MOLECULAR MECHANISM FOR CATALYSIS BY A NEW ZINC-ENZYME, DOPACHROME TAUTOMERASE

Dopachrome tautomerase (DCT; EC 5.3.3.12) catalyses the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid in the mammali an eumelanogenic biosynthetic pathway. This enzyme, also named TRP2, b elongs to a family of three metalloenzymes termed the tyrosinase-relat ed proteins (TRPs). It is well known that tyrosinase has copper in its active site. However, the nature of the metal ion in the active site of DCT is under discussion. Whereas theoretical predictions based on s imilarity between the protein sequences of the TRPs suggest the presen ce of copper, the different inhibition pattern of DCT with some metal chelators compared with that of tyrosinase suggests that the nature of the metal ion could differ. Direct estimations of the metal content i n purified DCT preparations show the presence of around 1.5 Zn atoms/m olecule and the absence of copper. Apoenzyme preparation by treatment of DCT with cyanide or o-phenanthroline followed by reconstitution exp eriments of tautomerase activity in the presence of different ions con firmed that the metal cofactor for the DCT active site is zinc. Our re sults are consistent with Zn2+ chelation by the highly conserved histi dine residues homologous to the histidines at the classical copper-bin ding sites in tyrosinase. This finding accounts for the reaction catal ysed by DCT, i.e. a tautomerization, versus the copper-mediated oxidat ions catalysed by tyrosinase. Based on the predicted tetrahedrical coo rdination of the zinc ions in the enzyme active site, a molecular mech anism for the catalysis of L-dopachrome tautomerization is proposed. F rom the present data, the existence of additional ligands for metal io ns other than zinc in the DCT molecule, such as the proposed cysteine iron-binding sites, cannot be completely ruled out. However, if such s ites exist, they could be subsidiary binding sites, whose function wou ld be likely to stabilize the protein.