Kinetic study of the oxidation of 4‐hydroxyanisole catalyzed by tyrosinase

Abstract

Despite the importance of the substrate 4‐hydroxyanisole in melanoma therapy, the kinetics of its oxidation catalyzed by tyrosinase has never been properly characterized. This approach is reported here for the first time. The applicability to 4‐hydroxyanisole of the reaction mechanism of tyrosinase previously proposed for other monophenols has been corroborated. The Michaelis constant for the oxidation of 4‐hydroxyanisole catalyzed by mushroom tyrosinase was (62±1.5jt]μM at pH 7 and increased when the pH decreased, reaching a value of (195±5jt]μM at pH 5.5. However the maximum steady‐state rate, whose value was (0.54±0.01jt]μM/min, did not change with the pH. The apparent catalytic constant was (184±5jt]s‐1, around twenty three times higher than that previously described for L‐tyrosine (8 s‐l).

4-Hydroxyanisole: The Most Suitable Monophenolic Substrate for Determining Spectrophotometrically the Monophenolase Activity of Polyphenol Oxidase from Fruits and Vegetables

“…Very recently, the kinetic study of the oxidation of 4HA catalyzed by mushroom PPO has been reported (20). The high catalytic constant of mushroom PPO toward this monophenol was reported in this paper.…”

“…Therefore, this assay method measures half of the steady-state rate. However, when the oxygen consumption is measured then the whole steady-state rate is determined and it is 1.5 times higher than that when the formation of NQH is measured (3O 2 : 2NQH) (15,20,51). NMR assays.…”

“…2 Abbreviations used: AB, sodium acetate buffer; C4, carbon atom in the 4-position of the aromatic ring of monophenols; D 2 O, deuterium oxide (heavy water); DMF, N,NЈ-dimethylformamide; ␦ 4 , chemical displacement value for C4; DOBA, 3,4-dihydroxybenzaldehyde; DOMA, 3,4-dihydroxy mandelic acid; 4HA, 4-hydroxyanisole; EDTA, ethylendiaminetetraacetic acid; K m M , apparent Michaelis constant of PPO toward monophenols; LOD, limit of detection; LOQ, limit of quantitation; MBTH, 3-methyl-2-benzothiazolinone hydrazone; [MBTH] sat , saturating MBTH concentration regarding the reaction of MBTH with the enzyme generated o-quinones; NADH, reduced nicotinamine adenine dinucleotide; NMR, nuclear magnetic resonance; PB, sodium phosphate buffer; PHPAA, p-hydroxyphenyl acetic acid; PHPPA, p-hydroxyphenyl propionic acid; PPO, polyphenol oxidase; [PPO] activity of PPO has been widely characterized (9 -14). Monophenolase activity of PPO from several sources has been recently characterized (15)(16)(17)(18)(19)(20). The scarce information about the monophenolase activity of PPO up to date is mainly due to the poor sensitivity of the assay methods used for measuring this activity.…”

Kinetic Study of the Oxidation of γ-l-Glutaminyl-4-hydroxybenzene Catalyzed by Mushroom (Agaricus bisporus) Tyrosinase

“…This stoichiometry predicts that V max O 2 /V max ) 1.5 for the monophenolase activity and V max O 2 /V max ) 1 for the diphenolase activity. These ratios were verified experimentally for the oxidation of GHB, L-Tyr, GDHB, and L-DOPA (Table 2) (Rodríguez-López et al, 1992a;Ros et al, 1994a;Espín et al, 1997b).…”

“…The determination of these Scheme 1. Sequence of Reactions for the Monophenolase and Diphenolase Activities of Tyrosinase on GHB and GDHB in the Presence of MBTH a a AH + , protonated p-quinoid adduct (tautomery between AH1 and AH2 in an equilibrium shifted toward AH2); A, deprotonated p-quinoid adduct; B, oxidized MBTH-o-quinone adduct after AH + evolution (Espín et al, 1997b). Reactions for monophenolase and diphenolase activities of tyrosinase are detailed in Scheme 2.…”