Mutational mapping of the catalytic activities of human tyrosinase.
Tyrosinase (EC 1.14.18.1) is a copper-containing metalloglycoprotein that catalyzes several steps in the melanin pigment biosynthetic pathway; the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (dopa) and the subsequent oxidation of dopa to dopaquinone. It has been proposed that tyrosinase is also able to oxidize 5,6-dihydroxyindole (DHI), a later product in the melanogenic pathway, to indole-5,6-quinone. Tyrosinase enzymatic activity is deficient in patients with classic type I oculocutaneous albinism (OCA), and more than 50 distinct mutations have now been identified in the tyrosinase genes of such patients. To determine the effects of the various tyrosinase gene mutations on the catalytic activities of the enzyme, we carried out site-directed mutagenesis of human tyrosinase cDNA, transiently expressed the mutant cDNAs in transfected HeLa cells, and assayed the resultant encoded proteins for tyrosine hydroxylase, dopa, and DHI oxidase activities, and resulting melanin production. The tyrosine hydroxylase activity of normal tyrosinase is thermostable, whereas its dopa oxidase and DHI oxidase activities are temperature-sensitive. Although all amino acid substitutions tested generally affected the dopa oxidase and DHI oxidase activities in parallel, several exerted distinctly different effects on the tyrosine hydroxylase activities. Together, these results confirm the DHI oxidase activity of mammalian tyrosinase and suggest that the dopa oxidase and DHI oxidase activities of tyrosinase share a common catalytic site, whereas the tyrosine hydroxylase catalytic site is at least partially distinct in the tyrosinase polypeptide.
Tripathi
R K
RK
Department of Medical Genetics, University of Wisconsin-Madison 53706.
Hearing
V J
VJ
Urabe
K
K
Aroca
P
P
Spritz
R A
RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase