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New aspects of the reactivity of tyrosinase
Micron 35 (2004) 141–142 www.elsevier.com/locate/micron
New aspects of the reactivity of tyrosinase Luigi Casellaa,*, Alessandro Granataa, Enrico Monzania, Roberta Pievoa, Luca Pattarellob, Luigi Bubaccob a
Dipartimento di Chimica Generale, Universita` di Pavia, Via Taramelli 12, 27100 Pavia, Italy b Dipartimento di Biologia, Universita` di Padova, Via Ugo Bassi 58b, 35121 Padova, Italy
Abstract Tyrosinase was found to be active in the sulfoxidation of thioanisol, producing the (R)-sulfoxide with high enantiomeric excess. The activity of the enzyme with phenolic and diphenolic substrates in a mixed aqueous Hepes buffer pH 6.8-methanol– glycerol solvent was also investigated over a range of temperatures. These experiments enabled us to deduce the thermodynamic parameters associated with substrate binding to the enzyme and the activation parameters associated with the rate determining step of the enzymatic reaction. q 2003 Elsevier Ltd. All rights reserved. Keywords: Tyrosinase; Sulfoxidation; Kinetics; Enzymes in organic solvents
Tyrosinase is strongly active on substrates carrying phenolic functions (Fenoll et al., 2001) but little is known on its potential activity towards different substrates. Organic sulfides have been shown to be oxidized by monooxygenases like P450 (Casella and Colonna, 1994) and dopamine b-hydroxylase (May et al., 1981). Therefore, we thought it of interest to assay the activity of tyrosinase in the sulfoxidation reaction. Preliminary experiments using thioanisole as representative substrate showed that indeed mushroom tyrosinase is capable of supporting oxygenation at sulfur using a catechol such as L -dihydroxyphenylalanine (L -dopa) as reducing agent (Scheme 1). Ascorbate is unable to support the reaction while the more sterically hindered 3,5-di-tert-butylcatechol acts as a very slow reducing agent in the sulfoxidation. Yields of sulfoxide are limited (, 10%), due to enzyme inactivation by the quinone products derived from the catechol, but it is interesting to note that methyl phenyl sulfoxide is obtained with high degree of enantioselectivity (the (R) isomer being favored, . 80% e.e.), showing the important role of the enzyme in the stereochemical control of the reaction. Another aspect of the reactivity of tyrosinase that we recently started to investigate is the behavior of
* Corresponding author. Tel.: þ 39-382-507-331; fax: þ39-382-528-544. E-mail address: [email protected] (L. Casella). 0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.10.045
the enzyme in cryosolvents, because these media may open the possibility to trap and characterize reactive enzyme intermediates at low temperatures. In fact, in spite of extensive research efforts, important details of the mechanisms by which tyrosinase oxidizes monophenolic and diphenolic substrates remain completely unknown (Fenoll et al., 2001). The mode of substrate binding and product release from the enzyme, and the mode of dioxygen activation and cleavage at the dinuclear copper center are, in particular, still subject to speculation (Decker et al., 2000; Siegbahn, 2003). Since the crystal structure of tyrosinase is not available, the structural models of its active site make use of the structural details of the other members of the type-3 copper protein family, namely hemocyanin (Cuff et al., 2000) and catechol oxidase (Gerdeman et al., 2002). Reports on the activity of tyrosinase in organic solvents have appeared (Kermasha et al., 2001), but the main scope of these studies was to reduce the extent of product polymerization and enzyme inactivation in the organic medium. A variety of cryosolvents with suitable viscosity characteristics were preliminarily tested, and good performance of the enzymes from mushroom and S. antibioticus in the range from 2 20 to þ 30 8C was found using a mixture of 34.4% methanol/glycerol (7/1 v/v) and 65.6% (v/v) aqueous 50 mM Hepes buffer at pH 6.8. The effect of the cryosolvent on enzyme activity was assessed on the catalytic oxidation of L -dopa. A weak, mixed type inhibition effect was found by
142
L. Casella et al. / Micron 35 (2004) 141–142
Scheme 1.
studying the dependence of the L -dopa oxidation rate as a function of the concentration of the organic component of the cryosolvent, due to slight variation in substrate binding affinity and local conformational changes of the protein in the aqueous/organic medium. The kinetics of enzymatic oxidation of the substrates L-dopa, dopamine and tyramine were studied in the range from about 0 to 30 8 C, while L -tyrosine could not be studied for its limited solubility. The thermodynamic parameters associated with substrate binding to the enzyme and the activation parameters associated with the r.d.s. of the enzymatic reaction were obtained for the substrates studied. In all cases, the enzymatic reactions were found to be monophasic and independent on dioxygen concentration. The activation energy is characterized by rather similar enthalpic and entropic components for the three phenolic substrates. The large positive DH – values (ranging from 54 to 67 kJ mol21) are probably associated with cleavage of the bound peroxide O – O bond in the transition state. This is in agreement with predictions from theoretical calculations (Siegbahn, 2003) and seems also confirmed by the reduced DS– values (from 2 12 to þ 30 J K21 mol21), which are consistent with little structural rearrangement upon O –O bond cleavage.
References Casella, L., Colonna, S., 1994. Biological oxidations: stereochemical aspects. In: Montanari, F., Casella, L. (Eds.), Metalloporphyrins Catalyzed Oxidations, Kluwer, Dordrecht, pp. 307 –340. Cuff, M.E., Miller, C., van Holde, K.E., Hendrickson, W.A., 2000. Crystal structure of a functional unit from Octopus hemocyanin. J. Mol. Biol. 278, 855–870. Decker, H., Dillinger, R., Tuczek, F., 2000. How does tyrosinase work? Recent insights from model chemistry and structural biology. Angew Chem. Int. Ed. 39, 1591–1595. Fenoll, L.G., Rodriguez-Lopez, J.N., Garcia-Sevilla, F., Garcia-Ruiz, P.A., Varon, R., Garcia-Canovas, F., Tudela, J., 2001. Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones. Biochim. Biophys. Acta 1548, 1 –22. Gerdemann, C., Eicken, C., Krebs, B., 2002. The crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins. Acc. Chem. Res. 35, 183–191. Kermasha, S., Bao, H., Bisakowski, B., 2001. Biocatalysis of tyrosinase using catechin as substrate in selected organic solvent media. J. Mol. Catal. B 11, 929 –938. May, S.W., Phillips, R.S., Mueller, P.W., Herman, H.H., 1981. Dopamine b-hydroxylase. Comparative specificities and mechanisms of the oxygenation reactions. J. Biol. Chem. 256, 8470–8475. Siegbahn, P.E.M., 2003. The catalytic cycle of tyrosinase: peroxide attack on the phenolate ring followed by O–O bond cleavage. J. Biol. Inorg. Chem. 8, 567–576.
New aspects of the reactivity of tyrosinase Luigi Casellaa,*, Alessandro Granataa, Enrico Monzania, Roberta Pievoa, Luca Pattarellob, Luigi Bubaccob a
Dipartimento di Chimica Generale, Universita` di Pavia, Via Taramelli 12, 27100 Pavia, Italy b Dipartimento di Biologia, Universita` di Padova, Via Ugo Bassi 58b, 35121 Padova, Italy
Abstract Tyrosinase was found to be active in the sulfoxidation of thioanisol, producing the (R)-sulfoxide with high enantiomeric excess. The activity of the enzyme with phenolic and diphenolic substrates in a mixed aqueous Hepes buffer pH 6.8-methanol– glycerol solvent was also investigated over a range of temperatures. These experiments enabled us to deduce the thermodynamic parameters associated with substrate binding to the enzyme and the activation parameters associated with the rate determining step of the enzymatic reaction. q 2003 Elsevier Ltd. All rights reserved. Keywords: Tyrosinase; Sulfoxidation; Kinetics; Enzymes in organic solvents
Tyrosinase is strongly active on substrates carrying phenolic functions (Fenoll et al., 2001) but little is known on its potential activity towards different substrates. Organic sulfides have been shown to be oxidized by monooxygenases like P450 (Casella and Colonna, 1994) and dopamine b-hydroxylase (May et al., 1981). Therefore, we thought it of interest to assay the activity of tyrosinase in the sulfoxidation reaction. Preliminary experiments using thioanisole as representative substrate showed that indeed mushroom tyrosinase is capable of supporting oxygenation at sulfur using a catechol such as L -dihydroxyphenylalanine (L -dopa) as reducing agent (Scheme 1). Ascorbate is unable to support the reaction while the more sterically hindered 3,5-di-tert-butylcatechol acts as a very slow reducing agent in the sulfoxidation. Yields of sulfoxide are limited (, 10%), due to enzyme inactivation by the quinone products derived from the catechol, but it is interesting to note that methyl phenyl sulfoxide is obtained with high degree of enantioselectivity (the (R) isomer being favored, . 80% e.e.), showing the important role of the enzyme in the stereochemical control of the reaction. Another aspect of the reactivity of tyrosinase that we recently started to investigate is the behavior of
* Corresponding author. Tel.: þ 39-382-507-331; fax: þ39-382-528-544. E-mail address: [email protected] (L. Casella). 0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.10.045
the enzyme in cryosolvents, because these media may open the possibility to trap and characterize reactive enzyme intermediates at low temperatures. In fact, in spite of extensive research efforts, important details of the mechanisms by which tyrosinase oxidizes monophenolic and diphenolic substrates remain completely unknown (Fenoll et al., 2001). The mode of substrate binding and product release from the enzyme, and the mode of dioxygen activation and cleavage at the dinuclear copper center are, in particular, still subject to speculation (Decker et al., 2000; Siegbahn, 2003). Since the crystal structure of tyrosinase is not available, the structural models of its active site make use of the structural details of the other members of the type-3 copper protein family, namely hemocyanin (Cuff et al., 2000) and catechol oxidase (Gerdeman et al., 2002). Reports on the activity of tyrosinase in organic solvents have appeared (Kermasha et al., 2001), but the main scope of these studies was to reduce the extent of product polymerization and enzyme inactivation in the organic medium. A variety of cryosolvents with suitable viscosity characteristics were preliminarily tested, and good performance of the enzymes from mushroom and S. antibioticus in the range from 2 20 to þ 30 8C was found using a mixture of 34.4% methanol/glycerol (7/1 v/v) and 65.6% (v/v) aqueous 50 mM Hepes buffer at pH 6.8. The effect of the cryosolvent on enzyme activity was assessed on the catalytic oxidation of L -dopa. A weak, mixed type inhibition effect was found by
142
L. Casella et al. / Micron 35 (2004) 141–142
Scheme 1.
studying the dependence of the L -dopa oxidation rate as a function of the concentration of the organic component of the cryosolvent, due to slight variation in substrate binding affinity and local conformational changes of the protein in the aqueous/organic medium. The kinetics of enzymatic oxidation of the substrates L-dopa, dopamine and tyramine were studied in the range from about 0 to 30 8 C, while L -tyrosine could not be studied for its limited solubility. The thermodynamic parameters associated with substrate binding to the enzyme and the activation parameters associated with the r.d.s. of the enzymatic reaction were obtained for the substrates studied. In all cases, the enzymatic reactions were found to be monophasic and independent on dioxygen concentration. The activation energy is characterized by rather similar enthalpic and entropic components for the three phenolic substrates. The large positive DH – values (ranging from 54 to 67 kJ mol21) are probably associated with cleavage of the bound peroxide O – O bond in the transition state. This is in agreement with predictions from theoretical calculations (Siegbahn, 2003) and seems also confirmed by the reduced DS– values (from 2 12 to þ 30 J K21 mol21), which are consistent with little structural rearrangement upon O –O bond cleavage.
References Casella, L., Colonna, S., 1994. Biological oxidations: stereochemical aspects. In: Montanari, F., Casella, L. (Eds.), Metalloporphyrins Catalyzed Oxidations, Kluwer, Dordrecht, pp. 307 –340. Cuff, M.E., Miller, C., van Holde, K.E., Hendrickson, W.A., 2000. Crystal structure of a functional unit from Octopus hemocyanin. J. Mol. Biol. 278, 855–870. Decker, H., Dillinger, R., Tuczek, F., 2000. How does tyrosinase work? Recent insights from model chemistry and structural biology. Angew Chem. Int. Ed. 39, 1591–1595. Fenoll, L.G., Rodriguez-Lopez, J.N., Garcia-Sevilla, F., Garcia-Ruiz, P.A., Varon, R., Garcia-Canovas, F., Tudela, J., 2001. Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones. Biochim. Biophys. Acta 1548, 1 –22. Gerdemann, C., Eicken, C., Krebs, B., 2002. The crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins. Acc. Chem. Res. 35, 183–191. Kermasha, S., Bao, H., Bisakowski, B., 2001. Biocatalysis of tyrosinase using catechin as substrate in selected organic solvent media. J. Mol. Catal. B 11, 929 –938. May, S.W., Phillips, R.S., Mueller, P.W., Herman, H.H., 1981. Dopamine b-hydroxylase. Comparative specificities and mechanisms of the oxygenation reactions. J. Biol. Chem. 256, 8470–8475. Siegbahn, P.E.M., 2003. The catalytic cycle of tyrosinase: peroxide attack on the phenolate ring followed by O–O bond cleavage. J. Biol. Inorg. Chem. 8, 567–576.