Study of umbelliferone hydroxylation to esculetin catalyzed by polyphenol oxidase

Abstract

We characterize umbelliferone, a derivative of 2,4-dihydroxycoumaric acid, as a substrate of polyphenol oxidase. This enzyme hydroxylates umbelliferone to esculetin, its o-diphenol, and then oxidizes it to o-quinone. The findings show that umbelliferone, an intermediate in one of the coumarin biosynthesis pathways, may be transformed into its o-diphenol, esculetin, which is also an intermediate in the same pathway. The activity of the enzyme on umbelliferone was followed by measuring the consumption of oxygen, spectrophotometrically and by HPLC. Kinetic constants characterizing the hydroxylation process were: k cat=0.09±0.02 s-1 and Km=0.17±0. 06 mm. The o-diphenol, esculetin, was a better substrate and when its oxidation was followed spectrophotometrically, the kinetic constants were: k cat=1.31±0.25 s-1 and Km=0.035±0. 002 mM. Both compounds therefore can be considered as alternative substrates to l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA), since both indirectly inhibit melanogenesis. © 2013 The Pharmaceutical Society of Japan.