title: Catalytic oxidation of 2,4,5-trihydroxyphenylalanine by tyrosinase: identification and evolution of intermediates.
abstract: The oxidation of 3,4-dihydroxyphenylalanine (dopa) by O2 catalyzed by tyrosinase yields 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaquinone-H+). This evolves non-enzymatically through two branches (cyclization and/or hydroxylation), whose respective operations are determined by pH. The hydroxylation branch of o-dopaquinone-H+ only operates significantly at pH < or = 5.0 and involves the accumulation of 2,4,5-trihydroxyphenylalanine (topa), which has been detected by high-performance liquid chromatography (HPLC). This last compound is also a substrate of tyrosinase. The oxidation of topa by both tyrosinase and periodate yields 5-(2-carboxy-2-aminoethyl)-4-hydroxy-1,2-benzoquinone, with its amino group protonated (o-topaquinone-H+), which is red (RTQH) (lambda max 272-485 nm) at pH 7.0 and yellow (TTQH) (lambda max 265-390 nm) at pH 3.0. This is based on pKa 4.5 of the 2-OH group of the benzene ring of o-topaquinone-H+, as derived from spectrophotometric and HPLC assays. At physiological pH, RTQH undergoes deprotonation of the ammonium group of the side chain to yields RTQ, which cyclize into 2-carboxy-2,3-dihydroxyindolen-5,6-quinone (dopachrome), with a 1:1 stoichiometry and first-order kinetics. The evolution of RTQH has been analyzed by spectrophotometry, HPLC, cyclic voltammetry and constant potential electrolytic assays. From HPLC assays, the value of the first-order constant for the evolution of RTQH at pH 7.0 (kRTQHapp 4.83 x 10(-5) s-1), as well as of the rate constant for the cyclization step of RTQ (kRTQc 2.53 x 10(-3) s-1) were determined.
authors.affiliation: Departamento de Química-Física, E.U. Politécnica de Albacete, Universidad de Castilla-La Mancha, Spain.
authors.forename: J N
authors.initials: JN
authors.lastname: Rodríguez-López
authors.forename: M
authors.initials: M
authors.lastname: Bañón-Arnao
authors.forename: F
authors.initials: F
authors.lastname: Martinez-Ortiz
authors.forename: J
authors.initials: J
authors.lastname: Tudela
authors.forename: M
authors.initials: M
authors.lastname: Acosta
authors.forename: R
authors.initials: R
authors.lastname: Varón
authors.forename: F
authors.initials: F
authors.lastname: García-Cánovas
mesh_terms: D000818:Animals; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: C001388:6-hydroxydopa; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D014442:Monophenol Monooxygenase