The influence of catechol structure on the suicide-inactivation of tyrosinase

Article abstract of DOI:10.1039/b910500j

3,6-Difluorocatechol, which cannot act as a monooxygenase tyrosinase substrate, is an oxidase substrate, and, in contrast to other catechols, oxidation does not lead to suicide-inactivation, providing experimental evidence for an inactivation mechanism in

Full text of DOI:10.1039/b910500j

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The influence of catechol structure on the suicide-inactivation of tyrosinase
Christopher A. Ramsden,*a Michael R. L. Stratfordb and Patrick A. Rileyc
Received 3rd June 2009, Accepted 30th June 2009
First published as an Advance Article on the web 9th July 2009
DOI: 10.1039/b910500j
3,6-Difluorocatechol, which cannot act as a monooxyge-
nase tyrosinase substrate, is an oxidase substrate, and, in
contrast to other catechols, oxidation does not lead to
suicide-inactivation, providing experimental evidence for an
inactivation mechanism involving reductive elimination of
Cu0 from the active site.
prediction (i), the catechol oxidase extracted from bananas (Musa
cavendishii), which is deficient in monooxygenase activity, is not
inactivated.8 In accord with prediction (ii), we now demonstrate
that 3,6-difluorocatechol 5 is a substrate for oxy-tyrosinase but
does not cause deactivation of the enzyme.
3,6-Difluorocatechol
5 was prepared from 5,8-difluoro-
2,3-dihydro-1,4-benzodioxin using the method of Ladd and
Weinstock.9,10 Tyrosinase (ex Agaricus bisporus) was made up in
phosphate buffer (0.1 M, pH 6.3) and catechol oxidation was mon-
itored using combined enzyme oximetry and spectrophotometry,
using the apparatus previously described.11
The kinetics of oxygen uptake for 3,6-difluorocatechol 5 and
4-fluorocatechol 6 are shown in Fig. 1. The enzymatic oxidation
of 3,6-difluorocatechol 5 proceeds at a nearly exponential rate,
dependent on the amount of enzyme added. The oxidation rate
per unit of tyrosinase (2.5 nmoles O2 min-1) is considerably lower
than that of 4-fluorocatechol (66 nmoles O2 min-1). However,
the kinetic characteristics significantly differ from those for the
oxidation of 4-fluorocatechol 6, which demonstrates suicide-
inactivation and premature termination of oxygen uptake. The
Vmax for 3,6-difluorocatechol oxidation by mushroom tyrosinase
was calculated as 250 nmol substrate min-1 and the Michaelis
constant as 483 mM.
Tyrosinase (EC 1.14.18.1) is a mixed-function oxidase that is
widely distributed in nature. It is involved in several biologically
significant processes including pigmentation, cuticular hardening
in insects, and protection of seeds.1 Tyrosinase is a metalloen-
zyme with a strongly conserved active centre containing two
copper atoms that bind dioxygen to form oxy-tyrosinase 1.2,3
Oxy-tyrosinase is able to catalyze ortho-quinone formation by
the oxidation of both catechols (oxidase activity) and phenols
(monooxygenase activity) (Scheme 1). Phenols, including tyrosine,
are oxidised to ortho-quinones with formation of deoxy-tyrosinase
2, which then binds dioxygen to reform oxy-tyrosinase 1 (path a,
Scheme 1). Oxidation of catechols (path b, Scheme 1) leads to an
ortho-quinone plus met-tyrosinase 3. Reduction of met-tyrosinase
by a second molecule of catechol gives deoxy-tyrosinase 2 which
in turn binds dioxygen to reform oxy-tyrosinase 1 and complete
the cycle.
It has been known for more than half a century that tyrosinase
is subject to reaction-inactivation (suicide-inactivation), but only
when catalysing the oxidation of catechols.4,5 Based on a structure–
activity study of a variety of dihydroxy tyrosinase substrates, we
recently proposed a novel mechanism to rationalise this previously
unexplained feature of catechol oxidation.6 In particular, we
proposed that, in addition to acting as oxidase substrates for oxy-
tyrosinase (path b), catechols can also act as monooxygenase sub-
strates, and that under these circumstances the hydroxy substituent
in the bound substrate can deprotonate, leading to reductive
elimination of copper and irreversible formation of inactivated
tyrosinase 4 (path c, Scheme 1). This mechanism accounts for the
50% loss of the copper from the active site reported by Dietler and
Lerch.7
The inactivation mechanism shown in Scheme 1 leads to two
predictions: (i) catechol oxidases lacking monooxygenase activity
will not exhibit suicide-inactivation, and (ii) catechols having
substituents that block oxygenation at positions 3 and 6 cannot
act as monooxygenase substrates, and will not exhibit suicide-
inactivation. Subsequently we have shown that, in agreement with
Fig. 1 Oximetric data for tyrosinase-catalysed oxidation of 3,6-difluo-
rocatechol 5 compared to 4-fluorocatechol 6 at 30 ◦C. Oxidation of 5
(960 mM) (᭹), shown for additions of 7.5, 10, 15 and 30 units enzyme
(in 3.65 mL), fits a set of exponential curves (mean correlation coefficient
0.987), in contrast to the kinetics of oxidation of equimolar 6 (ꢀ), in
which tyrosinase (15 units) is subject to suicide-inactivation. The data are
shown as a semi-logarithmic plot of the residual oxygen as a function of
incubation time.
aLennard-Jones Laboratories, School of Physical and Geographical Sciences,
Keele University, Staffordshire, ST5 5BG, UK
In separate experiments we have shown that prior exposure
(10 min) of tyrosinase (20 units) to 3,6-difluorocatechol 5 (225 mM)
does not diminish the oxidation of 4-methylcatechol (790 mM)
relative to a control experiment using fresh tyrosinase.
bGray Institute for Radiation Oncology & Biology, University of Oxford, Old
Road Campus Research Building, Oxford, OX3 7DQ, UK
cTotteridge Institute for Advanced Studies, The Grange, Grange Avenue,
London, N20 8AB, UK
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Scheme 1
The spectrophotometric data show a similar kinetic distinction
between 3,6-difluorocatechol 5 and 4-fluorocatechol 6 oxidation.
3,6-Difluorocatechol 5 has a strong absorbance peak at 220 nm
and a shoulder at 260 nm. On oxidation by tyrosinase there is an
initial rise in absorbance at 400 nm, consistent with the formation
of the corresponding ortho-quinone 7, and a rapidly increasing
absorbance in the 260–320 nm region (Fig. 2). The final spectrum
is very broad, extending to above 600 nm suggesting the formation
of melanoid polymers.
None of the products identified exhibited a quinone absorbance,
indicating that any quinones formed (e.g. 7–9) are highly reactive.
Fluoroquinones are known to undergo substitution with release of
fluoride,12 and we examined the kinetics of fluoride release by ion
chromatography. Fig. 3 shows the time-dependent yield of fluoride
and Fig. 4 shows the change of the products identified by LC-MS
with time.13,14
Fig. 3 Time course of fluoride release from 3,6-difluorocatechol 5
(700 mM) and tyrosinase (3 units in 0.1 M phosphate buffer, pH 6.3)
(1 mL).
Fig. 2 Successive spectral scans at 30-second intervals during oxidation
of 960 mM 3,6-difluorocatechol 5 by tyrosinase (30 units).
Data from LC-MS examination of an oxidation mixture
in phosphate buffer (0.1 M, pH 6.3) (1 mL) containing
3,6-difluorocatechol 5 (700 mM) and tyrosinase (3 units) indicated
the formation of several products of which four major components
were clearly identified by their molecular ions at m/z (M-H) 289
(RT 6.2 min), 159 (RT 3.7), 305 (RT 6.4), and 429 (RT 6.8).
The major product (m/z 289), which rapidly forms, is the diaryl
ether 10, formed from the ortho-quinone 7 and its precursor 5
(Scheme 2). The second initial product (m/z 159) is a tetrahydroxy
product 11 and/or 12, formed with fluoride release from the initial
ortho-quinone 7 and water. The secondary products are formed by
further oxidation, substitution and addition reactions of the initial
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there is no inactivation of tyrosinase. The rate of oxidation is slow
in comparison with 4-fluorocatechol and 3-fluorocatechol,6 but
the kinetics of oxygen utilization differ significantly from these
substrates. Both the oximetric and spectrophotometric data show
that the oxidation kinetics of 3,6-difluorocatechol 5 fit a pseudo-
first-order function and resemble those found using catechol
oxidase extracted from bananas.8 They differ from the usual
kinetics of catechol oxidation by tyrosinase, which exhibit suicide-
inactivation.6 We attribute this result to the fluoro substituents
flanking the catecholic group which prevent the difluorocatechol
5 from being processed by the monooxygenase route (path c,
Scheme 1). This provides further evidence for the inactivation
mechanism that we have proposed to account for the suicide-
inactivation of tyrosinase by catecholic substrates.
Fig. 4 Time-dependent changes of major oxidation products identified
by LC-MS. The products illustrated are those with m/z 289 (ꢀ), 159 (ꢀ),
305 (ꢀ), and 429 (᭺).
Notes and references
1 P. A. Riley, in Melanin: Its Role in Human Photoprotection, ed. L. Zeise,
M. R. Chedekel and T. B. Fitzpatrick, Valdenmar Publishing Co.,
Overland Park, KS, 1995, pp 1–10.
2 H. Decker and N. Terwilliger, J. Exp. Biol., 2000, 203, 1777.
3 E. J. Land, C. A. Ramsden and P. A. Riley, Acc. Chem. Res., 2003, 36,
300.
4 J. M. Nelson and C. R. Dawson, Adv. Enzymol. Relat. Areas Mol. Biol.,
1944, 4, 99.
5 I. Asimov and C. R. Dawson, J. Am. Chem. Soc., 1950, 72, 820.
6 E. J. Land, C. A. Ramsden and P. A. Riley, Tohoku J. Exp. Med., 2007,
212, 341.
7 C. Dietler and K. Lerch, in Oxidases and Related Redox Systems, ed.
T. E. King, H. S. Mason and M. Morrison, Pergamon Press, New York,
1982, pp. 305–317.
8 E. J. Land, C. A. Ramsden, P. A. Riley and M. R. L. Stratford,
Tohoku J. Exp. Med., 2008, 216, 231.
9 D. L. Ladd and J. Weinstock, J. Org. Chem., 1981, 46, 203.
10 After Kugelrohr distillation the catechol 5 was purified by chroma-
tron chromatography (eluent: petroleum ether–EtOH, 4 : 1) to give
colourless plates, mpt 50–75 ◦C (lit.9 white solid); nmax/cm-13384, 1636,
1520, 1502, 1363, 1304, 1224, 1144, 1033, 952, 785 and 745; lmax(0.1 M
phosphate buffer): pH 6.3, 218 (e 3327) and 260sh (560) nm; dH(CDCl3)
5.54 (2H, br s, OH) and 6.54 (2H, pseudo t, J 7.2 Hz, aromatic H).
11 C. J. Cooksey, P. J. Garratt, E. J. Land, S. Pavel, C. A. Ramsden, P. A.
Riley and N. P. M. Smit, J. Biol. Chem., 1997, 272, 26226.
12 R. S. Phillips, J. G. Fletcher, R. L. Von Tersch and K. L. Kirk, Arch.
Biochem. Biophys., 1990, 276, 65.
Scheme 2
13 LC-MS studies used a similar system to that previously described,8 but
using an Atlantis dC18 column, 150 ¥ 3 mm (Waters), the organic
modifier was MeCN, and the gradient was 15–60% (flow rate of
0.5 mL min-1). Electrospray ionisation was carried out in negative
mode, capillary voltage 2.1 kV, cone voltage 21 V.
product 10. Fluoride release is slower than formation of the ether
10 and concomitant with the rate of formation of the monofluoro
products 11 and 12.
The data show that 3,6-difluorocatechol 5 is a substrate for
tyrosinase and forms 3,6-difluoro-ortho-quinone 7 via the oxidase
mechanism (path b, Scheme 1). In contrast to other catechols,
14 Separation of fluoride was carried out on an AS12 column (Dionex)
using water and 37.5 mM NaOH, 50 mM sodium tetraborate as eluents.
Initial conditions were 15% buffer, with a linear gradient to 75% from
1–8 min (flow rate 1.5 mL min-1). Detection was by conductivity using
a Dionex ED40 with external water suppression.
3390 | Org. Biomol. Chem., 2009, 7, 3388–3390
This journal is
The Royal Society of Chemistry 2009
©