Laccase analysis

Tatiana V, Pegasova P, Zwart O, Koroleva V, Stepanova EV, Rebrikovc DV and Lamzinb V. 2003. Crystallization and preliminary X-ray analysis of a four-copper laccase from Coriolus hirsutus. Acta Crystallogr... 

Bourbonnais R, Leech D, Paice MG (1998) Electrochemical analysis of the interaction of laccase with lignin model compounds. Biochim Biophys Acta 1379 381-390... 

To elucidate some enzymatic characteristics of the isolated laccases I, II, and III, substrate specificities for several simple phenols, electrophoresis patterns, ultraviolet spectra, electron spin resonance spectra, copper content, and immunological similarities were investigated. Tyrosine, tannic acid, g c acid, hydroquinone, catechol, pyrogallol, p-cresol, homocatechol, a-naphthol, -naphthol, p-phenylenediamine, and p-benzoquinone as substrates. No differences in the specificities of these substrates was found. The UV spectra for the laccases under stucfy are shown in Figure 4. Laccase III displays three adsorption bands (280, 405, and 600nm), laccase II shows one band 280nm), and laccase I shows two bands (280 and 405 nm). These data appear to indicate differences in chemical structure. The results of the copper content analysis (10) and two-dimensional electrophoresis also indicate that these fractions are completely different proteins (10), Therefore, we may expect differences in substrate specificities between the three laccase fractions for more lignin-like substrates, yet no difference for some simple phenolic substrates. 

Figure 2. Analysis of laccase I and laccase HI by SDS-PAGE (10% polyacrylamide slab gel electophoresis).

In order to investigate the active sites of these proteins, laccases I and III were subjected to ESR (electron spin resonance) spectroscopic analysis. The ESR spectra shown in Figure 5 indicate clear differences in peaks 2 and 6 which support the concept that the copper atoms in laccases I and III have different conformations in each molecule. Furthermore, immunological similarity between laccases I and III was also investigated. Antibody specific for laccase III was prepared from rabbit serum by conventional methods. When applied to Ouchterlony diffusion plates containing laccase I, no precipitation lines developed (Figure 6). This result showed that there were no conserved epitopes on the surfaces laccases I and III. 

When the complete amino acid sequence of ceruloplasmin was determined, an internal threefold repeat suggested gene triplication (Ta-kahashi et al., 1983). Moreover, sequence similarity to the small blue copper domains suggested that there were at least two domains with blue copper-binding sites. Analysis of fragments of laccase sequence indicated that there might be a relationship of this to small blue proteins and ceruloplasmin as well (Ryden, 1988). 

It is noteworthy that the proximity of the copper sites in ceruloplasmin, and, indeed, the involvement of most of the correct ligand histidines, were predicted some time ago by Ryden (1982, 1984) strictly on the basis of sequence homologies to plastocyanin. A similar prediction was made for laccase based on sequence similarities around the cysteine regions (Briving et al, 1980). Proximity of the type II site to the type III site (e.g., a trinuclear site) was also predicted by Solomon and co-workers (Allen-dorf et al., 1985 Spira-Solomon et al, 1986) on the basis of spectroscopic analysis of azide binding to laccase. What could not have been foreseen... 

In the discussion of the biochemistry of copper in Section 62.1.8 it was noted that three types of copper exist in copper enzymes. These are type 1 ( blue copper centres) type 2 ( normal copper centres) and type 3 (which occur as coupled pairs). All three classes are present in the blue copper oxidases laccase, ascorbate oxidase and ceruloplasmin. Laccase contains four copper ions per molecule, and the other two contain eight copper ions per molecule. In all cases oxidation of substrate is linked to the four-electron reduction of dioxygen to water. Unlike cytochrome oxidase, these are water-soluble enzymes, and so are convenient systems for studying the problems of multielectron redox reactions. The type 3 pair of copper centres constitutes the 02-reducing sites in these enzymes, and provides a two-electron pathway to peroxide, bypassing the formation of superoxide. Laccase also contains one type 1 and one type 2 centre. While ascorbate oxidase contains eight copper ions per molecule, so far ESR and analysis data have led to the identification of type 1 (two), type 2 (two) and type 3 (four) copper centres. 

Yaropolov Al et al. (1995) Flow-injection analysis of phenols at a graphite electrode modified with co-immobilized laccase and tyrosinase. Anal Chim Acta 308 137-144... 

R Bourbonnais, D Leech, MG Paice. Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochim. Biophys. Acta (BBA) General Subjects 1379(3) 381-390, 1998. 

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