Evidence that dopachrome tautomerase is a ferrous iron-binding glycoprotein.

Dopachrome tautomerase (DT) (EC 5.3.2.3) is a melanocyte-specific, membrane-associated, heat-labile, non-dialyzable, protease-sensitive factor which catalyzes the isomeric rearrangement of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), apparently through a tautomerization reaction. Metal ions such as Cu, Ni, Co, Zn, Mn, Ca, Al, and Fe can also catalyze the dopachrome/DHICA isomerization. How is the reaction regulated in vivo? An attractive possibility would be that DT is a metalloenzyme. Here we present evidence that this may indeed be the case. Purified preparations of DT and tyrosinase, obtained from Cloudman S91 mouse melanoma cells, were assayed in the presence of a variety of metal chelators including EDTA (predominantly Ca and Mg), EGTA (predominantly Ca), phenylthiourea (PTU) (predominantly Cu), 2,2'-dipyridyl (predominantly Fe); 1,10-phenanthroline (predominantly Fe), and 2,3-dihydroxybenzoic acid (predominantly Fe). In addition, DT activity was assayed in the presence of two non-chelating structural analogs of 1,10-phenanthroline. Results were as follows: (i) iron chelators inhibited DT activity with no effects on tyrosinase activity; (ii) inhibition by the chelators was reversible with the addition of ferrous iron; (iii) 1,10-phenanthroline pre-complexed to ferrous iron was not inhibitory to DT; (iv) non-chelating analogs of phenanthroline were not inhibitory to DT; (v) PTU was inhibitory to tyrosinase but not DT; (vi) Ca2+ and Mg2+ chelators had little effect on either enzyme activity. Finally, studies with glycosylation inhibitors, glycosylase enzymes, and immobilized lectins, indicated that DT is a glycoprotein. The results suggest that DT is a metal-containing glycosylated enzyme, possibly with ferrous iron at its catalytic center.