The Relationship between the IC(50) Values and the Apparent Inhibition Constant in the Study of Inhibitors of Tyrosinase Diphenolase Activity Helps Confirm the Mechanism of Inhibition

Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fruits and vegetables. Control of its activity can be carried out using inhibitors, which is interesting in terms of quantitatively understanding the action of these regulators. In the study of the inhibition of the diphenolase activity of tyrosinase, it is intriguing to know the strength and type of inhibition. The strength is indicated by the value of the inhibition constant(s), and the type can be, in a first approximation: competitive, non-competitive, uncompetitive and mixed. In this work, it is proposed to calculate the degree of inhibition ([Formula: see text]), varying the concentration of inhibitor to a fixed concentration of substrate, L-dopa (D). The non-linear regression adjustment of [Formula: see text] with respect to the initial inhibitor concentration [Formula: see text] allows for the calculation of the inhibitor concentration necessary to inhibit the activity by 50%, at a given substrate concentration (IC(50)), thus avoiding making interpolations between different values of [Formula: see text]. The analytical expression of the IC(50), for the different types of inhibition, are related to the apparent inhibition constant ([Formula: see text]. Therefore, this parameter can be used: (a) To classify a series of inhibitors of an enzyme by their power. Determining these values at a fixed substrate concentration, the lower IC(50), the more potent the inhibitor. (b) Checking an inhibitor for which the type and the inhibition constant have been determined (using the usual methods), must confirm the IC(50) value according to the corresponding analytical expression. (c) The type and strength of an inhibitor can be analysed from the study of the variation in [Formula: see text] and IC(50) with substrate concentration. The dependence of IC(50) on the substrate concentration allows us to distinguish between non-competitive inhibition ([Formula: see text] does not depend on [Formula: see text]) and the rest. In the case of competitive inhibition, this dependence of [Formula: see text] on [Formula: see text] leads to an ambiguity between competitive inhibition and type 1 mixed inhibition. This is solved by adjusting the data to the possible equations; in the case of a competitive inhibitor, the calculation of [Formula: see text] is carried out from the IC(50) expression. The same occurs with uncompetitive inhibition and type 2 mixed inhibition. The representation of [Formula: see text] vs. n, with [Formula: see text] , allows us to distinguish between them. A hyperbolic [Formula: see text] vs. n representation that passes through the origin of coordinates is a characteristic of uncompetitive inhibition; the calculation of [Formula: see text] is immediate from the IC(50) value. In the case of mixed inhibitors, the values of the apparent inhibition constant of meta-tyrosinase (Em) and oxy-tyrosinase (Eox), [Formula: see text] and the apparent inhibition constant of metatyrosinase/Dopa complexes (EmD) and oxytyrosinase/Dopa (EoxD), [Formula: see text] are obtained from the dependence of [Formula: see text] vs. n, and the results obtained must comply with the IC(50) value.