Some kinetic properties of deoxytyrosinase

Passing a nitrogen stream over a preparation of oxy-tyrosinase (Eox) gives rise to the relaxed deoxy-tyrosinase form ( E d R ) , which, under anaerobic conditions, slowly transforms into tense deoxy-tyrosinase ( E d T ) . In the presence of oxygen, regeneration of the form Eox from E d R is rapid but from E d T it is a slow process. However, when two substrates (oxygen/o-diphenol or oxygen/monophenol) are simultaneously added, both the E d R and E d T forms rapidly revert to the active Eox form, pointing to a synergistic effect of both substrates. However, the activity obtained in the case of E d T is less than that of the native enzyme and of the enzyme that can be generated rapidly by pre-incubation with oxygen of the E d R recently formed by passage of the nitrogen stream, or that generated slowly by pre-incubating the E d T form with oxygen. Although the Vmax of the forms E d R and E d T are very similar, the Michaelis constant of the latter is higher. The kinetic properties of E d R 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 E d R to E d T , which is more rapid at pH 6.5, at which value the kinetic properties of the native enzyme and of E d T are similar and the oxygenation step in which E d T regenerates Eox is more rapid. At pH values other than 6.5, the transition of E d R to E d T takes place slowly. From a study of the effect of pH on the transition of E d R to E d T and of the re-oxygenation of E d T 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.