The Relationship between the IC50 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 (iD), varying the concentration of inhibitor to a fixed concentration of substrate, L-dopa (D). The non-linear regression adjustment of iD with respect to the initial inhibitor concentration I0 allows for the calculation of the inhibitor concentration necessary to inhibit the activity by 50%, at a given substrate concentration (IC50), thus avoiding making interpolations between different values of iD. The analytical expression of the IC50, for the different types of inhibition, are related to the apparent inhibition constant (KIapp). 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 IC50, 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 IC50 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 iD and IC50 with substrate concentration. The dependence of IC50 on the substrate concentration allows us to distinguish between non-competitive inhibition (iD does not depend on D0) and the rest. In the case of competitive inhibition, this dependence of iD on D0 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 KI1app is carried out from the IC50 expression. The same occurs with uncompetitive inhibition and type 2 mixed inhibition. The representation of iD vs. n, with n=D0/KmD, allows us to distinguish between them. A hyperbolic iD vs. n representation that passes through the origin of coordinates is a characteristic of uncompetitive inhibition; the calculation of KI2app is immediate from the IC50 value. In the case of mixed inhibitors, the values of the apparent inhibition constant of meta-tyrosinase (Em) and oxy-tyrosinase (Eox), KI1app and the apparent inhibition constant of metatyrosinase/Dopa complexes (EmD) and oxytyrosinase/Dopa (EoxD), KI2app are obtained from the dependence of iD vs. n, and the results obtained must comply with the IC50 value

Enzymatic oxidation of oleuropein and 3-hydroxytyrosol by laccase, peroxidase and tyrosinase.

The oxidation of oleuropein and 3‐hydroxytyrosol by oxidases laccase, tyrosinase, and peroxidase has been studied. The use of a spectrophotometric method and another spectrophotometric chronometric method has made it possible to determine the kinetic parameters Vmax and KM for each enzyme. The highest binding affinity was shown by laccase. The antioxidant capacities of these two molecules have been characterized, finding a very similar primary antioxidant capacity between them. Docking studies revealed the optimal binding position, which was the same for the two molecules and was a catalytically active position. Practical applications: One of the biggest environmental problems in the food industry comes from olive oil mill wastewater with a quantity of approximately 30 million tons per year worldwide. In addition, olive pomace, the solid residue obtained from the olive oil production, is rich in hydroxytyrosol and oleuropein and the action of enzymatic oxidases can give rise to products in their reactions that can lead to polymerization. This polymerization can have beneficial effects because it can increase the antioxidant capacity with potential application on new functional foods or as feed ingredients. Tyrosinase, peroxidase, and laccase are the enzymes degrading these important polyphenols. The application of a spectrophotometric method for laccase and a chronometric method, for tyrosinase and peroxidase, allowed us to obtain the kinetic information of their reactions on hydroxytyrosol and oleuropein. The kinetic information obtained could advance in the understanding of the mechanism of these important industrial enzymes

Kinetic characterization of the oxidation of catecolamines and related compounds by laccase

The pathways of melanization and sclerotization of the cuticle in insects are carried out by the action of laccases on dopamine and related compounds. In this work, the laccase action of Trametes versicolor (TvL) on catecholamines and related compounds has been kinetically characterized. Among them, dopamine, L-dopa, L-epinephrine, L-norepinephrine, DL-isoprenaline, L-isoprenaline, DL-α-methyldopa, L-α-methyldopa and L-dopa methylester. A chronometric method has been used, which is based on measuring the lag period necessary to consume a small amount of ascorbic acid, added to the reaction medium. The use of TvL has allowed docking studies of these molecules to be carried out at the active site of this enzyme. The hydrogen bridge interaction between the hydroxyl oxygen at C-4 with His-458, and with the acid group of Asp-206, would make it possible to transfer the electron to the T1 Cu-(II) copper centre of the enzyme. Furthermore, Phe-265 would facilitate the adaptation of the substrate to the enzyme through Π-Π interactions. To kinetically characterize these compounds, we need to take into consideration that, excluding L-dopa, L-α-methyldopa and DL-α-methyldopa, all compounds are in hydrochloride form. Because of this, first we need to kinetically characterize the inhibition by chloride and, after that, calculate the kinetic parameters K M and V max S. From the kinetic data obtained, it appears that the best substrate is dopamine. The presence of an isopropyl group bound to nitrogen (isoprenaline) makes it especially difficult to catalyse. The formation of the ester (L-dopa methyl ester) practically does not affect catalysis. The addition of a methyl group (α-methyl dopa) increases the rate but decreases the affinity for catalysis. L-Epinephrine and L-norepinephrine have an affinity similar to isoprenaline, but faster catalysis, probably due to the greater nucleophilic power of their phenolic hydroxyl

Spectrophotometric Characterization of the Action of Tyrosinase on p-Coumaric and Caffeic Acids: Characteristics of o-Caffeoquinone

New methods are proposed to determine the activity of tyrosinase on caffeic and p-coumaric acids. Because o-quinone from caffeic acid is unstable in its presence, it has been characterized through spectrophotometric measurements of the disappearance of coupled reducing agents, such as nicotinamide adenine dinucleotide reduced form. It has also been characterized by a chronometric method, measuring the time that a known concentration of ascorbic acid takes to be consumed. The activity on p-coumaric acid has been followed by measuring the formation of o-quinone of caffeic acid at the isosbestic point originated between caffeic acid and o-caffeoquinone and measuring the formation of o-quinone at 410 nm, which is stable in the presence of p-coumaric acid (both of them in the presence of catalytic amounts of caffeic acid, maintaining the ratio between p-coumaric acid and caffeic acid constant; R = 0.025). The kcat value of tyrosinase obtained for caffeic acid was higher than that obtained for p-coumaric acid, while the affinity was higher for p-coumaric acid. These values agree with those obtained in docking studies involving these substrates and oxytyrosinase

Structural and kinetic considerations on the catalysis of deoxyarbutin by tyrosinase.

Deoxyarbutin, a potent inhibitor of tyrosinase, could act as substrate of the enzyme. Oxytyrosinase is able to hydroxylate deoxyarbutin and finishes the catalytic cycle by oxidizing the formed o-diphenol to quinone, while the enzyme becomes deoxytyrosinase, which evolves to oxytyrosinase in the presence of oxygen. This compound is the only one described that does not release o-diphenol after the hydroxylation step. Oxytyrosinase hydroxylates the deoxyarbutin in ortho position of the phenolic hydroxyl group by means of an aromatic electrophilic substitution. As the oxygen orbitals and the copper atoms are not coplanar, but in axial/equatorial position, the concerted oxidation/reduction cannot occur and the release of a copper atom to bind again in coplanar position, enabling the oxidation/reduction or release of the o-diphenol from the active site to the medium. In the case of deoxyarbutin, the o-diphenol formed is repulsed by the water due to its hydrophobicity, and so can bind correctly and be oxidized to a quinone before being released. Deoxyarbutin has been characterized with: [Formula: see text] = 1.95 ± 0.06 s-1 and [Formula: see text] = 33 ± 4 μM. Computational simulations of the interaction of β-arbutin, deoxyarbutin and their o-diphenol products with tyrosinase show how these ligands bind at the copper centre of tyrosinase. The presence of an energy barrier in the release of the o-diphenol product of deoxyarbutin, which is not present in the case of β-arbutin, together with the differences in polarity and, consequently differences in their interaction with water help understand the differences in the kinetic behaviour of both compounds. Therefore, it is proposed that the release of the o-diphenol product of deoxyarbutin from the active site might be slower than in the case of β-arbutin, contributing to its oxidation to a quinone before being released from the protein into the water phase

Action of tyrosinase on alpha and beta-arbutin: A kinetic study

<div><p>The known derivatives from hydroquinone, α and β-arbutin, are used as depigmenting agents. In this work, we demonstrate that the <i>oxy</i> form of tyrosinase (oxytyrosinase) hydroxylates α and β-arbutin in <i>ortho</i> position of the phenolic hydroxyl group, giving rise to a complex formed by <i>met</i>-tyrosinase with the hydroxylated α or β-arbutin. This complex could evolve in two ways: by oxidizing the originated <i>o</i>-diphenol to <i>o</i>-quinone and <i>deoxy</i>-tyrosinase, or by delivering the <i>o</i>-diphenol and <i>met</i>-tyrosinase to the medium, which would produce the self-activation of the system. Note that the quinones generated in both cases are unstable, so the catalysis cannot be studied quantitatively. However, if 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate is used, the <i>o</i>-quinone is attacked, so that it becomes an adduct, which can be oxidized by another molecule of <i>o</i>-quinone, generating <i>o</i>-diphenol in the medium. In this way, the system reaches the steady state and originates a chromophore, which, in turn, has a high absorptivity in the visible spectrum. This reaction allowed us to characterize α and β-arbutin kinetically as substrates of tyrosinase for the first time, obtaining a Michaelis constant values of 6.5 ± 0.58 mM and 3 ± 0.19 mM, respectively. The data agree with those from docking studies that showed that the enzyme has a higher affinity for β-arbutin. Moreover, the catalytic constants obtained by the kinetic studies (catalytic constant = 4.43 ± 0.33 s^-1 and 3.7 ± 0.29 s^-1 for α and β-arbutin respectively) agree with our forecast based on 13 C NMR considerations. This kinetic characterization of α and β-arbutin as substrates of tyrosinase should be taken into account to explain possible adverse effects of these compounds.</p></div

Considerations about the kinetic mechanism of tyrosinase in its action on monophenols: A review

The mechanism of action of tyrosinase on monophenols is complex, several processes overlap in time, such as the hydroxylation of monophenols to o-diphenols, the oxidation of these to o-quinones and the evolution of the latter towards melanin. The enzyme's mechanism of action is unique but depending on the chemical nature of the substrate it may show different exceptions. In this review we want to dissect the kinetic mechanism for the action of the enzyme on: a) L-tyrosine, the physiological substrate for mammalian tyrosinase, and related compounds, whose o-quinones in their chemical evolution accumulate o-diphenol in the medium (Type A). b) Substrates that cannot accumulate o-diphenol in the medium because it is easily oxidized and they need the presence of hydrogen peroxide for the enzyme to show activity, such as hydroquinone and related compounds (Type B). c) Substrates that release o-diphenol into the medium and the enzyme oxidizes it generating a stable o-quinone and therefore does not generate more o-diphenol in the medium, as is the case of 4‑tert-butylphenol and related compounds (Type C). d) Substrates that do not release or generate o-diphenol in the medium, as is the case with deoxyarbutin, which produces a stable o-quinone (Type D). The different mechanisms that explain the enzymatic activity are proposed, a kinetic analysis is established for each mechanism and by means of numerical integration results are obtained that are discussed and compared with experimental data. To help and support the results and discussion, molecular docking for substrates (L-tyrosine, hydroquinone, 4‑tert-butylphenol, and deoxyarbutin) to both the oxy and met forms of tyrosinase was carried out