Ascorbate oxidase catalytic mechanism

E. X-Ray Structure of the Azide Form of Ascorbate Oxidase The Catalytic Mechanism... 

X-ray crystal structures of four functional derivatives of ascorbate oxidase were determined (149,150). The results of these investigations and implications for the catalytic mechanism of the blue oxidases will be outlined in the next section. 

A tentative catalytic mechanism of ascorbate oxidase has been proposed based on the refined X-ray structure and on spectroscopic and mechanistic studies of ascorbate oxidase and the related laccase. The results of these studies have been discussed in detail (74). The X-ray structure determinations of the fully reduced and peroxide derivatives define two important intermediate states during the catalytic cycle. A proposal for the catalytic mechanism incorporating this new information is given in Messerschmidt et al. (150) and presented in Fig. 14. This scheme should be valid in principle also for laccase due to the close similarities of both blue oxidases. 

X-ray structures of functional derivatives of ascorbate oxidase provided pictures of intermediate states, which will probably be passed during the catalytic cycle. A catalytic mechanism that is based on the available mechanistic data and these new results has been proposed. 

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). 

Figure 35 Proposal for the catalytic mechanism of ascorbate oxidase. ...

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... 

Messerschmidt A. Spatial structures of ascorbate oxidase, laccase, and related proteins implications for the catalytic mechanism. In Messerschmidt A (ed.), Multi-Copper Oxidases, World Scientific, Singapore, 1997. 

Multicopper oxidases are typically active in the catalytic one-electron oxidation of a variety of diphenolic, polyphenolic, enediolic, and aminophe-nolic substrates 1,53,166,167). The mechanism of these reactions is complex and, as discussed in Section I, it involves a sequence of four one-electron oxidations of substrate molecules. The radical products of these reactions undergo dismutation, as shown in Scheme 21 for the oxidation of ascorbate to semidehydroascorbate radical 168,169). The substrate binds to the enzymes close to type 1 Cu, whereas the trinuclear cluster is only accessible to dioxygen, or other small molecules. This situation is clearly difficult to reproduce in a model system and for this reason the type of model oxidation reactions that have been studied so far using synthetic trinuclear copper complexes is more related to the activity of type 3 Cu enzymes than multicopper oxidases. Nevertheless, such trinuclear complexes open new perspectives in stereoselective catalysis, because one of the metal centers... 

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