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.
Chakraborty
A K
AK
Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510.
Orlow
S J
SJ
Pawelek
J M
JM
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005296:Ferrous Compounds / Q000378:metabolism* / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000494:pharmacology; D045563:Indolequinones; D007211:Indoles / Q000378:metabolism; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008546:Melanoma, Experimental / Q000201:enzymology; D051379:Mice; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011809:Quinones / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002614:Chelating Agents; D005296:Ferrous Compounds; D006023:Glycoproteins; D062385:Hydroxybenzoates; D045563:Indolequinones; D007211:Indoles; D010618:Phenanthrolines; D011809:Quinones; C001123:dopachrome; D015082:2,2'-Dipyridyl; C009135:2,3-dihydroxybenzoic acid; D004492:Edetic Acid; D014442:Monophenol Monooxygenase; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase; C025205:1,10-phenanthroline