Laccase copper content

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. 

The number and type of copper centers, EPR parameters, and the two relevant absorption bands in the visible region for several representative members of the blue oxidases are listed in Table II. All laccases except that of the P. radiata enzyme contain four coppers per molecule with one type-1, one type-2, and one type-3 copper center. The EPR and absorption parameters resemble each other very much. Phlebia radiata laccase is supposed to contain only two coppers with one type 1, one type 2, and one PQQ per mole (70). This is quite unusual and will be discussed critically below, since it is possible that the copper content determined for this enzyme is inaccurate. Ascorbate oxidases have eight coppers per homodimer with two type-1, two type-2, and two type-3 copper centers. Ceruloplasmin typically contains six to seven copper ions per molecule with three type-1, one type-2, and one type-3 copper centers. It has also been proposed that there are only two type-1 copper ions and a new type-4 copper that is presumed to exhibit no EPR signal. In addition there is a variable content of chelatable copper. It is responsible for copper contents exceeding 6 coppers/mol but does not seem to be required for catalysis. It is now generally accepted that ceruloplasmin has three type-1 copper centers and the reason for this will be discussed 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... 

Another problem to be elucidated is the role of copper in cytochrome a. In the known copper enzymes such as tyrosinase and laccase, copper is an important component of the prosthetic group, but is released from the protein moiety by dialysis against potassium cyanide. In the case of cytochrome a, however, the mode of combination of copper must be different, since very little copper is released from the protein moiety by dialysis. The best known method of releasing the copper is by acid treatment. The role of copper in the electron trasnferring system is still obscure, though Cohen and Elvehjem (1934), Yoshikawa (1937), Schultze (1939, 1941), Gallagher et al. (1956), and Gubler et al. (1957) observed, from dietary experiments, that copper-deficient tissues and yeast have a low cytochrome oxidase activity and a decreased content of hemin a. 

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