Oxidation of monohydric phenol substrates by tyrosinase. An oximetric study.
The purity of commercially available mushroom tyrosinase was investigated by non-denaturing PAGE. Most of the protein in the preparation migrated as a single band under these conditions. This band contained both tyrosinase and dopa oxidase activity. No other activity of either classification was found in the preparation. Oxygen consumption by tyrosinase during oxidation of the monohydric phenol substrates tyrosine and 4-hydroxyanisole (4HA) was monitored by oximetry in order to determine the stoichiometry of the reactions. For complete oxidation, the molar ratio of oxygen: 4HA was 1:1. Under identical conditions, oxidation of tyrosine required 1.5 mol of oxygen/mol of tyrosine. The additional oxygen uptake during tyrosine oxidation is due to the internal cyclization of dopaquinone to form cyclodopa, which undergoes a redox reaction with dopaquinone to form dopachrome and dopa, which is then oxidized by the enzyme, leading to an additional 0.5 mol of oxygen/mol of original substrate. Oxygen consumption for complete oxidation of 200 nmol of 4HA was constant over a range of concentrations of tyrosinase of 33-330 units/ml of substrate. The maximum rate of reaction was directly proportional to the concentration of tyrosinase, whereas the length of the lag phase decreased non-linearly with increasing tyrosinase concentration. Activation of the enzyme by exposure to citrate was not seen, nor was the lag phase abolished by exposure of the enzyme to low pH. Michaelis-Menten analysis of tyrosinase in which the lag phase is abolished by pre-exposure of the enzyme to a low concentration of dithiothreitol gave Km values for tyrosine and 4HA of 153 and 20 microM respectively.
Naish-Byfield
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Department of Biology and Biochemistry, Brunel University, Uxbridge, Middx, U.K.
Riley
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PA
D000840:Anisoles / Q000378:metabolism; D001487:Basidiomycota / Q000201:enzymology; D016227:Benzoquinones / Q000378:metabolism; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010636:Phenols / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D000840:Anisoles; D016227:Benzoquinones; D002951:Citrates; D010636:Phenols; D019343:Citric Acid; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; C009760:mequinol; C035157:dopaquinone; D014442:Monophenol Monooxygenase