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.