Kinetic analyses of two-steps oxidation from l-tyrosine to l-dopaquinone with tyrosinase by capillary electrophoresis/dynamic frontal analysis

• Published in: Analytical biochemistry Vol. 655; p. 114856
• Main Authors: Mine, Masanori, Mizuguchi, Hitoshi, Takayanagi, Toshio
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 15.10.2022
• Subjects: Capillary electrophoresis; Dynamic frontal analysis; enzyme kinetics; Kinetic analysis; L-dopa; oxidation; Two-steps oxidation; Tyrosinase; tyrosine; Kinetic analysis; Dynamic frontal analysis; Capillary electrophoresis; Two-steps oxidation; Tyrosinase
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2022.114856

Tyrosinase catalyzes the oxidation of l-tyrosine in two stages to produce l-dopa and l-dopaquinone stepwise, and l-dopaquinone is subsequently converted to dopachrome. Most of the conventional analyses subjected only one-step reaction from l-tyrosine to l-dopa or from l-dopa to l-dopaquinone. In this study, kinetic analyses of two-steps oxidation of l-tyrosine with tyrosinase were made by capillary electrophoresis/dynamic frontal analysis (CE/DFA). When l-dopa was introduced into a capillary as a sample plug in a CE/DFA format, the enzymatic oxidation continuously occurred during the electrophoresis, and the product l-dopaquinone was subsequently converted to dopachrome which was detected as a plateau signal. A Michaelis-Menten constant of the second-step kinetic reaction, Km,Do, was determined as 0.45 ± 0.03 mmol L−1. In the analysis of the first-step kinetic reaction from l-tyrosine to l-dopa, l-dopa was not resolved by CE/DFA because both l-tyrosine and l-dopa are electrically neutral. The l-dopa formed and co-migrated at the l-tyrosine zone was calibrated beforehand with the final product of dopachrome detected as a plateau signal. Constantly formed l-dopa was successfully detected as a plateau signal of dopachrome, and a Michaelis-Menten constant of Km,Ty was also determined as 0.061 ± 0.009 mmol L−1 by the CE/DFA. CE/DFA is applicable to two-steps enzymatic reactions. [Display omitted] •Two-steps enzymatic oxidation with tyrosinase was analyzed by CE/DFA.•A final product of dopachrome was continuously resolved from the substrate zone.•Plateau signal under the enzymatic oxidation was provided for the kinetic analysis.•The two-steps reactions were independently analyzed without substrate inhibition.