Studies on enzymatic oxidation of 3′,4′-dihydroxy-l-phenylalanine to dopachrome using kinetic isotope effect methods

We report the studies on the mechanism of oxidation of 3',4'-dihydroxy-l-phenylalanine (l-DOPA) to neurotoxic dopachrome catalyzed by enzyme horseradish peroxidase (EC 1.11.1.7) using the kinetic (KIE), and solvent (SIE), isotope effect methods. For kinetic studies two specifically deuterated isotopomers: [2',5',6'-2H3]-l-DOPA was synthesized by the acid catalyzed isotopic exchange between native l-DOPA and heavy water, and [5'-2H]-l-DOPA was synthesized in two step reaction. The first step involved acid catalyzed isotopic exchange between l-tyrosine and deuterated water and resulting product [3',5'-2H2]-l-tyrosine was hydroxylated by enzyme tyrosinase (EC 1.14.18.1). The values of deuterium KIEs and SIE's in the enzymatic oxidation of l-DOPA and its isotopomers are determined using non-competitive spectrophotometric method. The measured values were: KIE on Vmax (1.1 and 2.2) and KIE on Vmax/KM (1.7 and 3.2) for [2',5',6'-2H3]-l-DOPA and [5'-2H]-l-DOPA, respectively, while the corresponding values of SIE were: SIE on Vmax (2.1, 2.4, and 2.1) and SIE on Vmax/KM (1.3. 1.6, and 1.1) for l-DOPA, [2',5',6'-2H3]-l-DOPA, and [5'-2H]-l-DOPA, respectively. The size of KIE and SIE, typical for secondary isotope effects indicate that both the solvent and presence of deuterium at the 2'-, 5', and 6'-positions of l-DOPA has the little impact on the enzymatic oxidation of this compound.