Regulation of tyrosinase by tetrahydropteridines and H2O2

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-L-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6) M 6BH4 to a specific domain 28 amino acids away from the Cu-A active site of the enzyme. Alternatively it was then shown that 10(-3) M 6BH4 inhibit the reaction by the reduction of the product dopaquinone back to L-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of L-dopaquinone back to L-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6) M after preactivation with L-tyrosine and this mechanism uncouples the enzyme reaction producing H2O2 from O-2. Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate L-tyrosine was demonstrated. The enzyme was activated by low concentrations of H2O2 (<0.3 x 10(-3) M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3) M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation. (C) 2004 Elsevier Inc. All rights reserved.