Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

[oculocutaneous albinism]

Tyrosinase (TYR ) catalyzes the rate-limiting , first step in melanin production and its gene (TYR ) is mutated in many cases of oculocutaneous albinism (OCA 1 ), an autosomal recessive cause of childhood blindness . Patients with reduced TYR activity are classified as OCA 1 B ; some OCA 1 B mutations are temperature-sensitive . Therapeutic research for OCA 1 has been hampered , in part , by the absence of purified , active , recombinant wild-type and mutant human enzymes .T he intra-melanosomal domain of human tyrosinase (residues 19 -469 ) and two OCA 1 B related temperature-sensitive mutants , R 422 Q and R 422 W were expressed in insect cells and produced in T . ni larvae . The short trans-membrane fragment was deleted to avoid potential protein insolubility , while preserving all other functional features of the enzymes . Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass . The protein was a monomeric glycoenzyme with maximum enzyme activity at 37 °C and neutral pH . The two purified mutants when compared to the wild-type protein were less active and temperature sensitive . These differences are associated with conformational perturbations in secondary structure .The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function . This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes ; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA 1 .