title: Deuterium isotope effect on the suicide inactivation of tyrosinase in its action on o-diphenols.
abstract: A solvent deuterium isotope effect on the inactivation suicide of tyrosinase in its action on o-diphenols (catechol, 4-methylcatechol, and 4-tert-butylcatechol) was observed. This isotope effect, observed during kinetic studies in the transition phase, was higher than that described previously in the steady state, indicating that there is an additional slow step in the suicide inactivation mechanism, which we believe to be responsible for the inactivation. In a proton inventory study of oxidation of o-diphenols, the representation of λmax(D,fn)/λmax(D,f0) versus n (atom fractions of deuterium), where λmax(D,fn) is the maximum apparent inactivation constant for a molar fraction of deuterium (n) and λmax(D,f0) is the corresponding kinetic parameter in a water solution, was linear for all substrates. This suggests that only one of the protons transferred from the two hydroxyl groups of the substrate, which are oxidized in one turnover, is responsible for the isotope effects. We propose that this proton could be the proton transferred from the hydroxyl group of C-2 to the hydroperoxide of the oxytyrosinase form (Eox ) and that it probably causes enzyme inactivation through the reduction of the Cu(2+) A to Cu(0) and its subsequent release from the active site.
authors.affiliation: GENZ: Grupo de Investigación Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, Espinardo, Murcia, Spain.
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Muñoz-Muñoz
authors.forename: María del Mar
authors.initials: Mdel M
authors.lastname: García-Molina
authors.forename: Francisco
authors.initials: F
authors.lastname: Garcia-Molina
authors.forename: Pedro Antonio
authors.initials: PA
authors.lastname: Garcia-Ruiz
authors.forename: Francisco
authors.initials: F
authors.lastname: Garcia-Sevilla
authors.forename: Jose N
authors.initials: JN
authors.lastname: Rodriguez-Lopez
authors.forename: Francisco
authors.initials: F
authors.lastname: Garcia-Canovas
mesh_terms: D000364:Agaricus / Q000201:enzymology; D003903:Deuterium / Q000737:chemistry; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D010636:Phenols / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D010636:Phenols; D003903:Deuterium; D014442:Monophenol Monooxygenase
keywords: isotopic effect; steady state; suicide inactivation; transition phase; tyrosinase