Inhibitor binding to the binuclear active site of tyrosinase: temperature, pH, and solvent deuterium isotope effects.
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.
Conrad
J S
JS
Department of Chemistry, Drew University, Madison, New Jersey 07940.
Dawso
S R
SR
Hubbard
E R
ER
Meyers
T E
TE
Strothkamp
K G
KG
D001665:Binding Sites; D002264:Carboxylic Acids / Q000494:pharmacology; D003903:Deuterium; D006571:Heterocyclic Compounds / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011485:Protein Binding; D012997:Solvents; D013696:Temperature; D013816:Thermodynamics; D014050:Toluene / Q000494:pharmacology
D002264:Carboxylic Acids; D006571:Heterocyclic Compounds; D012997:Solvents; D014050:Toluene; D003903:Deuterium; D014442:Monophenol Monooxygenase