Análisis y discriminación entre sustratos e inhibidores de tirosinasa

Zusammenfassung

ABSTRACT OBJECTIVES: The main objectives of this report are (1) to investigate further the mechanisms of action and inhibition of tyrosinase enzyme, considering the monophenolase and diphenolase activities, (2) to establish a methodology in order to distinguish phenolic inhibiting molecules from alternative substrates of the enzyme, and finally, (3) to identify and kinetically characterize as substrates some molecules described as inhibitors in the scientific literature and used as such in the cosmetic and food industries. METHODOLOGY: Spectrophotometric assays, to measure the monophenolase and diphenolase activities of tyrosinase and study its inhibition; oxymetric assays, to measure tyrosinase activity by following the oxygen consumption (oxygen is a cosubstrate of the enzyme); RMN assays, to determine carbon atom chemical shift values of the molecule of interest; simulation assays, to test the validity of the experimental results; computational docking, to understand the binding modes of tyrosinase with different ligands; protein chromatography in FPLC equipment, to purify commercial tyrosinase; lineal and non-lineal regression. CONCLUSIONS: An experimental design has been established which permits to distinguish inhibiting phenolic molecules from alternative substrates of tyrosinase, considering the monophenolase and diphenolase activities of the enzyme. The measurements of the monophenolase activity should be made by adding the quantity of o-diphenol, needed to abolish the lag period at the beginning of the activity recordings, to the reaction medium. The compounds p-hydroxybenzyl alcohol, tyrosol, phloretin and phloridzin have been identified as alternative substrates of tyrosinase, by following this methodology. The compounds 4-hexylresorcinol, described as inhibitor and used in the cosmetic and food industries as a depigmenting and anti-browning agent, and oxyresveratrol, proposed for the same purposes due to the described inhibitory effect on this enzyme, have been identified as tyrosinase substrates. These molecules are hydroxylated by the enzyme and subsequently oxidized to an o-quinone, which rapidly isomerizes to a more stable p-quinone. Moreover, these substrates have been kinetically characterized, obtaining the kinetic parameters KM (Michaelis constant) and kcat (catalytic constant). Ellagic acid, a compound described as inhibitor and used as a depigmenting agent in the cosmetic industry, has been identified as a tyrosinase substrate by spectrophotometric methods. Its kinetic characterization as a substrate has been carried out, obtaining the KM and kcat values. It has been determined that ellagic acid inhibits the melanin biosynthesis pathway due to its antioxidative effect, by reacting with superoxide anions, o-quinones and semiquinones produced during the process. The action mechanism of inhibitors which are analogous to phenolic substrates was studied. We concluded that those inhibitors which produce the same degree of inhibition on the monophenolase and diphenolase activities, bind to the same enzymatic species in the two activities, being the type and the apparent inhibition constants the same too. A methodology was established in order to study tyrosinase inhibitors. It was observed that values show a slight dependence on the substrate nature, through the constants k2 and k6. Finally, the pH effect on the catalysis of tyrosinase was studied. It was proposed that a group with a pKa value of 4.63 ± 0.04 might be responsible for this effect. This group could correspond to the glutamic acid E322, which would control the substrate access to the active site depending on its protonation state. Protons act as competitive inhibitors, binding to the met-tyrosinase and oxy-tyrosinase forms. The pKa value of the phenolic hydroxyl groups and the charge of the R group are important too.