Physicochemical and Kinetic Properties of Mushroom Tyrosinase

Abstract

Abstract Tyrosinase (o-diphenol:O2 oxidoreductase, EC 1.10.3.1) has been purified from a commercial lyophilized powder prepared from edible mushrooms. Specific activities toward dl-dihydroxyphenylalanine and catechol, homogeneity, molecular weight, absorption spectrum, amino acid composition, and copper content of the preparation are comparable to other purified samples of tyrosinase prepared from fresh mushrooms. Circular dichroism of the enzyme reveals an unusually shaped envelope of negative ellipticity bands in the deep ultraviolet (196 to 250 mµ), which is difficult to explain by a combination of the usual peptide conformational forms. Small positive ellipticity bands ([θ] per 32,000 mol wt ≅ 30,000) are associated with the near ultraviolet absorption of the aromatic amino acid side chains. Kinetic constants for the oxidation of a homologous series of catechol substrates substituted in position 4 by groups —H, —SCN, —COCH3, —CHO, —CN, and —NO2 have been determined. As the electron-withdrawing ability of the substituent increases, Km and kcat decrease in the order H g SCN g COCH3 g CHO g CN g NO2. Except for the kcat values for the two most slowly oxidized substrates, both Km and kcat conform to Hammett relationships when the substituent parameter σ- is used. The ρ values are -1.01 ± 0.09 for Km and -2.49 ± 0.11 for kcat. The oxidation of pyrocatechol, 4-COCH3, and 4-CHO-catechol give a satisfactory isokinetic relationship between ΔH‡ and ΔS‡. These findings suggest that these substrates are oxidized by the same basic mechanism. Studies of the dependence of catechol oxidation on the concentration of oxygen show that Km for oxygen varies with the nature of the catechol substrate. With the exception of the pyrocatechol complex, the enzyme-substrate complexes for this series of catechols are saturated in air (20.95% O2). The oxygen kinetics suggest a sequential mechanism for the binding of catechol and oxygen. Detailed studies of the pH dependence of Km and kcat show the presence of a group in the enzyme whose pKa changes upon the binding of a catechol molecule. Inhibition of tyrosinase by benzoic acid and cyanide show that the former is competitive with catechol and noncompetitive with oxygen, while the latter is competitive with oxygen and noncompetitive with catechol. These results are taken to indicate two distinct substrate binding sites on the enzyme, one with a high affinity for aromatic compounds including phenolic substrates, the other for metal-binding agents and oxygen. The latter site presumably involves enzyme copper. Binding studies with 14C-benzoic acid suggest that there are two binding sites for benzoic acid per mole of tetramer (mol wt = 128,000). The equilibrium constant for this binding is the same as the Ki measured for competitive inhibition of catechol oxidation by benzoic acid. Catechols are activators of the phenolase reaction of tyrosinase, and tyrosine is shown to be an inhibitor of this activation of its own oxidation in a manner suggesting that there is a third binding site on the enzyme for an activator catechol or phenol.