Abstract
The conversion of dopachrome to dihydroxyindole(s), a key reaction in eumelanin biosynthetic pathway, has been shown to be under the control of dopachrome conversion factor. Dopachrome conversion factor isolated from the hemolymph of Manduca sexta larvae, which is devoid of any tyrosinase activity, exhibits a narrow substrate specificity and readily bleaches the iminochromes derived from the oxidation of L-dopa, L-dopa methyl ester, and alpha-methyl-L-dopa, but failed to attack the corresponding D-isomers. The product formed in the case of L-dopachrome was identified to be 5,6-dihydroxyindole. Therefore, aromatization of dopachrome seems to accompany its decarboxylation as well. However, the enzyme also converts L-dopachrome methyl ester to an indole derivative indicating that it can deprotonate the alpha-hydrogen when the carboxyl group is blocked. These results are accounted for by the transient formation and further transformation of a reactive quinone methide intermediate during the dopachrome conversion factor-catalyzed reaction. The fact that the enzyme-catalyzed conversion of alpha-methyl dopachrome methyl ester (where both decarboxylation and deprotonation are blocked) resulted in the generation of a stable quinone methide in the reaction mixture confirms this contention and supports our recent proposal that quinone methide and not indolenine is the key transient intermediate in the conversion of dopachrome to dihydroxyindole observed during melanogenesis.
Highlights
The conversion odfopachromteo dihydroxyin- Mason [2, 3]
The dopachrome conversion step is an important reaction during melanogenesis, and yet only recently have various groups started investigating the detailed biochemistry of this reaction
The DCF from mammalian sources seems to catalyze the conversion of dopachrome to DHICA [6,7,8], while the DCF from insect cuticle seems to catalyze the conversion of dopacrome toDHI [9]
Summary
Fig. 3shows the HPLC analysis of the reaction mixture containing L-dopachrome and DCF. Assay of DCF-A reactionmixture (0.5 ml)containing enzyme protein and dopachrome was incubated a t room temperature and the chromic shift to 324 nm in 5% sodiumborate From these spectral data and the above mentioned HPLC studies, the product of the dopachrome conversion catalyzed by DCF was identified t o be DHI. Preparation of Zrninochrornes-Iminochromes and quinones were prepared by the silver oxide oxidation of parent catechols indeionized dopachrome methyl ester is the best substrate for DCF, and the product formed, in this case, was identified to be DHI-2carboxymethyl ester.
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