Kinetic characterization of substrate-analogous inhibitors of tyrosinase.

The development of effective tyrosinase inhibitors has become increasingly important in the cosmetic, medicinal, and agricultural industries for application as antibrowning and depigmenting agents. The kinetic mechanisms of action of tyrosinase on monophenols and o-diphenols are complex, particularly in the case of monophenols because of the lag period that occurs at the beginning of the reaction. When enzyme inhibitors are studied, the problem becomes more complicated because the lag period increases, which has led to erroneous identification of the type of inhibition that many compounds exert on the monophenolase activity and the inaccurate determination of their inhibition constants. When the degrees of inhibition of an inhibitor which is analogous to tyrosinase substrates are the same for both monophenolase and diphenolase activities, this means that the inhibitor binds to the same enzymatic species and so the inhibition constants should be similar for both activities. In this study, we demonstrate this typical behavior of substrate-analogous inhibitors and propose a methodology for determining the type of inhibition and the inhibition constants for the monophenolase and diphenolase activities of the enzyme. Benzoic acid and cinnamic acid were used as inhibitors and the monophenol/o-diphenol pairs l-tyrosine/l-dopa and α-methyl-L-tyrosine/α-methyl-L-dopa as substrates.