Endo-Hydrolase

Hydrolysis of end-labeled 3-hydroxybutyrate oligomers by purified A. faecalis T poly(3HB) depolymerase showed that the enzyme mainly cleaved the second and third ester linkage from the hydroxyl terminus [69]. However, since the enzyme also hydrolyzes cyclic oligomers, the A. faecalis depolymerase has endo-hydrolase activity in addition to exo-hydrolase activity [18, 70]. Results of... 

Brandi et al. [71] using culture fluid of Acidovorax delafieldii and cyclic 3HB oligomers were in agreement with the presence of endo-hydrolase activity of poly(3HB) depolymerases. Similar results were obtained by de Koning et al. [72] who demonstrated that covalently cross-linked poly(HAMCL) was hydrolyzed completely by P. fluorescens. It is assumed that most - if not all - extracellular poly(HA) depolymerases have endo- and exo-hydrolase activity. Depending on the depolymerase the hydrolysis products are only monomers, monomers and dimers, or a mixture of oligomers (mono- to trimers). 

Schematic drawing of monosaccharide binding sites in various glycosyl hydrolases, (a) Subsites of an endo-hydrolase, e. g., hen egg white lysozyme, (b) subsites of a disaccharide hydrolase, e. g., ((x-glucosidase, (c) subsites of a monosaccharide-releasing exo-hydrolase, e. g., glucoamylase, (d) subsites of a disaccharide-releasing exe-hydrolase, e.g., / -amylase (after [18])...

Positional isomers of a tetrasaccharide on a five subsite endo-hydrolase. Of the eight possible positional isomers only IV, V, and VI are productive, i. e., lie over the catalytic site. The cleavage products are shown subsite on enzyme position of catalytic site monosaccharide residue residue bearing the reducing hemiacetal in substrate residue bearing the reducing hemiacetal in products (after [28])... 

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. 

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... 

These enzymes differ from other exo-hydrolases by not hydrolyzing their substrates at unique sites, at least with the / -l,4-oligoglucosides of CL up to 6. When swollen cellulose was the substrate, cellobiose was an important product for each of the enzyme fractions, but there is no information as to whether cellotriose was also a product. The polarity of attack and the inversion of the configuration of the oligosaccharides released, distinguish this enzyme from endo-hydrolases. 

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... 

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. 

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). 

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... 

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. 

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. 

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