This paper reports a quantitative study of the effect of ring substituents in the 1-position of the aromatic ring on the rate of monophenol hydroxylation and o-diphenol oxidation catalyzed by tyrosinase. A possible correlation between the electron density of the carbon atom supporting the oxygen from the monophenolic hydroxyl group and the V M max values for each monophenol was found. In the case of o-diphenols the same effect was observed but the size of the side-chain became very important. NMR studies on the monophenols justified the sequence of the V M max values obtained. As regards the o-diphenols, on the other hand, only a fair correlation between NMR and V D max values was observed due to the effect of the molecular size of the ring substituent. From these data, it can be concluded that the redox step k 33 is not the rate-determining step of the reaction mechanism. Thus, the monophenols are converted into diphenols, but the order of specificities towards monophenols is different to that of o-diphenols. The rate-limiting step of the monophenolase activity could be the nucleophilic attack k 5 1 of the oxygen atom of the hydroxyl group on the copper atoms of the active site of the enzyme. This step could also be similar to or have a lower rate of attack than the electrophilic attack (k 5 2 ) of the oxygen atom of the active site of oxytyrosinase on the C-3 of the monophenolic ring. However, the rate-limiting step in the diphenolase activity of tyrosinase could be related to both the nucleophilic power of the oxygen atom belonging to the hydroxyl group at the carbon atom in the 3-position k 32 and to the size of the substituent side-chain. On the basis of the results obtained, kinetic and structural models describing the monophenolase and diphenolase reaction mechanisms for tyrosinase are proposed.Keywords: diphenolase; enzyme kinetics; 3-methyl-2-benzothiazolinone hydrazone (MBTH); monophenolase; mushroom.The enzyme tyrosinase or polyphenol oxidase, PPO (monophenol, o-diphenol:oxygen oxidoreductase, EC 1.14.18.1) is of central importance in vertebrate melanin pigmentation. Enzymatic browning in vegetables and fruits is caused by the activity of tyrosinase in plant tissues. Tyrosinase plays an important role in fruit and vegetable processing and during the storage of processed foods. This enzyme catalyzes the hydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase activity). These o-quinones evolve nonenzymatically to yield several unstable intermediates, which polymerize to render melanins [1,2]. The active site of tyrosinase consists of two copper atoms and three states:`met',`deoxy', and`oxy' [3±10]. Structural models for the active site of these three forms of tyrosinase have been proposed [11±15]. Recent advances on structural homology [16] and on the active-site conformation [17,18] have been reported. Nevertheless, the complete spatial structure of the active site is still unknown.The conversion of p-monophenols into o-diphenols is an interesti...

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.

No abstract

Browning reactions in fruits and vegetables are a serious problem for the food industry. In mushrooms, the principal enzyme responsible for the browning reaction is polyphenoloxidase (PPO). Microwaves have recently been introduced as an alternative for the industrial blanching of mushrooms. However, the direct application of microwave energy to entire mushrooms is limited by the important temperature gradients generated within the samples during heating, which can produce internal water vaporization and associated damage to the mushrooms texture. A microwave applicator has been developed, whereby irradiation conditions can be regulated and the heating process monitored. Whole edible mushrooms (Agaricus bisporus) were blanched by conventional, microwave, and combined heating methods to optimize the rate of PPO inactivation. A combined microwave and hot-water bath treatment has achieved complete PPO inactivation in a short time. Both the loss of antioxidant content and the increase of browning were minor in the samples treated with this combined method when compared to the control. This reduction in processing time also decreased mushroom weight loss and shrinkage.