Kinetic and paramagnetic NMR investigations of the inhibition of Streptomyces antibioticus tyrosinase

A scaled-up isolation and purification procedure is described for tyrosinase from Streptomyces antibioticus. Kinetic studies of the enzyme catalysed conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) into DOPAchrome show that kojic acid, l-mimosine, p-toluic acid and benzoic acid exhibit competitive inhibition with inhibition constants of 3.4, 30, 1.9×102 and 8.0×102 μM, respectively. Paramagnetic NMR techniques appear well suited to study the binding of inhibitors to the active site. From the variation of the NMR shifts with temperature a value of −2J=156±6 cm−1 is derived for the exchange coupling between the unpaired spins on the two Cu(II) ions in the active site of the met-tyrosinase/kojic acid complex.