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Mechanistic Insight into the Activity of Tyrosinase from Variable‐Temperature Studies in an Aqueous/Organic Solvent
Author(s) -
Granata Alessandro,
Monzani Enrico,
Bubacco Luigi,
Casella Luigi
Publication year - 2006
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.200501097
Subject(s) - chemistry , aqueous solution , activation energy , enthalpy , substrate (aquarium) , carboxylate , active site , stereochemistry , solvent , gibbs free energy , entropy of activation , arrhenius equation , organic chemistry , enzyme , reaction rate constant , kinetics , thermodynamics , physics , oceanography , quantum mechanics , geology
Abstract The activity of mushroom tyrosinase towards a representative series of phenolic and diphenolic substrates structurally related to tyrosine has been investigated in a mixed solvent of 34.4 % methanol–glycerol (7:1, v/v) and 65.6 % (v/v) aqueous 50 m M Hepes buffer at pH 6.8 at various temperatures. The kinetic activation parameters controlling the enzymatic reactions and the thermodynamic parameters associated with the process of substrate binding to the enzyme active species have been deduced from the temperature variation of the k cat and K M parameters. The activation free energy is dominated by the enthalpic term, the value of which lies in the relatively narrow range of 61±9 kJ mol −1 irrespective of substrate or reaction type (monophenolase or diphenolase). The activation entropies are small and generally negative and contribute no more than 10 % to the activation free energy. The substrate binding parameters are characterized by large and negative enthalpy and entropy contributions, which are typically dictated by polar protein–substrate interactions. The substrate 4‐hydroxyphenylpropionic acid exhibits a strikingly anomalous temperature dependence of the enzymatic oxidation rate, with Δ H ≠ ≈150 kJ mol −1 and Δ S ≠ ≈280 J K −1 mol −1 , due to the fact that it can competitively bind to the enzyme through the phenol group, like the other substrates, or the carboxylate group, like carboxylic acid inhibitors. A kinetic model that takes into account the dual substrate/inhibitor nature of this compound enables rationalization of this anomalous behavior.