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

Abstract

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, Lepinephrine, L-norepinephrine, L-isoprenaline L-α-methyldopa and L-dopa methylester. A chronometric method has been used for this characterization, with the help of a small amount of ascorbic acid. 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 copper centre of the enzyme. 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 Lnorepinephrine have a similar affinity to isoprenaline, but faster catalysis, probably due to the greater nucleophilic power of their phenolic hydroxyl.

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

“…A 2D view of these results is shown in Figures S6 and S7. Our docking results with laccase are in good agreement with previously reported works, where participation of His458 and Asp206 in hydrogen bonds formation with substrates of laccase (Madzak et al., 2006; Manzano‐Nicolas, Taboada‐Rodriguez, et al., 2020; Piontek et al., 2002; Polyakov et al., 2019), and Π‐interactions of substrates with Phe265 have been suggested (Manzano‐Nicolas, Taboada‐Rodriguez, et al., 2020).…”

“…The products of the reaction of these three enzymes (laccase, peroxidase, and tyrosinase) when acting on OL and HT are semiquinones that evolve towards o‐ quinones, in the case of the first two (Manzano‐Nicolas, Marin‐Iniesta, et al., 2020; Manzano‐Nicolas, Taboada‐Rodriguez, et al., 2020; Rodríguez‐López, Gilabert, et al., 2000) and directly o‐ quinones in the case of tyrosinase (Espín et al., 2001; Muñoz et al., 2006; Rodríguez‐López, Fenoll, et al., 2000). It is known that o‐ quinones are unstable, especially at high pH values.…”

“…It is known that o‐ quinones are unstable, especially at high pH values. Thus, o‐ quinones absorb in the visible region of the spectrum and make it possible to measure the activity of these enzymes; however, their instability means that only laccase can be measured at pH = 4.0, at its optimal pH (Manzano‐Nicolas, Marin‐Iniesta, et al., 2020; Manzano‐Nicolas, Taboada‐Rodriguez, et al., 2020). The other two enzymes peroxidase (Rodríguez‐López, Gilabert, et al., 2000) and tyrosinase (Rodríguez‐López, Fenoll, et al., 2000), which have an optimal pH of 5.5 and 6.8, respectively, cannot be correctly measured, as shown below, in which case a spectrophotometric chronometric method is proposed, as it is described below.…”

“…The enzymatic activity of peroxidase and tyrosinase on OL and HT was followed spectrophotometrically in the visible zone, measuring the formation of the corresponding products after the consumption of a determined amount of AH 2 (micromolar) by the reaction with the different quinones and semiquinones generated by the enzymes. Since in all cases the product absorbs in the visible area, the classic chronometric method is used (Muñoz et al., 2006), since the AH 2 spectrum does not influence the measurement (Manzano‐Nicolas, Marin‐Iniesta, et al., 2020; Manzano‐Nicolas, Taboada‐Rodriguez, et al., 2020; Rodríguez‐López, Fenoll, et al., 2000; Rodríguez‐López, Gilabert, et al., 2000). Except where otherwise indicated, the experimental conditions were as follows: pH 5.5 and 50‐mM acetate buffer for peroxidase, while pH 6.8 and 50‐mM phosphate buffer for tyrosinase; temperature was maintained at 25°C.…”

“…In previous works, we have kinetically characterized the action of laccase, peroxidase, and tyrosinase on a series of substrates (Manzano‐Nicolas, Marin‐Iniesta, et al., 2020; Manzano‐Nicolas, Taboada‐Rodriguez, et al., 2020; Rodríguez‐López, Fenoll, et al., 2000; Rodríguez‐López, Gilabert, et al., 2000). Due to the instability of the products, the activity measurement method used consists of avoiding following these molecules, and this is achieved by using a small amount of ascorbic acid (AH 2 ) (µM), achieving the reversion of free radicals or o‐ quinones to their original substrate and ascorbic acid is oxidized to dehydroascorbic (Manzano‐Nicolas, Marin‐Iniesta, et al., 2020; Manzano‐Nicolas, Taboada‐Rodriguez, et al., 2020; Rodríguez‐López, Fenoll, et al., 2000; Rodríguez‐López, Gilabert, et al., 2000).…”