Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates.
Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (MT). Using diazo derivatives of phenol, 4-[(4-methoxybenzo)azo]-phenol, 4-[(4-methylphenyl)azo]-phenol, 4-(phenylazo)-phenol, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide, and diazo derivatives of catechol 4-[(4-methylbenzo)azo]-1,2-benzenediol, 4-(phenylazo)-1,2-benzenediol, and 4-[(4-sulfonamido)azo]-1,2 benzenediol (SACat), as substrates allows measurement of the rates of the corresponding enzymatic reactions through recording of the depletion rates of substrates at their lambda(max)(s) with the least interference of the intermediates' or products' absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of MT were comparable approaches. Based on the ensuing data, the electronic effect of the substituent on the substrate activity and the affinity of the enzyme for the substrate are reviewed. Kinetic parameters extracted from the corresponding Lineweaver-Burk plots and advantages of these substrates over the previously used substrates in similar assays of tyrosinases are also presented.
Haghbeen
Kamahldin
K
National Research Center for Genetic Engineering and Biotechnology, PO Box 14155-6343, Tehran, Iran. kamahl@nrcgeb.ac.ir
Wue Tan
Eng
E
D000363:Agaricales / Q000201:enzymology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D013379:Substrate Specificity
D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D014442:Monophenol Monooxygenase