A NEW ENZYMATIC FUNCTION IN THE MELANOGENIC PATHWAY - THE 5,6-DIHYDROXYINDOLE-2-CARBOXYLIC ACID OXIDASE ACTIVITY OF TYROSINASE-RELATED PROTEIN-1 (TRP1)

Since the characterization of 5,6 dihydroxyindole-2-carboxylic acid (D HICA) as a major melanogenic intermediate, the fate of this compound a nd the mechanisms of its incorporation into the melanin polymer have b ecome major issues in the study of melanogenesis. DHICA is a stable di hydroxyindole with a low rate of spontaneous oxidation, suggesting tha t enzymatic mechanism(s) might contribute to its evolution. The most o bvious candidates are the melanosomal tyrosinases. We have recently sh own that mouse melanosomes contain two electrophoretically distinct ty rosinase isoenzymes, termed low electrophoretic mobility tyrosinase (L EMT) and high electrophoretic mobility tyrosinase (HEMT), that can be resolved and purified. In this study, we report immunological evidence indicating that LEMT corresponds to the protein encoded by the brown locus (termed tyrosinase-related protein-1, TRP1), while HEMT correspo nds to the tyrosinase encoded by the albino locus. We have compared th e ability of both isoenzymes to catalyze DHICA evolution as determined by high performance liquid chromatography; although LEMT is a relativ ely poor tyrosine hydroxylase and DOPA oxidase as compared to HEMT, it was readily able to accelerate DHICA consumption concomitant with the production of a brownish product. However, the DHICA conversion activ ity of HEMT was barely detectable. The ability of purified LEMT to cat alyze DHICA conversion could be almost completely abolished by treatme nt with heat or trypsin, and was inhibited in a concentration dependen t way by the tyrosinase inhibitor 2-phenylthiourea and by L-tyrosine, Moreover, in the presence of low concentration of ascorbate, the DHICA conversion activity of LEMT displayed a lag period which was progress ively longer at higher ascorbate concentrations. Based on the relation ship between ascorbate added, enzyme activity, and lag period, it is v ery likely that the DHICA converting activity is indeed a DHICA oxidas e activity. This was further proven by the demonstration that the prod uct reacts rapidly and efficiently with the quinone trapping reagent 3 -methyl-2-benzothiazolinone hydrazone, yielding a colored adduct simil ar to the one obtained with DOPAquinone. The DHICA oxidase activity of LEMT displayed a K-m for DHICA of about 0.8 mM, as compared to 1.9 mM for L-DOPA and 0.23 mM for L tyrosine. These results suggest that TRP 1, the product of the brown locus, is indeed a tyrosinase with DHICA o xidase activity. However, as opposed to the tyrosinase encoded by the albino locus, TRP1's role in melanogenesis could be more directly rela ted to DHICA metabolism than to the first steps of the pathway.