLC was equipped with a
Pursuit® C18 column (100 mm×2.0 mm i.d., particle size 5 μm). An
isocratic mobile phase (100% methanol) at flow rate of 0.2 mL/min
was used. Settings used were as follows: needle voltage 5.7 kV;
nebulising gas (N2) 45.0 psi; shield voltage 0.6 kV; drying gas 200 °C;
20.0 psi; capillary voltage 40 V. The LC–MS system was controlled by
Varian MS Workstation software, and data were collected with the
same software.
2.3. Molecular modelling
The primary structure of Agaricus bisporus tyrosinase was obtained
from the Swiss-Prot database (accession number O42713). The identi-
fication of homologues of tyrosinase was performed using the protein–
protein BLAST algorithm (Basic Local Alignment Search Tool) [31] on the
Protein Data Bank (BLOSUM62 matrix). The programme compares
protein sequences to sequence databases and calculates the statistical
significance of matches (E value). Vitis vinifera polyphenol oxidase [32]
(PDB code 2P3XA) was selected as the most appropriate template
(sequence identity 23%; amino acid positives 38%). An automated
homology modelling programme, ESyPred3D [9] was used to build the
three-dimensional (3D) structure of tyrosinase from Agaricus bisporus.
The amino acid sequence of A. bisporus tyrosinase and the sequence of the
polyphenol oxidase template were submitted to the programme. This
programme used a new alignment methodology: by comparing the
results from various multiple alignment algorithms, it derived a
“consensus” alignment of the target sequence and the template. The 3D
models were visualised using the Mercury view programme (v. 2.3,
http://www.ccdc.cam.ac.uk./free_services/mercury/).
Fig. 1. Absorption spectra of aminophenolic compounds 2(a–d) with tyrosinase.
Reaction media contained the aminophenolic compound (200 μM), tyrosinase (130 U),
and potassium phosphate buffer (100 mM) in a final volume of 1 mL. Scans were taken
every 5 min over a total time of 20 min. In all cases, possible autoxidation was ruled out
before scanning.
3. Results
The synthesised aminophenols represented in Table 1 were tested
as tyrosinase substrates. All compounds 1(a–d) showed a negligible

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Fig. 4. Spectrophotometric measurement at 400 nm of the effect of compounds 1b and
2b (200 μM) on monophenolase activity of tyrosinase in the presence of 500 μM tert-
butylphenol (BP) as substrate. The reaction media contained enzyme (130 units) and
potassium phosphate buffer (100 mM) in a final volume of 1 mL.
Fig. 2. Quantitative comparison of compounds 2(a–d) as tyrosinase substrates. Activity
was given as μMol phenoxazinone derivatives versus reaction time. All other reaction
conditions as described in Fig. 1.
fact, these ε values ranged from 3950 (2b) and 5100 (2a) M−1 cm−1
[24,34]. The corresponding KM values are 17.31, 0.17, 0.81, and
1.76 mM for the four compounds 2(a–d) respectively. The kcat are 0.6,
12.5, 3.6, and 3.4 s−1 respectively. These latter values show that
compounds 2(a–d) are on the whole very poor substrates in
comparison to o-diphenols.
Compounds 1(a–d) were essentially inert as tyrosinase substrates as
confirmed by experiments aimed at more thoroughly understanding their
action on monophenolase activity. Therefore, their ability to influence
the lag time shown by the enzyme when acting on monophenols was
tested.
Compound 2b was chosen as a representative of the 2-HMA series
because it is the best substrate and was compared to its analogue,
compound 1b (which was inactive as a substrate).
Fig. 4 shows that 2b exerted a marked effect on tert-butylphenol
oxidation whereas 1b was almost ineffective.
Compound 1b was also tested for effects, particularly inhibitory
ones, on the catecholase activity of the enzyme. In this case, to
minimise cross-reactions, tert-butylcatechol was chosen as the
substrate, and the production of 4-tert-butyl-o-benzoquinone was
monitored as described above. Inspection of Fig. 5 shows that 200 μM
1b did not have any inhibitory effect on the speed of oxidation of tert-
butylcatechol. In the presence of 2b, however, a green adduct quickly
formed. A deeper investigation of the reaction has shown that the blue
adduct would arise anyway, but it non-enzymatically reacted with 2b
leading to the observed green compound.
4. Discussion
In general, aromatic amines, o-aminophenols, and even o-diamines
enter and interact with tyrosinase active site and behave as enzyme
substrates; amines are hydroxylated to the corresponding o-aminophe-
nols, and o-aminophenols are oxidised to the corresponding o-quinonei-
mines [15,18]. A very similar affinity pattern is also observed when
comparing o-diamines, o-aminophenols, and o-diphenols. Very different
effects are observed when it comes to reaction rates: substituting nitrogen
for oxygen (e.g., converting monophenols to monoamines, diphenols to
aminophenols, or aminophenols to diamines) causes a dramatic drop in
reaction speed. This has been ascribed to the need for phenol
deprotonation [18] as a condicio sine qua non for reaction; while such a
deprotonation is favourable for phenols (pKa≈10), it is quite unfavour-
able for aromatic amines (pKaN25). Also, the significant influence of meta
and para substitutions in o-aminophenols on tyrosinase activity has been
stressed, in particular for some amino-hydroxybenzoic acids [15].
Fig. 5. Spectrophotometric measurement at 625 nm of the effect of compound 1b
(200 μM) on catecholase activity of tyrosinase in the presence of 5 mM tert-
butylcatechol (BC) as substrate. The reaction medium contained enzyme (13 U) and
potassium phosphate buffer (100 mM) in a final volume of 1 mL.
Fig. 3. Mass spectrum (positive electrospray ionisation mode) of the reaction mixture
obtained by the tyrosinase mediated oxidation of compound 2b.

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Previous interest in the transformation of o-aminophenols by
by the presence of sulphur could form an obstacle that prevents the
compound from entering the active site. In contrast, the carboxyl
moiety of 3-HAA can easily become coplanar with the aromatic ring,
allowing 3-HAA to gain access to the enzyme cleft containing the
dicopper cluster. This consideration cannot be split from an insight on
the proper interaction mode between the dicopper cluster and the o-
aminophenol functionality of the studied compounds. As shown
below, CuA and CuB are not equivalent in their ability to bind with
phenol or amine moieties (vide infra).
Testing compounds 2(a–d) as tyrosinase substrates proved that the
sulfonyl substituents are not capable of preventing the interaction with
tyrosinase per se; in fact, all of these compounds were converted to the
corresponding quinoneimines and in turn to phenoxazinone derivatives
according to the reaction depicted in Scheme 3. Most of the expected
products have been isolated and unambiguously characterised by means
of NMR, MS, UV, HPLC techniques [25]. More recently, other aminophe-
nols have been studied and their attitude to produce phenoxazinones
upon enzymatic oxidation has been assessed and exploited [34].
The affinities of the four substrates for the active site and their
oxidation speeds have been compared. Inspection of KM values (see
Results) shows that affinities increase in the order 2abb2d≤2cb2b.
Somewhat surprisingly, 2a—which bears the smallest substituent—
shows the lowest affinity, whereas 2d and 2c are much more
efficiently recognised by the enzyme in spite of their bulky
alkylsulfonamide substituents; 2b is the most favoured. This behav-
iour is clearly related to the fact that only 2a bears a net negative
charge which is independent on pH (because ―SO3H corresponds to a
strong acid). Therefore, an electrostatic repulsion between 2a and an
tyrosinase [19] and the fact that some synthetic vic-aminohydrox-
ybenzenesulfonic derivatives have recently become available [24,25]
prompted this study on possible interactions between these com-
pounds and mushroom tyrosinase. Two series of such molecules are
available, deriving from 3-hydroxy-orthanilic acid (2-amino-3-hy-
droxy-benzenesulfonic acid, 3-HOA) and 2-hydroxy-metanilic acid
(3-amino-2-hydroxy-benzenesulfonic acid, 2-HMA). The structures of
the studied substances are reported in Table 1. It is worth noting that
1a is the sulphur-containing analogue of 3-hydroxyanthranilic acid
(3-HAA), which is well known as both a substrate for and an activator
of mushroom tyrosinase [16]. We were surprised, therefore, to find
that 1a was not a substrate for the enzyme and that an almost
complete lack of activity was also found for its derivatives 1(b–d).
Whatever the case, the possible lack of activity towards phenols,
catechols, and o-aminophenols bearing electron-withdrawing sub-
stituents does not exclude the possibility that these compounds could
interact with the dicupric cluster at the active site of tyrosinase [33–
35]. In such a case, competitive inhibition of the enzyme would be
observed, as in the case of 4-nitrophenol [40]. In contrast, neither the
monophenolase nor the diphenolase activity of tyrosinase is influ-
enced by compounds 1(a–d); in particular, no shortening of the lag
time was observed. The following important conclusion can therefore
be drawn: compounds 1(a–d) likely cannot enter the active site. 3-
HAA has been previously recognised as both a substrate and as an
activator for mushroom tyrosinase [16]; so why can it enter the active
site while its sulphur analogue 1a cannot? The major difference
between the two compounds is that the tetrahedral geometry induced
Scheme 3. The biotransformation of compounds 2(a–d) mediated by tyrosinase. Spontaneous hydrolysis of the iminogroup (before or after condensation) leads to 2-hydroxy-
phenoxazin-3-one (the product detected by HPLC-MS).

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Fig. 6. Modelling of Agaricus bisporus tyrosinase CuA domain. The figure illustrates tyrosinase CuA domain with the three copper-binding histidine residue (H57, H81, H90) showed
in red. The D 286 residue is showed in green. The theoretical 3D structure of the CuA domain from A. bisporus was calculated with the homology-modelling programme ESyPred3D
(http://www.fundp.ac.be/sciences/biologie/urbm/bioinfo/esypred/), using as the template the structure of Vitis vinifera polyphenol oxidase (PDB code 2P3XA).
anionic residue at the active site is most probably responsible for the
comparatively low affinity.
two-electron redox reaction leading from catechols to their o-quinone
counterparts, albeit with different reaction rates [3], and this piece of
evidence would lend some support to the idea that an adduct
involving both the copper ions together with the two phenolic
hydroxyls is formed, at least for the oxy form [43].
A conserved aspartate residue (D 286) in the vicinity of CuA might
exert this electrostatic repulsion (Fig. 6). The distances between the
carboxylate moiety and the nitrogen atoms (of the three histidines)
engaged in the bonding to CuA have been estimated to fall between 5
and 10 Å.
This residue is close enough to the substrate cleft to exert its
electrostatic repulsion towards the sulfonate anion, thus substantially
lowering the affinity for the substrate. On the other hand, it is far enough
away from the active site that it does not completely forbid 2a access to
the cleft. The finding also helps to exclude the residue as the acceptor of
the proton derived from the dissociation of the phenolic hydroxyl from
the substrate [35,36]. With respect to the E 247 residue, the computa-
tional model used shows that it is too far away to exert a noticeable
electrostatic effect; moreover, it is in an orientation opposite to that of the
active site. Consequently, the fact that 2b is the best substrate is not
surprising if one considers that its unsubstituted sulfonamide group is
both small and uncharged.
With respect to the oxy form, the hypothetical substrate adduct
with one phenolic oxygen bridging the two copper ions must be
indeed ruled out. Obviously, no peroxide bridge is present in the met
form, where the redox reaction results in reduction of the two cupric
ions to their cuprous, deoxy form. Therefore, such an adduct is most
likely formed when met tyrosinase acts on o-diphenols, as suggested
by growing experimental and computational evidence [34,38].
With concern to o-aminophenols, the amino group—owing to its
electronic structure—simply cannot act as a chemical bridge between
the two copper ions, not even in the met form. This is another indirect
proof that the enzyme recognises firstly the amino group of o-
aminophenols, and later the o-phenoxide ion could exert its bridging
role in the met form of the enzyme (Fig. 7).
Experimental evidence also shows that the same reaction pathway
observed for catechols must take place for o-aminophenols; this
invariably generates the expected o-quinoneimines as (transient)
oxidation products. It is also worth noting that—at least for Neurospora
tyrosinase—amines show affinities for the enzyme that are comparable
to or even higher than those of phenols. Apart from the substantially
lower kcat values [18], the most obvious difference between vic-
substituted o-aminophenols and catechols is the presence of the amino
For the sake of completeness, 3-amino-2-hydroxybenzoic acid (3-
aminosalicylic acid) was also tested as a tyrosinase substrate. It was a
somewhat less efficient substrate than the isomeric 3-HAA, most
likely because its ―OH group is part of a quasi-aromatic ring with the
adjacent carboxylate ion, as is well documented for o-hydroxybenzoic
acids.
Comparison of the behaviour of the two sets 1(a–d) and 2(a–d),
together with the information available about the structures of related
proteins containing the same type of dicopper cluster (haemocyanins,
catechol oxidases, and—recently—tyrosinases [2,37,38]), could sug-
gest some hypotheses about the structure of tyrosinase's active site
and its chemistry throughout the catalytic cycle. Although similar in
their coordinative spheres, the two cupric ions do not necessarily
behave identically. It is still debatable whether a mono- or di-phenolic
substrate must bind to CuA or to CuB, even if a growing consent on CuB
as the primary binding site exists [2,3,7,37,39–42]. Recently, struc-
tural studies on some tyrosinases [2] have shown that CuA is more
sterically restricted than CuB. Although it has been suggested that CuB
would be the primary site for monophenol interaction with the
enzyme prior to its o-hydroxylation by the oxytyrosinase, the exact
bonding mode of o-diphenols remains to be ascertained. Of particular
interest is the question of whether these molecules form a mono-
cupric (most probably, with CuB) or a dicupric catechol/enzyme
adduct. It is worth noting that both met and oxy forms can perform the
Fig. 7. The proposed mode of interaction of the compounds 2(a–d) with the dicupric
cluster at the active site of met-tyrosinase, showing the specific bond of the amino
group with CuB, and the phenolate ion bridging the two cupric ions.

806
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group, which allows two vic-isomers to exist, as in the case of the two
series 1(a–d) and 2(a–d). An important conclusion of our study is
therefore that although it is wide, the substrate-binding cleft of tyrosinase
is capable of clearly discriminating between the two isomeric series. Even
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small sulfonylated substituent and is not (negatively) charged under
the testing conditions. Therefore, a substrate interaction with the
dicopper cluster, in the manner depicted here, that causes leaning of
the sulfonyl substituents toward CuA, is in full agreement with the
electrostatic effect hypothesised for the substrate series 2(a–d). On
the other hand, this hypothesis explains why the compounds
belonging to the series 1(a–d) behave as nonsubstrates: this is due
to the presence of the “axial” histidine 260 hindering the approach of
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