Kinetic Evaluation of Phenolase Activity of Tyrosinase Using Simplified Catalytic Reaction System

Article abstract of DOI:10.1021/ja036425d

A very simple tyrosinase reaction system has been developed using borate anion as a trapping agent of catechols and hydroxylamine as an external reductant to evaluate the phenolase activity without the interference of catecholase activity. Reactivities of variously para-substituted phenols in this system were compared directly to those of the phenols in the model reactions, demonstrating that the enzymatic oxygenation reaction of phenols proceeds via the same mechanism as the model reaction, that is, electrophilic aromatic substitution mechanism. Copyright

Full text of DOI:10.1021/ja036425d

Published on Web 10/07/2003
Kinetic Evaluation of Phenolase Activity of Tyrosinase Using Simplified
Catalytic Reaction System
Shin-ichi Yamazaki and Shinobu Itoh*
Department of Chemistry, Graduate School of Science, Osaka City UniVersity 3-3-138,
Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
Received May 30, 2003; E-mail: shinobu@sci.osaka-cu.ac.jp
Tyrosinase is a copper monooxygenase that catalyzes oxy-
genation of phenols to catechols (phenolase activity) and oxida-
tion of catechols to the corresponding o-quinones (catecholase
Scheme 1
1,2
activity). The enzyme has a dinuclear copper active site, where
2
2
a (µ-η :η -peroxo)dicopper(II) species (oxy-form) has been sug-
gested to perform the above oxidation reactions, producing the
corresponding oxidation products and the oxo-bridged dicopper-
II) form of the enzyme (met-form).1 Met-tyrosinase also reacts
-3
(
with catechols to generate deoxy-tyrosinase (dicopper(I) form), from
1,4-7
2
which oxy-tyrosinase is regenerated by the reaction with O .
2
tyrosinase as indicated in Scheme 1. In fact, O -consumption by
Among the catalytic processes of tyrosinase, the phenol oxy-
genation step (phenolase reaction) has attracted much interest not
only in bioinorganic chemistry but also in the fields of coordination
chemistry and synthetic organic chemistry. To clarify the mecha-
nism of the phenol oxygenation reaction, substituent effects of
phenols in the enzymatic catalytic reaction have been examined in
the catechol oxidation was successfully suppressed when tyrosinase
and exogenous catechols were treated in 0.5 M borate buffer (pH
)
9.0) (data not shown). More importantly, the oxygenation of
phenols was also completely prohibited in the borate buffer [line
a) in Figure 1A]. Thus, the catalytic cycle may stop at the stage
(
of met-tyrosinase as we expected, since the reductant of met-
tyrosinase (free catechol) is excluded from the catalytic cycle by
the complex formation with borate anion (Scheme 1).
5
detail. However, the reported data contain the substituent effects
not only on the phenolase reaction but also on the catecholase reac-
tion. Thus, the discussion based on such data might be problematic.
Model studies on the phenolase activity of tyrosinase have
provided profound insights into the aromatic hydroxylation mech-
Addition of hydroxylamine (NH
solution containing tyrosinase and p-fluorophenol drastically en-
hanced the O -consumption rate [line (b) in Figure 1A], but no
-consumption was observed when NH OH and tyrosinase were
treated without the substrate. Thus, NH OH functions as an external
reductant for met-tyrosinase but not as a substrate for oxy-tyrosinase
Scheme 1). Product analysis of the reaction mixture by HPLC
indicated a formation of the corresponding catechol (42% based
2
OH) into the borate buffer
2
2
2
8-11
anism by the (µ-η :η -peroxo)dicopper(II) complex.
Since the
O
2
2
pioneering work by Karlin and co-workers was reported in the early
2
8
1
980s, several examples of intramolecular aromatic ligand hy-
droxylation reaction in a dinuclear copper(I) complex by O have
2
(
been reported.9 Recently, our group and others have achieved
the intermolecular oxygenation of phenolates by the peroxo
complex, providing more insights into the phenolase activity of
tyrosinase.1 Detailed kinetic studies of the model reaction have
suggested that electrophilic aromatic substitution is the most
plausible reaction mechanism.10
,12
15
on oxygen consumption). The observed O
exhibits little lag phase at the high NH
2
-consumption profile
OH and low phenol
2
0,11
concentrations (Figure 1A). Thus, the modified enzymatic reaction
system is suitable for the kinetic studies of the phenolase activity.
The initial rates of O -consumption were then plotted against
2
In this study, we present a very simple enzymatic reaction system
using borate anion as a trapping agent of catechols and hydroxy-
lamine as an external reductant (Scheme 1). With this simplified
enzymatic reaction system, we succeeded to examine the phenolase
activity without the interference of catecholase reactions and
perform the first direct comparison of the reactivities of variously
para-substituted phenols between in the enzymatic reaction and in
the model reaction.
the concentrations of p-fluorophenol to afford a Michaelis-Menten
type saturation curve as shown in Figure 1B. No dependence on
the concentrations of NH OH, O , and borate was observed under
2
2
the present experimental conditions, indicating that these three
components are kinetically saturated. Thus, the rate equation can
P
be simply drawn as V ) Vmax /(1 + K /[P]), where [P] denotes
M
16
the concentrations of the phenol.
The kinetic parameters (KMP and Vmax) for several para-substituted
phenols were determined similarly by the Hanes-Woolf plot, and
are listed in Table S2. The parameter Vmax might reflect not only
Met-tyrosinase was purified from mushroom by following the
reported procedures with some modifications as described in
Supporting Information. The catalytic reaction of tyrosinase was
the phenol oxygenation process but also the NH OH-assisted
2
2
monitored by following O -consumption using a Clark-type oxygen
reduction process of met-tyrosinase (also see Supporting Informa-
electrode.13 As mentioned above, catechols affect the phenol
oxygenation process in a complicated manner. Therefore, we first
intend to minimize the influence of catechols by using a borate
buffer as the reaction media, since the borate anion is well-known
to form a stable complex with catechols.14 Such a complex
formation may exclude the catechols from the catalytic cycle by
preventing the reaction with oxy-tyrosinase as well as with met-
tion). However, the reduction process is relatively fast and common
in each reaction. Thus, we can use Vmax as an indicator of the
phenolase activity of tyrosinase. As seen in Table S2, the Vmax value
increases as the electron donor ability of the para-substituent
increases. This clearly indicates that the peroxo intermediate of
tyrosinase exhibits an electrophilic nature as suggested by the model
studies.1
2,17
Then, the para-substituent effect of the enzymatic
13034
9
J. AM. CHEM. SOC. 2003, 125, 13034-13035
10.1021/ja036425d CCC: $25.00 © 2003 American Chemical Society

C O MMU N I C A T I O N S
Science, Sports, and Culture of Japan (15350105), and Research
Fellowships of Japan Society for the Promotion of Science for
Young Scientists. We also thank Sumitomo Chemical Foundation
for the financial support.
Supporting Information Available: Experimental details including
tyrosinase purification, the spectrum data of oxy-tyrosinase (S1), kinetic
data of the phenolase reaction (S2), and the kinetic equations based on
the detailed reaction scheme (S3) (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
References
Figure 1. (A) Time course of O2-consumption in the tyrosinase-catalyzed
reaction in 0.5 M borate buffer (pH 9.0) (a) in the absence of NH2OH and
(1) (a) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. ReV. 1996,
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Res. 2003, 36, 300-308.
C6H4OH] ) 30 µM, under air-saturated conditions at 25 °C. (B) Plot of V
vs [p-F-C6H4OH]. Inset: Hanes-Woolf plot ([p-F-C6H4OH]/V vs [p-F-
C6H4OH]).
(
2) (a) Solomon, E. I.; Chen, P.; Metz, M.; Lee, S.; Palmer, A. E. Angew.
Chem., Int. Ed. 2001, 40, 4570-4590. (b) Decker, H.; Tuczek, F. Trends
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(
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(
5) For recent kinetic studies on the phenolase and catecholase activities,
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(
b) Espin, J. C.; Var o´ n, R.; Fenoll, L. G.; Gilabert, M. A.; Garc ´ı a-Ruiz,
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(
6) For spectroscopic approaches to the catalytic intermediates, see: (a) van
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J. W.; Vijgenboom, E.; Canters, G. W. FEBS Lett. 1999, 442, 215-220.
Figure 2. Plots of [log Vmax] (enzymatic reaction, closed circle) and [log
+
(7) For geometric discussions on each step in the catalytic mechanism, see:
a) Gerdemann, C.; Eicken, C.; Krebs, B. Acc. Chem. Res. 2002, 35, 183-
k] (model reaction, open circle) against σ values of the p-substituents.
(
1
91. (b) Klabunde, T.; Eicken, C.; Sacchettini, J. C.; Krebs, B. Nat. Struct.
Biol. 1998, 5, 1084-1090.
reaction has been evaluated by comparing the Hammett plot ([log
(
8) Karlin, K. D.; Hayes, J. C.; Gultneh, Y.; Cruse, R. W.; McKown, J. W.;
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9) Holland, P. L.; Rodgers, K. R.; Tolman, W. B. Angew. Chem., Int. Ed.
+
k] vs σ ) with that of the model reaction (Figure 2). The F value
(
(
the slope of the Hammett plot) of the enzymatic reaction (-2.4,
1
999, 38, 1139-1142.
2
10
R ) 0.98) is fairly close to that of our model reaction (-1.8)
(
10) (a) Itoh, S.; Kumei, H.; Taki, M.; Nagatomo, S.; Kitagawa, T.; Fukuzumi,
S. J. Am. Chem. Soc. 2001, 123, 6708-6709. (b) Itoh, S.; Fukuzumi, S.
Bull. Chem. Soc. Jpn. 2002, 75, 2081-2095.
and that of the aromatic ligand hydroxylation reaction reported by
Karlin and co-workers (-2.1).17 The negatively small F values of
both the enzymatic and model reactions clearly demonstrated that
the phenolase reaction of tyrosinase proceeds via the same
mechanism as that of the model reaction, that is, an electrophilic
aromatic substitution mechanism.5,18
In summary, we have successfully constructed a very simple
enzymatic reaction system that allows us to perform a quantitative
evaluation of the phenolase activity of tyrosinase for the first time.
One of the key features of the present system is the exclusion of
catechols from the catalytic cycle by using borate anion as the
trapping agent (Scheme 1). In the absence of borate anion (in a
nonborate buffer), catechols, the primary oxidation product of
phenols, may be further oxidized to the corresponding o-quinones
at the active site of met-tyrosinase as reported previously.1b In the
presence of high concentration of borate anion (0.5 M), however,
the primary oxidation product catechol may be withdrawn from
the active site before being oxidized to the o-quinone. This means
that the catechol release by the complex formation with borate anion
is fast enough to prevent such the over oxidation of catechols.
(
11) (a) Santagostini, L.; Gullotti, M.; Monzani, E.; Casella, L.; Dillinger, R.;
Tuczek, F. Chem. Eur. J. 2000, 6, 519-522. (b) Mirica, L. M.; Vance,
M.; Rudd, D. J.; Hedman, B.; Hodgson, K. O.; Solomon, E. I.; Stack, T.
D. P. J. Am. Chem. Soc. 2002, 124, 9332-9333.
(12) Itoh, S. In ComprehensiVe Coordination Chemistry-II; Que, L., Jr., Tolman,
W. B., Eds; Elsevier: Oxford, 2003. In press.
(
13) When hydrogen peroxide was added into a solution of met-tyrosinase,
the absorption band at 345 nm due to oxy-tyrosinase was generated (Figure
S1). However, the absorption band readily disappeared when the solution
was degassed. Thus, we could not perform single-turnover kinetic analysis
on the reaction between oxy-tyrosinase and phenols.
(
14) (a) For reviews, see: James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S.
Supramol. Chem. 1995, 6, 141-157. (b) Mochizuki, M.; Yamazaki, S.;
Kano, K.; Ikeda, T. Biochim. Biophys. Acta 2002, 1569, 35-44.
15) The low yield of catechol product may be due to nonenzymatic side
reactions such as a quinone-induced polymerization of catechols and a
(
reaction of the product (and/or the substrate) with oxidized NH
2
OH. It is,
however, apparent that the enzymatic over oxidation of catechols to the
quinones is not involved, since the catechol oxidation does not proceed
at all in the present borate buffer system.
(16) The detailed kinetic analysis is presented in Supporting Information (S3).
17) Nasir, M. S.; Cohen, B. I.; Karlin, K. D. J. Am. Chem. Soc. 1992, 114,
482-2494.
(
2
(18) Rate-dependence on the oxidation potentials of phenols in the present
enzymatic reaction is totally different from that observed in the oxidation
reaction of neutral phenols (phenoxyl radical formation) by the peroxo
complex which proceeds via a proton-coupled electron transfer (PCET)
mechanism: Osako, T.; Ohkubo, K.; Taki, M.; Tachi, Y.; Fukuzumi, S.;
Itoh, S. J. Am. Chem. Soc. 2003, 125, 11027-11033.
Acknowledgment. This work was supported in part by Grants-
in-Aid for Scientific Research from the Ministry of Education,
JA036425D
J. AM. CHEM. SOC.
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VOL. 125, NO. 43, 2003 13035