Inhibitory Effect of Ammonium Tetrathiotungstate on Tyrosinase and Its Kinetic Mechanism

Abstract

Tyrosinase (monophenol, o-diphenol : oxygen oxidoreductase, EC 1.14.18.1) is a binuclear copper enzyme, which is fairly ubiquitously distributed throughout organisms, and is known to play an important role in melanin synthesis. [1][2][3][4] Tyrosinase has been demonstrated to catalyze the hydroxylation of phenols to catechols, as well as the oxidation of catechols to quinones. The active site of tyrosinase consists of two copper ions, each coordinated by histidine residues within the active sites. These two coppers are known to be essential for the catalytic activities of this enzyme.5) The binuclear copper active sites exist in all tyrosinases, regardless of source. 6,7) Therefore, the chelation of copper ions in this enzyme by flavonoid compounds or any other agents has been targeted as a strategy for the attenuation of tyrosinase activity, an objective relevant to cosmetic concerns, darkening problems in agricultural products, and certain medicinal reasons. [8][9][10][11][12][13][14] Melanin pigments are synthesized by melanocytes, and are deposited throughout the epidermis. This process determines skin color. [15][16][17][18] Tyrosinase is involved in two different ratelimiting steps in melanin synthesis; namely, the hydroxylation of tyrosine to 3,4-dihyroxyphenylalanine (DOPA), and the oxidation of DOPA to dopaquinone.19) The abnormal accumulation of melanin pigment can induce hyperpigmentations, such as freckles, melasma, and senile lentigines. These symptoms have responded, in the past, to treatment with depigmenting agents, including hydroquinone, ascorbic acid derivatives, retinoids, arbutin, and kojic acid. In particular, kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) is a copper-chelator, which has been used as a cosmetic agent in skin-whitening formulations. 20) However, safer and more effective depigmenting agents are clearly needed, as kojic acid is not only carcinogenic, but its associated whitening effects are also somewhat weak. 21,22) We have, in previous studies, applied several copper-specific chelating agents to mushroom tyrosinase, in an effort to screen an effective inhibitor, with the strategy of copper chelation at the active enzyme site.23) Among these compound, ammonium tetrathiomolybdate (ATTM) has been shown to exert a significant inhibitory effect, as compared to the effect of other tested substances. In this study, we have attempted to characterize the effects of ammonium tetrathiotungstate (ATTT) on tyrosinase. ATTT has been identified as an effective decoppering reagent for copper storage-related diseases in both rats and sheep. 24,25) However, the kinetics of its inhibition in copper-containing enzymes has been the subject of only minimal exploration. Thus, we have attempted to evaluate the inhibition kinetics of tyrosinase by ATTT. Our kinetic studies revealed that ATTT was capable of the inactivation of tyrosinase in vitro. We have also verified the inhibitory effects of ATTT on tyrosinase in human melanocyte cells, as compared with those evidenced by other known inhibi...

Kinetic, structural and molecular docking studies on the inhibition of tyrosinase induced by arabinose

“…We monitored such changes using spectrofluorometry. The inhibitory mechanism of arabinose resembled that of a copper chelator, but differs in that arabinose did not directly bind to copper atoms at the active site [34][35][36][37].…”

Tyrosinase inhibition by isophthalic acid: Kinetics and computational simulation

“…In this context, we determined that IPA, with two hydroxyl groups, also inhibits the enzyme. However, the inhibitory mechanism of IPA differs from those of simple copper chelators [28][29][30][31][32]. This is evident in that IPA competes with substrate l-DOPA in the active site pocket in a complex manner (Scheme 1) without inducing changes in the tertiary structure.…”

Potent inhibitory effects of benzyl and p-xylidine-bis dithiocarbamate sodium salts on activities of mushroom tyrosinase

“…On the other hand, the absorbance spectra of the inhibitors did not show considerable absorbance at the intrinsic emission area of MT (data not shown). So, the conformation of inhibitor-bound MT was stable and more rigid than that of uninhibited MT 62 . In recent studies, we have considered the inhibitory effects of some thiol compounds such as n-alkyl dithiocarbamates, n-alkyl xanthates, and benzylthiol 48,49,60 .…”