Kinetics study of the oxidation of 4‐tert‐butylphenol by tyrosinase

Abstract

The reaction between 4-tert-butylphenol (BuPhOH) and mushroom tyrosinase was investigated by following 4-tert-butyl-ortho-benzoquinone, whose high stability permits the reaction to be used as a model for the study of the monophenolase acfivity of tyrosinase. The system evolves to a pseudo-steady state through an induction period (z), the pseudo-steady-state rate (VJ decreasing when the (BuPhOH) concentration increases. Increases in enzyme concentration result in a parabolic pattern with V,,, while t is shortened. The addition of increasing catalytic amounts of 4-tert-butylcatechol at the start of the reaction reduces z until it is totally abolished, an initial burst being observed at high 4-t-butylatechol concentrations. Initial bursts are also obtained at pH 4.5 or lower, indicating a lower affinity of the met-tyrosinase or oxidized form for the monophenol at low pH. These experimental results can be explained by the reaction mechanism of tyrosinase.Tyrosinase is a copper enzyme found in microorganisms, plants and animals. The active site of tyrosinase consists of two copper atoms and three states: met, deoxy and oxy [l, 21. Structural models for the active site of these three enzyme forms have been proposed [3-51. Mushroom tyrosinase has a molecular mass of 120 kDa. It is composed of two H subunits (43 kDa) and two L subunits (13 kDa) and contains two binuclear active sites/molecule [6]. Most of the enzyme in a fresh preparation is in the met-tyrosinase form, in which the active site is bicupric and unable to bind oxygen. Only a small proportion of the enzyme is present as the oxy-form, which is the form which acts on monophenols [7].Tyrosinase catalyses both the hydroxylation of monophenols to o-diphenols and the oxidation of these to o-quinones, which are unstable in aqueous solutions and can undergo non-enzymic reactions, such as cyclization to aminechrome, following a Michael intramolecular 1,4 addition (i.e. o-dopaquinone), or the addition of water to the benzene ring (i.e. o-benzoquinone). Both processes present an intermediate species, which is oxidized by one molecule of o-quinone to regenerate o-diphenol which, in turn, is accumulated in the medium. When cyclization of the o-quinones is possible, the system can reach the steady state, and a constant diphenol concentration, which is proportional to monophenol concentration, is maintained [8]. However, the systems in which o-quinones incorporate water into the aromatic ring cannot reach the steady state since this addition reaction is slow and the diphenol concentration necessary for the support of the steady state is not reached during the enzymic reaction time. The addition of nucleophiles, such as serine or proline, to the reaction medium results in an activation on the system due to the accumulation of diphenol caused by these reagents [9,

Substrate-dependent kinetics in tyrosinase-based biosensing: amperometry vs. spectrophotometry

“…This is a different behavior from that of the other monophenols studied and is not consistent with the MichaelisMenten kinetics model. This has been reported before by Ros et al [30] and Fennol et al [35], who related this behavior to both the suppressed autoactivation of tyrosinase from met to oxy form caused by binding of monophenols to the met form of the enzyme together with the high stability of quinone products.…”

“…The reactivity of tyrosinase decreases upon a transition of the substituent in the para position of phenols from electron donating to electron withdrawing [26], and tyrosinase does not react with bulky phenols [27]. There are few studies on kinetics of tyrosinase for different phenolic substrates with alkyl groups side chain such as methyl- [28], ethyl- [29], and tert-butylphenol [30]. To complete the series with the type of R-group substitutions, we have measured the turnover rate of tyrosinase for methyl-, ethyl-, isopropyl-, and tert-butylphenol using both UV-Vis spectrophotometry and amperometry techniques and show that the two methods are in good agreement.…”

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

“…These traces are very hard to detect by HPLC or other analytical methods but can drastically shorten the lag period. This has been reported for some (synthetic) commercial monophenols such as 4-tertbutylphenol (Ros et al, 1994b). The latter monophenol needed further purification (with a column of Al 2 O 3 activated by ammoniun acetate) to remove o-diphenol traces.…”

Proteolytic Activation of Latent Paraguaya Peach PPO. Characterization of Monophenolase Activity

“…An increase in enzyme concentration produced a linear increase in the monophenolase activity of latent peach PPO (Figure 4), as well as a shortening of the lag period (Figure 4). This behavior has been widely described in other PPOs (23)(24)(25).…”