Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase

Abstract

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...

Melanogenesis Inhibition Due to NADH

“…Thus, in the case of NADH, the attack can be carried out by an oxygen atom of the nicotinamide ring, while in the case of 6BH 4 it is a nitrogen atom (a stronger nucleophile) that carries out the attack. As has been found for the case of monophenols and diphenols, 28) the values of k cat reflect the power of the nucleophilic attack. The Fig.…”

The phenylthiourea is a competitive inhibitor of the enzymatic oxidation of DOPA by phenoloxidase

“…To elucidate the type of inhibition exactly, we used two other linearization methods, namely, the Dicson and one-curve method plots ( Figure 5A and 5B). The set of curves on these plots completely satisfied the competitive type of inhibition 34 . The inhibition constant for PTU was estimated from each of the used linearization plots.…”

“…To determine the type of inhibition, the Lineweaver-Burk, the Hanes-Woolf, and the Eadie-Hofstee linearizations were used. In the case of competitive type of inhibition, the intersection points of the curves must be on the axes on the Lineweaver-Burk and the Eadie-Hofstee plots 34 . All used linearizations presented on Figure 4B, 4C, and 4D indicate that the type of inhibition of PO by PTU is competitive or complex with dominance of competitive type, as the intersection points slightly deviate from ordinate axes on the plots ( Figure 4B and 4D) that could indicate the complex type of inhibition.…”

Dopamine- and tyramine-based derivatives of triazenes: Activation by tyrosinase and implications for prodrug design

“…These K m values compare favourably with those reported for tyramine (0.51 mM) and dopamine (2.2 mM). The V max values correspond to turnover numbers of 0.6 s-1 and 6 s-1 for 5c and 5f, respectively, which are rather smaller than the 25.9 s -1 and 439 s -1 observed for tyramine and dopamine substrates [18], though the relative sizes between the tyramine and dopamine derivatives are similar. This observation is consistent with the report that tyrosinase accepts mono-, di-and trihydroxyphenols as substrates but displays the highest turnover rates for dihydroxyphenols [19,20].…”