0-Glucan endo-hydrolases

At present none of the 0-glucan endo-hydrolases have been examined crystallographically, nor have their amino acid sequences been determined, so that their three-dimensional structures cannot yet be deduced. Without this information it is not possible to relate substrate or inhibitor specificity to amino acid orientation at the active site. 

An observation basic to the elucidation of the mechanism of hydrolysis of lysozyme substrates is that the glycosidic linkages are cleaved at the Ci—O bond (195). No similar information is available for the 0-glucan endo-hydrolases. Another key to the description of the hydrolytic mechanism of lysozyme is the observed retention of the configuration of the substrate by the products of transfer (40,182,197). Such retention... 

N-acetyl glucosamine is also an inhibitor of lysozyme, but in contrast to the oligosaccharide inhibitors, its action is due to binding across, rather than in, the cleft of the active site (24). Monosaccharides are known to inhibit some 0-glucan endo-hydrolases (202) and a physical explanation of this kind is possible, but would be difficult to confirm without recourse to crystallographic methods. 

This brief comparative resume shows clearly that a number of facets of the action of 0-glucan endo-hydrolases may be explained by reference to an enzyme model based on the structure found for lysozyme. The crystallographic data which provide the basis for the three-dimensional model of lysozyme have been supplemented by information on enzyme-substrate interactions in solution (4, 19, 39, 41, 47, 51, 52, 181, 196). Since it is not yet possible to examine the 0-glucan endo-hydrolases crystallographically, the methods which have been used to study lysozyme-substrate interactions in solution will be of primary importance in the elucidation of the action of these enzymes. Already certain 0-1,4-glucan endo-hydrolases have been examined using some of these techniques (57, 171, 172) but wider application of such methods is needed. 

The fi-glucan exo- and endo-hydrolases are discussed with reference to newer techniques for the investigation of their specificity and action pattern. Those exo-hydrolases which have been well characterized are described individually. The endo-hydrolases are examined from the point of view of their linkage specificity, action on substituted glucans and their specificity for various monomer units. The significance of more random and less random endo-action patterns is considered in relation to single or multiple attack mechanisms. Certain features of p-glucan endo-hydrolase catalyzed reactions are discussed in relation to current views on the three-dimensional structure and mechanism of action of lysozyme. 

Although many 0-1,4-glucan endo-hydrolases have been examined, the ability to hydrolyze the 1,3 1,4 mixed-linked glucans has been investigated in detail only for a Streptomyces preparation (163,169,193), for a purified Aspergillus niger enzyme (45, 46), and for a fraction from a Trichoderma viride preparation (97). In each case the nature of the products (see Table IV) makes it clear that 4-0-glucosyl residues are... 

Fleming, Manners, and Masson (62) found that a /M,3-glucan endo-hydrolase from a bacterial source was able to hydrolyze laminarin to glucose, laminaribiose, laminaritriose, and other oligosaccharides. The... 

Comparison of fi-Glucan Endo-hydrolases with Lysozyme... 

Lysozyme is unique among the polysaccharide hydrolases in that its chemical (107) and physical (24, 25, 173, 197) structure is accurately known and the mechanism of its hydrolytic action explained in considerable detail (182, 195, 221). Comparison of the characteristics of lysozyme action (Table V) with those of the -glucan endo-hydrolases (Table II) shows that these enzymes share many common features. 

Certain properties of lysozyme, which are common to / -glucan endo-hydrolases, have been explained following the elucidation of the structure of the active site. It is therefore relevant to discuss the current concepts of lysozyme structure and action and to compare these with the rather fragmentary observations so far made on the action of the various / -glucan endo-hydrolases. 

Binding constants for the substrates of / -glucan endo-hydrolases are not known, but the kinetics of the splitting of / -l,4-oligoglucosides are similarly dependent on CL, suggesting that multiple sub-site binding is a general property of these endo-hydrolases (81, 122, 229, 230). 

Competitive inhibition of lysozyme by potential oligosaccharide substrates has been explained as being caused by their non-productive binding at sites adjacent to, but not across, the bond-breaking site (24). The inhibition of certain 0-1,4-glucan endo-hydrolases by cellobiose and lactose (138) may have an analogous physical explanation. 

P. cinnamomi polysaccharide 500 iig/mL (O) 50 iig/mL (A) 5 g/mL (a) untreated control (b) after treatment with B. subtilis l,3 l,4- -glucan endo-hydrolase (c) after treatment with E. gracilis 1,3- -glucan exohydrolase (d) after treatment with R. arrhizus 1,3- -glucan endohydrolase. 

The distinction between exo-hydrolases and endo-hydrolases based on their action patterns during the hydrolysis of polymeric and oligomeric glucans is well established. As shown in Table II, there are also differences in the anomeric configuration of reducing groups released, although... 

The patterns of end products of hydrolysis of homogeneous glucans by various endo-hydrolases of known purity show differences which are dependent on the specificity of the enzymes. For example, purified 0-1,3-... 

Enzymic transglycosylation, which may accompany the action of glycoside hydrolases (102, 184), and in some circumstances exo- (165) and endo-hydrolase action (23, 91,176, 226), could produce oligosaccharide patterns during the hydrolysis of glucan substrates, which would lead to possible misinterpretation of enzyme specificity. 

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