Ascorbate oxidase function

S / V CONTENTS Preface, Robert W. Hay. Structure and Function of Manganese-Containing Biomolecules, David C. Weather-bum. Repertories of Metal Ions as Lewis Acid Catalysts in Organic Reactions, Junghan Suh. The Multicopper-Enzyme Ascorbate Oxidase, Albrecht Messerschmidt. The Bioinorganic Chemistry of Aluminum, Tomas Kiss and Etelka Farkas. The Role of Nitric Oxide in Animal Physiology, Anthony R. Butler, Frederick Flitney and Peter Rhodes. Index. 

This discussion of copper-containing enzymes has focused on structure and function information for Type I blue copper proteins azurin and plastocyanin, Type III hemocyanin, and Type II superoxide dismutase s structure and mechanism of activity. Information on spectral properties for some metalloproteins and their model compounds has been included in Tables 5.2, 5.3, and 5.7. One model system for Type I copper proteins39 and one for Type II centers40 have been discussed. Many others can be found in the literature. A more complete discussion, including mechanistic detail, about hemocyanin and tyrosinase model systems has been included. Models for the blue copper oxidases laccase and ascorbate oxidases have not been discussed. Students are referred to the references listed in the reference section for discussion of some other model systems. Many more are to be found in literature searches.50... 

Many enzymes require additional substances in order to function effectively. Conjugated enzymes require a prosthetic group before they are catalytically active, such groups being covalently or ionically linked to the protein molecule and remaining unaltered at the end of the reaction. Catalase (EC 1.11.1.6), for instance, contains a haem group while ascorbate oxidase (EC 1.10.3.3) contains a copper atom. 

The multi-copper oxidases include laccase, ceruloplasmin, and ascorbate oxidase. Laccase can be found in tree sap and in fungi ascorbate oxidase, in cucumber and related plants and ceruloplasmin, in vertebrate blood serum. Laccases catalyze oxidation of phenolic compounds to radicals with a concomitant 4e reduction of O2 to water, and it is thought that this process may be important in the breakdown of lignin. Ceruloplasmin, whose real biological function is either quite varied or unknown, also catalyzes oxidation of a variety of substrates, again via a 4e reduction of O2 to water. Ferroxidase activity has been demonstrated for it, as has SOD activity. Ascorbate oxidase catalyzes the oxidation of ascorbate, again via a 4e reduction of O2 to water. Excellent reviews of these three systems can be found in Volume 111 of Copper Proteins and Copper Enzymes (Lontie, 1984). 

A number of copper -containing protein compounds are enzymes with an oxidase function (ascorbic acid oxidase, urease, etc 1 and these play an important role in Ihe biological oxidation-reduction system. There is a definite relationship of copper with iron in connection with utilization of iron in hemoglobin function. 

The most common metal encountered in electron transfer systems is iron, although copper and manganese play vital functions. Merely to emphasise the complexity of the catalysts that are used in biology, the structures of the active sites of ascorbate oxidase (Fig. 10-11) and superoxide dismutase (Fig. 10-12) are presented. It is clear that we have only just begun to understand the exact ways in which metal ions are used to control the reactivity of small molecules in biological systems. 

Copper proteins are involved in a variety of biological functions, including electron transport, copper storage and many oxidase activities. A variety of reviews on this topic are available (Sykes, 1985 Chapman, 1991). Several copper proteins are easily identified by their beautiful blue colour and have been labelled blue copper proteins. The blue copper proteins can be divided into two classes, the oxidases (laccase, ascorbate oxidase, ceruloplasmin) and the electron carriers (plastocyanin, stellacyanin, umecyanin, etc.). 

Figure 8. Proposed electron transfer pathway in blue copper proteins. The plastocyanin wave function contours have been superimposed on the blue copper (type 1) site in ascorbate oxidase (40). The contour shows the substantial electron delocalization onto the cysteine Spir orbital that activates electron transfer to the trinuclear copper cluster at 12.5 A from the blue copper site. This low-energy, intense Cys Sp - Cu charge-transfer transition provides an effective hole superexchange mechanism for rapid long-range electron transfer between these sites (2, 3, 28).

Preliminary observation of additional electron density at this fourth coordination position of Cu-2 upon soaking crystals with N02 is consistent with this idea. Thus, from the structural data it would appear that Cu-1 is a type 1 center that functions to transfer electrons to the catalytic Cu-2 ion (See Note Added in Proof). It has been suggested, largely on the basis of electronic structural considerations (27, 28), that the Cys-136-His-135 link between Cu-1 and Cu-2 is a possible conduit for electron transfer between the two sites. An analogous dipeptide bridge between the type 1 center and the catalytic tricopper cluster in ascorbate oxidase (29, 30) may function similarly. Indeed, other close similarities between protein domains in ascorbate oxidase and NiR have been noted (17). 

Messerschmidt, A., Luecke, H., and Huber, R. (1993). X-ray structures and mechanistic implications of three functional derivatives of ascorbate oxidase from zucchini./. Mol. Biol 230, 997-1014. 

A few metalloenzymes are involved in AA metabolism or reqnire AA as a cofactor, including ascorbate oxidase and prolyl and lysyl hydroxylase. The structure and function of these enzymes are discussed in Section IV.C. 

Occurrence, Sequences, and Biological Function Molecular and Spectroscopic Properties X-Ray Structure of Ascorbate Oxidase... 

This chapter will concentrate mainly on structural and functional aspects of these enzymes with the major emphasis on ascorbate oxidase and laccase. Significant progress has been achieved in the last 10 years the determination of amino acid sequences of all three enzymes, each from several sources, and the X-ray structure of ascorbate oxidase. The new information forms the basis of a much deeper understanding of the function of the enzymes as will be demonstrated in this chapter. 

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. 

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. 

Two other copper enzymes possess ascorbate oxidase activity, human ceruloplasm and Polyporus laccase (70,71). Ceruloplasm may function as an AA oxidase in vivo. Both ceruloplasm and laccase are 10 times less active toward AA oxidation than is ascorbate oxidase. However, the reaction is definitely enzymic, and water is produced. 

The high-affinity pathway involves oxidation of Fe to Fe by the ferroxidase FET3 and subsequent transport of Fe " " across the plasma membrane by the permease FTRl. FET3p is a member of the family of multicopper oxidases, which include ascorbate oxidase, laccase, and ceruloplasmin (see Chapter 14), and does not become functional until it is loaded with copper intracellularly through the activities of the copper chaperone ATX Ip and the copper transporter CCC2p. It appears that Fe " " produced by FET3 is transferred directly to FTRl, and does not equilibrate with the bulk phase, as is illustrated in Fig. 7.13. This is almost certainly achieved by the classic metabolite-channeling mechanism, a common feature of multifunctional enzymes. 

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). 

Ascorbate oxidase (EC 1.10.3.3) occurs exclusively in higher plants [267]. Its physiological function is as yet unknown, although it may play a role in ripening [36, 268], growth control, or disease control [269]. Ascorbate is probably the enzyme s physiological electron-donor. 

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