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,
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