The activity of mushroom tyrosinase on a representative series of phenolic and diphenolic substrates structurally related to tyrosine has been investigated in the mixed solvent of 34.4% methanol-glycerol (7/1 v/v) and 65.6% (v/v) aqueous 50 mM Hepes buffer pH 6.8 at various temperatures. The kinetic activation parameters ruling the enzymatic reactions and the thermodynamic parameters associated with the substrate binding process to the enzyme active species have been deduced from the temperature variation of the kcat and KM parameters. The activation free energy is dominated by the enthalpic term, which occurs in the relatively narrow range of 61 kJ mol-1 independent of substrate and 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-hydroxyphenyl propionic acid exhibits a strikingly anomalous temperature dependence of the enzymatic oxidation rate, with deltaH 150 kJ mol-1 and deltaS 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 includes the dual nature of substrate/inhibitor of this compound enables to account for the anomalous behavior.
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