Laccases from Basidiomycetes: Physicochemical Characteristics and Substrate Specificity towards Methoxyphenolic Compounds

S. A. Smirnov^1*, O. V. Koroleva², V. P. Gavrilova³, A. B. Belova¹, and N. L. Klyachko¹

¹School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3429; E-mail: smirnov@enzyme.chem.msu.ru

²Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia; fax: (095) 954-2732; E-mail: alena_koroleva@hotmail.com

³Komarov Institute of Botany, Russian Academy of Sciences, ul. Popova 2, St. Petersburg, 198022 Russia; fax: (812) 234-4512; E-mail: Valeria@VG2438.spb.edu

^* To whom correspondence should be addressed.

Received December 6, 2000; Revision received April 4, 2001

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Laccases from the Basidiomycetes Coriolus hirsutus, Coriolus zonatus, Cerrena maxima, and Coriolisimus fulvocinerea have been isolated and purified to homogeneity and partially characterized. The kinetics of oxidation of different methoxyphenolic compounds by the fungal laccases has been studied. As laccase substrates, such methoxyphenolic compounds as 4-hydroxy-3,5-dimethoxycinnamic acid (sinapinic acid), 4-hydroxy-3-methoxycinnamic acid (ferulic acid), and 2-methoxyphenol (guaiacol) were used. The stoichiometries of the enzymatic reactions were determined: guaiacol and sinapinic acid are one-electron donors and their oxidation apparently results in the formation of dimers. It was established that k_cat/K_m, which indicates the effectiveness of catalysis, increases in the series guaiacol, ferulic acid, and sinapinic acid. This fact might be connected with the influence of substituents of the phenolic ring of the substrates. This phenomenon was established for fungal laccases with different physicochemical properties, amino acid composition, and carbohydrate content. This suggests that all fungal laccases possess the same mechanism of interaction between organic substrate electron donors and the copper-containing active site of the enzyme and that this interaction determines the observed values of the kinetic parameters.

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KEY WORDS: laccase, methoxyphenolic compounds, ferulic acid, sinapinic acid, guaiacol, catalytic efficiency

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