Laccases substrates

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. 

Fig. 60. Signal amplification by electrochemical regeneration of the laccase substrate, hydroquinone (H2QX BQ benzoquinone.

W Cai, R Martin, B Lemaure, J-L Leuha, V Petiard. Hydroxyindols A new class of laccase substrates. Plant Physiol Biochem 31 441 445, 1993. 

Fig 5.8. Rhizoctonia soiani challenged by viscosinamide producing bacteria (left). An agar overlay containing the laccase substrate ABTS revealed high laccase activity in the zone of interaction between the fungus and the bacteria (right). 

Laccase creates free radicals that can fiirther react with odier compounds. Ideally, conditions for each substrate would be optimized for the grafting reaction, which would require investigation of concentration, pH, affinity of the enzyme, and other reactants in the pulp. Because optimizing the conditions for each substrate is beyond the scope of a screening test, the concentration of substrate (3.75 mM), enzyme (5 units), and fiber were held constant. We incubated 12 bar loblolly pine pulp in the presence of laccase with and without various laccase substrates, as described in Methods. These pulps were treated with laccase, washed, and fi-eeze dried. High affinity laccase substrates were. oxidized once before 1 h had elapsed. The enzyme reaction was continued for several hours to allow for lower affinity substrates to react. 

Because methylene blue is a cationic dye, it would be expected to have activity as a Lewis acid. The correlation with methylene blue was weak but showed the expected trend, with increased methylene blue binding being negatively correlated to increased Phloroglucinol and isovanillic acid were the only laccase substrates that increased the ATp of the fiber. All substrates other than resorcinol, 4-hydroxyphenylacetic acid, 1,2,3-trihydroxybenzene, and 1,2,4-... 

Laccase active site with proposed mode of substrate / Intermediate binding... 

Figure 17.3 Anatomy of a redox enzyme representation of the X-ray crystallographic structure of Trametes versicolor laccase III (PDB file IKYA) [Bertrand et al., 2002]. The protein is represented in green lines and the Cu atoms are shown as gold spheres. Sugar moieties attached to the surface of the protein are shown in red. A molecule of 2,5-xyhdine that co-crystallized with the protein (shown in stick form in elemental colors) is thought to occupy the broad-specificity hydrophobic binding pocket where organic substrates ate oxidized by the enzyme. Electrons from substrate oxidation are passed to the mononuclear blue Cu center and then to the trinuclear Cu active site where O2 is reduced to H2O. (See color insert.)...

Figure 17.5 The protein environment around the Cu centers (gold spheres) of laccase from Melanocarpus albomyces (PDB file IGWO) showing a substrate O2 molecule bound in the trinuciear Cu site [Hakulinen et al., 2002], The protein is depicted in stick representation with atoms in their conventional coloring. (Courtesy of Armand W. J. W. Tepper.) (See color insert.)...

Bertrand T, Jolivalt C, Briozzo P, Caminade E, Joly N, Madzak C, Mougin C. 2002. Crystal structure of a four-copper laccase complexed with an arylamine Insights into substrate recognition and correlation with kinetics. Biochemistry 41 7325-7333. 

The low specificity of electron-donating substrates is remarkable for laccases. These enzymes have high redox potential, making them able to oxidize a broad range of aromatic compounds (e.g. phenols, polyphenols, methoxy-substituted phenols, aromatic amines, benzenethiols) through the use of oxygen as electron acceptor. Other enzymatic reactions they catalyze include decarboxylations and demethylations [66]. 

The catalytic cycle of laccase includes several one-electron transfers between a suitable substrate and the copper atoms, with the concomitant reduction of an oxygen molecule to water during the sequential oxidation of four substrate molecules [66]. With this mechanism, laccases generate phenoxy radicals that undergo non-enzymatic reactions [65]. Multiple reactions lead finally to polymerization, alkyl-aryl cleavage, quinone formation, C> -oxidation or demethoxylation of the phenolic reductant [67]. 

FIGURE 12.4 Proposed catalytic cycle for laccase, where S represents substrate. (From [44], with permission from the American Chemical Society.)... 

Baiocco P, Barreca AM, Fabbrini M, Galli C and Gentili P. 2003. Promoting laccase activity towards non-phenolic substrates a mechanistic investigation with some laccase—mediator systems. Org Biomol Chem 1(1) 191—197. 

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