MUTATIONAL ANALYSIS OF COPPER-BINDING BY HUMAN TYROSINASE

Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme that catalyzes several reactions in the biosynthesis of melanin pigments and is defi cient in patients with type I oculocutaneous albinism (OCA1), Tyrosina se is thought to bind two copper ions, one at each of two conserved se quence motifs, termed CuA and CuB, but to date this has been directly proved only for the Neurospora and mushroom enzyme. Here, we demonstra te that mammalian tyrosinase directly binds copper, and that the CuA a nd CuB sites are both required for copper binding and for catalytic ac tivity. We show that in human tyrosinase, copper binding by the CuB si te is most Likely coordinated by residues His363, His367, and His389, and that copper binding may be cooperative, with copper binding at one site facilitating copper binding by the other site. Furthermore, corr ect folding of the tyrosinase polypeptide appears to be necessary for copper binding, and a number of human OCA1 mutations disrupt copper bi nding and thus catalytic function of tyrosinase.