Kinetic characterization of the oxidation of esculetin by polyphenol oxidase and peroxidase.
Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.
Munoz-Munoz
Joseph Louis
JL
GENZ, Investigation Group of Enzymology, Department of Biochemistry and Molecular Biology A, Faculty of Biology, University of Murcia, Espinardo, Murcia, Spain.
Garcia-Molina
Francis
F
Varon
Raymond
R
Rodriguez-Lopez
Joseph Neptune
JN
Garcia-Canovas
Francis
F
Tudela
Joseph
J
D000363:Agaricales / Q000201:enzymology; D001205:Ascorbic Acid / Q000378:metabolism; D004156:Catechol Oxidase / Q000737:chemistry*; D006027:Glycosides / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry; D007202:Indicators and Reagents; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000737:chemistry; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000378:metabolism; D009195:Peroxidase / Q000737:chemistry*; D011284:Pregnenolone / Q000031:analogs & derivatives* / Q000737:chemistry; D011809:Quinones / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014815:Vitamins / Q000378:metabolism
D006027:Glycosides; D007202:Indicators and Reagents; D011809:Quinones; D014815:Vitamins; D010504:Periodic Acid; C081583:esculentin steroid; D011284:Pregnenolone; D004156:Catechol Oxidase; D006735:Horseradish Peroxidase; D009195:Peroxidase; D014442:Monophenol Monooxygenase; D001205:Ascorbic Acid