Inhibitor Binding to the Binuclear Active Site of Tyrosinase: Temperature, pH, and Solvent Deuterium Isotope Effects

Abstract

Competitive inhibition of the monophenolase reaction of tyrosinase by a variety of compounds was investigated with respect to temperature and pH. Derivatives of benzoic acid as well as p-nitrophenol were the best inhibitors. Toluene and several N-heterocycles were all weak inhibitors. Thermodynamic parameters of toluene inhibition were qualitatively different from those of aromatic acids and were consistent with a hydrophobic binding site for toluene. Inhibition by both aromatic acids and p-nitrophenol was strongly pH-dependent over the range 5.1-8.0, with inhibitor binding favored at lower pH. In contrast, toluene binding and indazole binding were pH-independent while benzimidazole binding was favored at higher pH. For both carboxylic acids and p-nitrophenol, inhibitor binding was quantitatively accounted for by assuming the protonated, electrically neutral form of the inhibitor is the reactive species. A solvent deuterium isotope effect on the binding of benzoic acid was observed. A proton inventory study indicated that a single proton transferred from benzoic acid to a site with a fractionation factor of 0.64 +/- 0.02 accounts for the isotope effect. The identity of the proton acceptor and possible mechanistic implications are discussed.

Spectroscopic Characterization of the Electronic Changes in the Active Site of Streptomyces antibioticus Tyrosinase upon Binding of Transition State Analogue Inhibitors

“…As we are dealing here with competitive inhibitors they must bind at or close to the copper site. However, the fact that nitrophenol can also act as a substrate, albeit a very poor one (45), is diagnostic of binding to the copper. The only inhibitor/enzyme complex of a type-3 copper protein of which a structure has been reported in the literature is catechol oxidase (8), where the thiourea inhibitor binds directly to the copper site.…”

Naturally occurring tyrosinase inhibitors: mechanism and applications in skin health, cosmetics and agriculture industries

“…It is interesting to note that electron-donating groups (isopropyl and methoxy) at the para position in cuminaldehyde provide stability to the Schiff base at the active site of the enzyme through inductive effect. In the case of (2E)-alkenals the hydrophobic alkyl chain length from the hydrophilic enal group seems to be related to their inhibitory potency, which may be due to better association of the longer alkyl chain with the hydrophobic protein pocket close to the binuclear copper site (Tanford, 1980;Wilcox et al, 1985;Conrad et al, 1994). Except for 2-hydroxy-4-methoxybenzaldehyde, the above-indicated aromatic aldehydes were described as noncompetitive tyrosinase inhibitors by .…”

Dual effects of aliphatic carboxylic acids on cresolase and catecholase reactions of mushroom tyrosinase

“…In the case of acid derivatives, the mechanism of inhibition involves the formation of copper-carboxylic acid complex at binuclear copper site of the enzyme [10,39]. P-coumaric acid and its corresponding dihydroxy derivative, caffeic acid bind to the free copper through their carboxylic terminal that is attached to a sp 2 carbon.…”