Studies on enzymatic oxidation of 30,40-dihydroxy-L-phenylalanine to dopachrome using kinetic isotope effect methods

Abstract We report the studies on the mechanism of oxidation of 30,40-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: [20,50,60-2H3]-L-DOPA was synthesized by the acid catalyzed isotopic exchange between native L-DOPA and heavy water, and [50-2H]-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 [30,50-2H2]-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 spec-trophotometric method. The measured values were: KIE on Vmax (1.1 and 2.2) and KIE on Vmax/KM (1.7 and 3.2) for [20,50,60-2H3]-L-DOPA and [50-2H]-L-DOPA, respectively, while the corresponding values of SIE were: SIE on Vma