Physicochemical and kinetic properties of mushroom tyrosinase.

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 mp), which is difficult to explain by a combination of the usual peptide conformational forms. Small positive ellipticity bands ([0] per 32,000 mol wt LZ 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 -I% -SCN, -COCHa, -CHO, -CN, and -NO2 have been determined. As the electron-withdrawing ability of the substituent increases, K, and keat decrease in the order H > SCN > COCHB > CHO > CN > NOz. Except for the keat values for the two most slowly oxidized substrates, both K, and kcat conform to Hammett relationships when the substituent parameter uis used. The p values are 1.01 f 0.09 for K, and -2.49 f 0.11 for &. The oxidation of pyrocatechol, 4-COCH, and 4-CHO-catechol give a satisfactory isokinetic relationship between AH$. and AS& 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 K, 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% 0~). The oxygen kinetics suggest a sequential mechanism for the binding of catechol and oxygen. Detailed studies of the pH dependence of K,,, and kcat show the presence of a group in the enzyme whose pK, changes upon the binding of a catechol molecule.