Catálisis y regulación de tirosinasa ante fenonas, arbutinas y ácidos hidroxicinámicos

Abstract

OBJECTIVES: For this thesis, they had been established as objectives to deepen in the mechanism of the action of tyrosinase and to develop a reliable method to distinguish the true alternative substrates, activators and inhibitors of the enzyme. METHODOLOGY: The following techniques were made: isolation, concentration and purification of tyrosinase, using FPLC or microgel electrophoresis, to obtain a purified isoenzyme; characterization of the activity of tyrosinase, and regulation of the enzyme by means of inhibition or activation, using cromogenic and fluorogenic substrates as well as absorbance essays with Ultraviolet/visible spectrophotometry; optimization of chromatographic methods with detection by using mass spectrometry (GC-MS and HPLC-MS), to identify and quantify biomolecules; NMR essays, to obtain chemical shift values and computational simulation of molecular coupling or docking, to understand the types of binding of tyrosinase with different ligands; and finally, quantification of the reliability of the experimental results obtained by means of parameters of descriptive statistics and tests of statistical significance. CONCLUSIONS: Thanks to the development of this thesis, the understanding of the kinetic and structural mechanism of tyrosinase has advanced, and kinetic criteria to distinguish between alternative substrates, activators and inhibitors of the enzyme has been established. Moreover, these findings have been supported and confirmed by docking studies. In this way, we confirmed that 3-4/-aminoacetophenones are really inhibitors, despite showing activation or inhibition depending on the quantity of L-dopa used in the essays, which is due to the formation of an adduct which absorbs more than dopachrome. 2,2',4,4'-tetrahydroxybenzophenone (Uvinul D50) was confirmed as an inhibitor of the enzyme, establishing the new constants of apparent inhibition and IC50 as 2.02 ± 0.09 mM y 3.82 ± 0.39 mM, respectively. We also studied the action of tyrosinase on caffeic and p-coumaric acids, by using new methods to avoid interferences caused by the instability of the generated compounds, so the kinetic characterization was correct. Subsequently, the depigmenting agents α y β-arbutin were studied as well as their derived desoxiarbutin, checking that are alternative substrates of the enzyme, so it has to be taken into account for its use in cosmetics. Moreover, desoxiarbutin was proved as the single discovered substrate that not release o-diphenol to the medium after the hydroxylation. The following research was about the action of tyrosinase on caffeic acid and its n-nonyl ester, n-nonyl caffeate, which were proved as suicide substrates that inactivate the enzyme. N-nonyl caffeate showed more potency to inactivate the enzyme than caffeic acid. Finally, the action of tyrosinase on cinnamic acid and some derivatives was studied, checking that cinnamic, 2-hydroxycinnamic, 2,3 and 4-methocycinnamic acids are inhibitors of tyrosinase, whereas 3-hydroxycinnamic, 4-hydroxycinnamic and 3,4-dihydroxycinnamic acids are substrates of the enzyme. Moreover, the kinetic constants of these compounds were characterized.