Deuterium isotope effect on the oxidation of monophenols and o-diphenols by tyrosinase.

A solvent deuterium isotope effect on the catalytic affinity (km) and catalytic constant (kcat) of tyrosinase in its action on different monophenols and o-diphenols was observed. The catalytic constant decreased in all substrates as the molar fraction of deuterated water in the medium increased, while the catalytic affinity only decreased for the o-diphenols with an R group in C-1 [-H, -CH3 and -CH(CH3)2]. In a proton inventory study of the oxidation of o-diphenols, the representation of kcat fn/kcat f0 against n (atom fractions of deuterium), where kcat fn is the catalytic constant for a molar fraction of deuterium (n) and kcat f0 is the corresponding kinetic parameter in a water solution, was linear for all substrates, indicating that only one of the four protons transferred from the hydroxy groups of the two molecules of substrate, which are oxidized in one turnover, is responsible for the isotope effects, the proton transferred from the hydroxy group of C-4 to the peroxide of the oxytyrosinase form (Eox). However, in the representation of Km fn/Km f0 against n, where Km fn represents the catalytic affinity for a molar fraction of deuterium (n) and Km f0 is the corresponding kinetic parameter in a water solution, a linear decrease was observed as n increased in the case of o-diphenols with the R group [-H, -CH3 and -CH(CH3)2], and a parabolic increase with other R groups, indicating that more than one proton is responsible for the isotope effects on substrate binding. In the case of monophenols with six protons transferred in the catalytic cycle, the isotope effect occurs in the same way as for o-diphenols. In the present paper, the fractionation factors of different monophenols and o-diphenols are described and possible mechanistic implications are discussed.