Some kinetic properties of deoxytyrosinase

Passing a nitrogen stream over a preparation of oxy-tyrosinase (Eox) gives rise to the relaxed deoxy-  tyrosinase form (EdR), which, under anaerobic conditions, slowly transforms into tense deoxy-  tyrosinase (EdT). In the presence of oxygen, regeneration of the form Eox from EdR is rapid but from EdT it is a slow process. However, when two substrates (oxygen/o  -diphenol or oxygen/monophenol) are simultaneously added, both the EdR and EdT forms rapidly revert to the active Eox form, pointing to a synergistic effect of both substrates. However, the activity obtained in the case of EdT is less than that of the native enzyme and of the enzyme that can be generated rapidly by pre-incubation with oxygen of the EdR recently formed by passage of the nitrogen stream, or that generated slowly by pre-incubating the EdT form with oxygen. Although the Vmax of the forms EdR and EdT are very similar, the Michaelis constant of the latter is higher. The kinetic properties of EdR are similar to those of the native enzyme. The behaviour of the monophenols is similar to that of the o-  diphenols, although, while the latter inactivate the enzyme under anaerobic conditions, the former protect it from inactivation. The pH affects the transition from EdR to EdT, which is more rapid at pH 6.5, at which value the kinetic properties of the native enzyme and of EdT are similar and the oxygenation step in which EdT regenerates Eox is more rapid. At pH values other than 6.5, the transition of EdR to EdT takes place slowly. From a study of the effect of pH on the transition of EdR to EdT and of the re-oxygenation of EdT to Eox, the possible existence of two apparent pKas, with approximate values of 6.0 and 6.8, may be surmised. At high pH, the enzyme contains two acid/base groups carrying negative charges, which repel (pH > 6.8) or two positive charges (at pH < 6.0), which also repel, while at ∼pH 6.5 one positive and one negative group exists, which prevents the separation of the two copper atoms.