Kinetic study of monophenol and o-diphenol binding to oxytyrosinase

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 32; no. 5; pp. 185 - 192
• Main Authors: García-Molina, F., Peñalver, M.J., Fenoll, L.G., Rodríguez-López, J.N., Varón, R., García-Cánovas, F., Tudela, J.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.03.2005; Elsevier Science
• Subjects: Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Dead-end complex; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Monophenol and o-diphenol; Oxytyrosinase; Dead-end complex; Monophenol and o-diphenol; Oxytyrosinase; Phenols; Kinetics; Substrate enzyme complex
• DOI: 10.1016/j.molcatb.2004.12.005
• ISSN: 1381-1177

The complex reaction mechanism of tyrosinase involves three enzymatic forms, two overlapping catalytic cycles and a dead-end complex. The deoxytyrosinase form binds oxygen with a high degree of affinity, K s O 2 = 46.6 ± 2.4 μM. The mettyrosinase and oxytyrosinase forms bind monophenols and o-diphenols, although the former is inactive on monophenols. Analytical expressions for the catalytic and Michaelis constants of tyrosinase towards phenols and o-diphenols have been derived. Thus, the Michaelis constant of tyrosinase towards monophenols ( K m M ) and o-diphenols ( K m D ) are related with the catalytic constants for monophenols ( k cat M ) and o-diphenols ( k cat D ) , respectively, and with the binding rate constants of the oxytyrosinase form with these substrates ( k +4 and k +6, respectively), by means of the expressions K m M = k cat M / k + 4 and K m D = k cat D / k + 6 . From these expressions, we calculate the values of the binding rate constant of oxytyrosinase to the substrates (monophenols and o-diphenols) for tyrosinases from different biological sources, and reveal that the o-diphenols bind more rapidly to oxytyrosinase than the monophenols. In addition, a new kinetic constant K m D ( M ) = k cat M / 2 k 6 (the Michaelis constant for o-diphenol in the monophenolase activity), is derived and determined. Thus, it has been shown that tyrosinase has apparently higher affinity towards o-diphenols in its monophenolase than in its diphenolase activity.