Abstract
Puerol A (1) from Amorpha fruticosa showed highly potent inhibition against both monophenolase (IC50 = 2.2 μM) and diphenolase (IC50 = 3.8 μM) of tyrosinase. We tried to obtain a full story of enzyme inhibitory behavior for inhibitor 1 because the butenolide skeleton has never been reported as a tyrosinase inhibitor. Puerol A was proved as a reversible, competitive, simple slow-binding inhibitor, according to the respective parameters; k3 = 0.0279 μM−1 min−1 and k4 = 0.003 min−1. A longer lag-phase and a reduced static-state activity of the enzyme explained that puerol A had a tight formation of the complex with Emet. Dose-dependent inhibition was also confirmed by high-performance liquid chromatography (HPLC) analysis using N-acetyl-l-tyrosine as a substrate, which was completely inhibited at 20 μM. A high binding affinity of 1 to tyrosinase was confirmed by fluorescence quenching analysis. Moreover, puerol A decreased melanin content in the B16 melanoma cell dose-dependently with an IC50 of 11.4 μM.
Highlights
Tyrosinase (EC 1.14.18.1) is the type-3 metalloenzyme, which is most highly related with the production of melanin
(1 and 2) accord with those previously published for puerol A (1) and kuzubutenolide A (2) [15,16]
The results proved that the potent inhibition of 1 arises from an effective binding affinity to tyrosinase
Summary
Tyrosinase (EC 1.14.18.1) is the type-3 metalloenzyme, which is most highly related with the production of melanin. Melanin production in the living organism is a natural adaption to the outside environment for protecting the skin from ultraviolet damage and reactive oxygen species (ROS). Most of the strategies of controlling melanin production have focused on the regulation of tyrosinase activity. Tyrosinase catalyzes two steps with monophenolase and diphenolase at the same active site as follows; the hydroxylation of l-tyrosine to l-3,4-dihydroxyphenylalanine (l-DOPA) and the oxidation of l-DOPA to dopaquinone. The subsequent steps from dopaquinone to melanin are extremely rapid and are non-enzyme catalyzed processes [3]. The inhibitors of tyrosinase are derived from phenolic compounds because they have structural similarity with the substrate, l-tyrosine.
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