Ascorbate oxidase fungal

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

Visible MCD spectra of plastocyanin, azurin, Rhus vernicifera laccase, ascorbate oxidase and ceruloplasmin are similar on a per copper basis, but show differences from those of stellacyanin and fungal laccase. This is of interest in view of the absence of methionine from the coordination sphere of copper in stellacyanin, and the very high redox potential of fungal laccase.925... 

B. Structural Model of the Copper Sites for Ceruloplasmin Fungal Laccases, Ascorbate Oxidases, and Related Proteins... 

Table V supplies insight into the relatedness within fungal laccases and ascorbate oxidases and to the other included proteins. Obviously both Tables Va and Vb show the same trend. The laccases of the narrowly related basiodomycetes P. radiata and C. hirsutus show differences of only 37%. The differences for the laccases from the ascomycetes N. crassa and C. parasitica are about 50%. The laccase of the deuteromy-cete A. nidulans shows very low identities to all partners of the alignment when all residues are included into the calculations (Table Va). The picture becomes clearer when badly aligning segments are omitted from the calculations (Table Vb). The differences from the other lac-...

Redox potentials for the different copper centers in the blue oxidases have been determined for all members of the group but in each case only for a limited number of species. The available data are summarized in Table VI 120, 121). The redox potentials for the type-1 copper of tree laccase and ascorbate oxidase are in the range of 330-400 mV and comparable to the values determined for the small blue copper proteins plastocyanin, azurin, and cucumber basic protein (for redox potentials of small blue copper proteins, see the review of Sykes 122)). The high potential for the fungal Polyporus laccase is probably due to a leucine or phenylalanine residue at the fourth coordination position, which has been observed in the amino-acid sequences of fungal laccases from other species (see Table IV and Section V.B). Two different redox potentials for the type-1 copper were observed for human ceruloplasmin 105). The 490-mV potential can be assigned to the two type-1 copper sites with methionine ligand and the 580-mV potential to the type-1 center with the isosteric leucine at this position (see Section V.B). The... 

The reaction of nitric oxide with laccase (76) and ascorbate oxidase (147) has been studied as well. Nitric oxide fully reduces fungal and tree laccase when it is added to the oxidized enzyme under anaerobic conditions. In addition the binding of one NO molecule to laccase can be detected. This is characterized by a new EPR signal and has been described as coordinated with the type-2 copper (76). Only the reduction of the type-1 copper has been observed when NO has been added to ascorbate oxidase under anaerobic conditions. 

In contrast to tree and fungal laccase, whose molecular parameters and mechanisms of action have been thoroughly investigated (8), few such studies have been reported for ascorbate oxidase. This is mainly because of the relatively diflScult isolation and purification procedure of ascorbate oxidase in comparison with laccase. Furthermore, this enzyme appears to be more sensitive to environmental factors such as ionic strength of the buffer medium, its pH, or the presence of extraneous metal ions. Consequently, many samples isolated over a long period were found to be homogeneous from the standpoint of the protein biochemist but appeared inhomogeneous with respect to the catalytically active copper sites (9). 

The blue copper oxidases are similar to cytochrome oxidase in their ability to catalyze reduction of Oj to HjO. Catalysis is centered upon the protein-bound copper ions that can be differentiated into three classes according to their physical, chemical, and functional properties. They are designated Types 1, 2, and 3 copper . In the blue copper proteins (tree and fungal laccases, ceruloplasmin, ascorbate oxidase) these three classes of copper appear in varying amounts the laccases contain the minimum amounts of each (one each of Types 1 and 2 and two Type 3 coppers). 

As previously mentioned, laccase is very closely related to ascorbate oxidase. The principal molecular architecture and arrangement of the mononuclear and trinuclear copper centers are the same. Furthermore, spectroscopic and kinetic properties are similar in many circumstances. Therefore, the catalytic mechanism of the dioxygen reduction should be the same for both. Kinetic studies on fungal and tree laccases have been... 

FIGURE 8.5 RR spectra of tree (12 K) and fungal laccases (77 K), human ceruloplasmin (277 K), and zucchini squash ascorbate oxidase (277 K). The overtone regions (>600 nm) ate scale-expanded to show finer features. Laser wavelengths are 647.1 nm (tree laccase) and 620 nm (fungal laccase, ceruloplasmin, and zucchini ascorbate oxidase). (Adapted with permission from Ref. [69]. Copyright 1985, American Chemical Society.)... 

BCOs exhibit variable substrate specificity plant and fungal laccases, for example, typieally exhibit wide substrate specificity and can oxidize a variety of aminophenols, diphenols, and aryl diamines. The mechanism for these enzymes is consistent with Mareus theory [37], and suggests that oxidation occurs in the outer sphere and there is likely no specific binding pocket Conversely, some BCOs such as ascorbate oxidase (specifieity toward L-ascorbate) [38,39] and ceruloplasmin (specificity toward Fe +) [40,41] are often stereospecilic and highly substrate specific [42]. 

Copper-Free Ascorbic Add Oxidase. A totally different specific ascorbic acid oxidase has been prepared from fungal spores. This enzyme is not sensitive to copper inhibitors but is inhibited by sulfhydryl reagents. It does not attack ascorbic acid analogs. Like the copper-containing enzyme, fungal ascorbic acid oxidase gives rates proportional to oxygen tension. 

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