2-Thiouracil (TU), an
antithyroid drug, is receiving growing interest as a specific
tumor marker for
malignant melanoma, owing to its capability of being selectively accumulated into active
melanin-producing tissues. However, up until now, the molecular mechanism of TU uptake by growing
melanin has remained largely unknown. In an attempt to fill this gap, we have investigated the effect of TU on the
tyrosinase catalyzed oxidation of
tyrosine. At a concentration of 0.5 mM, TU was found to totally inhibit
melanin formation by
tyrosinase catalyzed oxidation of 0.25 mM
tyrosine in
phosphate buffer at pH 6.8. Polarographical monitoring of oxygen consumption under conditions of complete suppression of melanogenesis revealed a significant
tyrosinase activity, with TU acting as a modest non-competitive inhibitor of the
enzyme (Ki = 0.6 mM). HPLC and TLC analysis of the
tyrosine-
tyrosinase reaction in the presence of excess TU showed that the substrate is progressively consumed and a major hitherto unknown product (lambda max = 284 nm), positive to
ninhydrin and
ferric chloride, is concomitantly formed. This was isolated by repeated gel filtration chromatography of the reaction mixture on
Sephadex G-10 and was formulated as the TU-
dopa adduct 3,4-dihydroxy-6-(4'-hydroxypyrimidinyl-2'-thio)phenylalanine by spectral analysis. These results suggest that selective TU incorporation in pigmented
melanomas and other
melanin-producing systems is due to the covalent binding to
dopaquinone, produced by
tyrosinase catalyzed oxidation of
tyrosine.