Indirect oxidation of 6-tetrahydrobiopterin by tyrosinase

Abstract

6-Tetrahydrobiopterin is known to bind to an allosteric site of tyrosinase to directly inhibit the enzyme. However, simultaneous measurements of ultraviolet–visible absorption spectra and oxygen consumption led us to conclude that the inhibition was due to oxidation of 6-tetrahydrobiopterin by dopaquinone. Immediately after addition of 6-tetrahydrobiopterin, tyrosinase stopped producing dopachrome from either tyrosine or dopa. Duration of inhibition was proportional to the concentration of added 6-tetrahydrobiopterin and the enzyme activity was fully restored after the inhibition. Surprisingly, there was a rapid consumption of oxygen during the inhibition period. In addition, absorption spectra indicated that the only reaction that occurred during the inhibition was oxidation of 6-tetrahydrobiopterin to 7,8-dihydrobiopterin. In the absence of tyrosine or dopa, tyrosinase did not oxidize 6-tetrahydrobiopterin, suggesting that a reaction intermediate between dopa and dopachrome was a target for the inhibition. We propose a new mechanism in which dopa is oxidized to dopaquinone and the latter, instead of producing dopachrome, is reduced back to dopa by 6-tetrahydrobiopterin.