Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase

This paper reports a quantitative study of the effect of ring substituents in the 1-position of the aromatic ring on the rate of monophenol hydroxylat ion and o-diphenol oxidation catalyzed by tyrosinase. A possible correlatio n between the electron density of the carbon atom supporting the oxygen fro m the monophenolic hydroxyl group and the V-max(M) values for each monophen ol was found. In the case of o-diphenols the same effect was observed but t he size of the side-chain became very important. NMR studies on the monophe nols justified the sequence of the V-max(M) values obtained. As regards the o-diphenols, on the other hand, only a fair correlation between NMR and Vm (ax)(D) values was observed due to the effect of the molecular size of the ring substituent. From these data, it can be concluded that the redox step (k(33)) is not the rate-determining step of the reaction mechanism. Thus, t he monophenols are converted into diphenols, but the order of specificities towards monophenols is different to that of o-diphenols. The rate-limiting step of the monophenolase activity could be the nucleophilic attack (k(51) ) of the oxygen atom of the hydroxyl group on the copper atoms of the activ e site of the enzyme. This step could also be similar to or have a lower ra te of attack than the electrophilic attack (k(52)) of the oxygen atom of th e active site of oxytyrosinase on the C-3 of the monophenolic ring. However , the rate-limiting step in the diphenolase activity of tyrosinase could be related to both the nucleophilic power of the oxygen atom belonging to the hydroxyl group at the carbon atom in the 3-position (k(32)) and to the siz e of the substituent side-chain. On the basis of the results obtained, kine tic and structural models describing the monophenolase and diphenolase reac tion mechanisms for tyrosinase are proposed.