Laccase inhibition

The discovery of ABTS as a laccase substrate mediating or enhancing the enzyme action was essential to increase the range of molecules that can be converted by laccases (Fig. 4.5). Such a mediator requires several conditions (1) it must be a good laccase substrate (2) its oxidized and reduced forms must be stable (3) it must not inhibit the enzymatic reaction and (4) its redox conversion must be cyclic. 

Laccase, 36 318, 329, 40 122 see also Blue copper oxidases amino-acid sequences, 40 141 anaerobic reduction, 40 158-160 biological function, 40 124 electrochemistry, 36 360 fungal, 40 145-152 evolution, 40 153-154 inhibition, 40 162 kinetic properties, 40 157-162 molecular and spectroscopic properties, 40 125-126... 

Catecholases and laccases may be differentiated by the use of substrate specificity tests and selective specific inhibitors (Walker and McCallion, 1980 Ferrar and Walker, 1996 Table C4.1.1). Salicylhydroxamic acid (SHAM), PVP, and/or cinnamic acids (cinnamic, p-coumaric, or ferulic) are probably the best choice for catecholase inhibitors, whereas cetyltrimethylammonium bromide (CETAB) has been found to inhibit most laccases. 

Ruggiero et al. (1989) investigated the ability of a natural silt loam soil and the clay minerals, montmorillonite (Mte) and kaolinite (Kte), to immobilize laccase. They compared the catalytic abilities of the soil-enzyme and clay-enzyme complexes to degrade 2,4-dichlorophenol. They found that the immobilized laccase remains active in removing the substrate even after 15 repeated cycles of substrate addition (Figure 2.24). However, Claus and Filip (1988) found that the activity of tyrosinase, laccase, and peroxidase is inhibited by immobilization on bentonite. The type of saturating cations on clay surfaces also substantially influences enzymatic activity (Claus and Filip, 1990). 

Few synthetic or natural Inhibitors of these enzymes have been elucidated. Some natural products such as curcurbitacin I and 0 can repress Induction of laccase by the pathogenic fungus of cucumber, Botrvtis cinerea (Si). It Is not known how this compound Is Involved In resistance or If these compounds are active Inhibitors that could reduce defense In plant species by causing PPO or PO Inhibition. Oxalates are natural product Inhibitors of chloroplastic PPO (SS) and... 

Laccases also become inhibited at higher pH values. Tree laccase is inhibited above pH 6.5. It appears that at pH 7.4 50% of the enzyme molecules are inhibited due to the binding of OH to the type-2 copper. The nature of this OH binding to the resting form of ascorbate oxidase will be demonstrated below. 

In the case of Polyporus laccase, Malkin et al. (95) have differentially removed the non-blue Cu(II) from the protein. This inactivates the enzyme but leaves the intense blue color intact. The activity and original copper content can be restored by adding Cu(II) and ascorbate (95). Anions such as F" and CN" appear to inhibit by reacting with the non-blue copper (66). Fluoride, for example, appears to react exclusively with the non-blue Cu(II) since the super hyperfine lines from the fluoride nucleus appear exclusively on the non-blue Cu(II) hyperfine lines in the ESR spectrum, and the blue Cu(II) hyperfine lines remain unaltered (Figure 6) (96). Figure 6 is an ESR spectrum taken at a... 

The first reports on a reversible DET between redox proteins and electrodes were published in 1977 showing that cytochrome c is reversibly oxidized and reduced at tin-doped indium oxide [30] and gold in the presence of 4,4 -bipyridyl [31]. Only shortly after these publications appeared, papers were published describing the DET between electrode and enzyme for laccase and peroxidase [32,33]. It was observed that the overpotential for oxygen reduction at a carbon electrode was reduced by several hundred millivolts compared to the uncatalyzed reduction when laccase was adsorbed. This reaction could be inhibited by azide. The term bioelectrocatalysis was introduced for such an acceleration of the electrode process by... 

The rapid initial reduction of the type 1 copper is very similar to that reported for tree laccase (49). The amplitude of this reduction increases with substrate concentration to a maximum value of approximately 50% of total absorbance change at 10°C. In laccase this effect is explained by the existence of two forms of the enzyme in an acid-base equilibrium. The active form allows rapid type 1 to type 3 electron transfer, whereas in the inactive form this process is inhibited. At higher substrate concentrations, the reduction of the type 1 copper is faster than the interconversion of inactive enzyme into its active form, leading to an increase in initial phase amplitude. Turnover-induced activation of ascorbate oxidase (67) could also be explained in terms of displacement of this inactive-active equilibrium. 

The finding from rapid-freeze-quench EPR experiments, that the reduction of the type 2 copper is slow compared with that of the type 1 copper, is analogous to the behavior noted for tree laccase at higher pH values (50). In this enzyme the slow reduction of the type 2 center is linked to the inhibition of the type 3 reduction. In ascorbate oxidase, however, reduction of the type 3 copper pairs proceeds despite the slow reduction of the type 2 copper, suggesting that the two electrons necessary for the proposed intramolecular reduction of the two type 3 copper pairs can be transferred from two of the three type 1 copper centers, without involving the type 2 center in any redox process. 

F Xu. Oxidation of phenols, anilines, and benzenethiols by fungal laccases Correlation between activity and redox potentials as well as halide inhibition. Biochemistry 35 7608-7615, 1996. 

E Xu. Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases. J. Biol. Chem. 272(2) 924-928, 1997. 

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