Glycan hydrolases

Generally, D-galacturonanases resemble other glycan hydrolases in having their pH optimum in a weakly acidic region. The pH optimum of most endo-(as well as exo-)D-galacturonanases lies between pH 4.0 and 6.5 (Refs. 16, 73, 115, 116, 119, 121, 124, 143,... 

R. V. Stick, The synthesis of novel enzyme inhibitors and their use in defining the active sites of glycan hydrolases, Top. Curr. Chem., 187 (1997) 187—213. 

In the mid-1980s, my research group in Perth began a collaboration with a team of biochemists at La Trobe University in Melbourne to study the mechanism of action of a range of -glycan hydrolases and cellulases found in various plants and bacteria. The approach was to involve synthetic enzyme inhibitors. 

In the classic studies by Sharon et al. [12] and Legler and Bause [13], epoxyalkyl glycosides were used to inhibit the action of various glycan hydrolases. In view of the almost certain presence of an acid residue at the active site and the two mechanisms which have been proposed for the action of glycan hydrolases, namely that involving hydrolysis with retention or inversion of configuration at the anomeric centre (Figs. 1 and 2) [14, 15], we decided to use epoxyalkyl... 

In view of the efficient inhibition of a particular crystalline glycan hydrolase by the epoxylbutyl -cellobioside 7 (n = 2), we decided to prepare the deoxy iodo derivative 21 to aid in the X-ray crystallographic analysis. We soon found that, although the alkene 22 was easily available as a direct precursor to our target, the epoxide functionality had to be introduced indirectly using bromo-hydrin 23 technology any direct oxidation of 22 invariably led to some loss of the iodine atom [23]. 

We had for some time considered that aziridines, because of their increased basicity and hence reactivity at the active site of a glycan hydrolase, would prove to be better inhibitors than the epoxides in many respects. Conversely, the less basic thiirans would be expected to be poorer inhibitors than the corresponding epoxides. We thus embarked on a synthesis of the aziridine 30 and the thiiran 31. 

Stick RV (1997) The Synthesis of Novel Enzyme Inhibitors and Their Use in Defining the Active Sites of Glycan Hydrolases. 187 187-213 Stutz AE, see de Raadt A (1997) 187 157-186 Stumpe R, see Kim JI (1990) 157 129-180 Suami T (1990) Chemistry of Pseudo-sugars. 154 257-283 Suppan P (1992) The Marcus Inverted Region. 153 95-130 Suzuki N (1990) Radiometric Determination of Trace Elements. 157 35-56 Tabakovic I (1997) Anodic Synthesis of Heterocyclic Compounds. 185 87-140 Takahashi Y (1995) Identification of Structural Similarity of Organic Molecules. 174 105-134 Tasi G, Palinkd I (1995) Using Molecular Electrostatic Potentid Maps for Similarity Studies. 174 45-72... 

The Synthesis of Novel Enzyme Inhibitors and Their Use in Defining the Active Sites of Glycan Hydrolases... 

Topology of Active Sites Most often the active site is confined to one domain but there are examples where the active site is contributed by amino acid residues from two different domains [195,196]. The topology of the active sites of glycan hydrolases may be described in one of three general shapes a pocket, a cleft or groove, or a tunnel, irrespective of whether the enzyme is inverting or retaining. Examples of the three t) es are shown in Fig. 18. 

The pi and M.W. values for several D-galactanases from B. subtilis are given in Table VII. These enzymes are considered to be neutral (pi 6.88) and alkaline (pi 7.96-8.90) glycan hydrolases, and their... 

The D-mannanase produced by Bacillus subtilis83 was found to show a dependency towards Ca2+ for the maintenance of activity and stability. In the presence of Ca2+, this enzyme preparation showed optimal activity at pH 6.0 and 60°, and was stable at pH values of 5.0-9.5. The enzyme was completely inactivated when kept at 70° for 15 min at pH 7.5. In the presence of EDTA, however, the enzyme showed optimal activity at pH 6.0 and 40°, was stable only at pH values of 6.0 to 7.5, and was completely inactivated at 55°. Temperature and pH properties of D-mannanases (from other sources) not dependent on metal cofactors are summarized in Table XII, together with the pi and M.W. values for several D-mannanases. These enzymes are considered to be acid glycan hydrolases, and their sizes (M.W. range, 22-47 X 104) are comparable to those of other polysaccharases (see, for example, Sections II, III, and VI). Other physical properties of D-mannanases that have been measured include the partial specific volume (0.72 cm3.g 1, for the Aspergillus niger84 enzyme) and the sedimentation coefficient (3.85, for the A. niger84 enzyme). 

Increases in levels of glycan hydrolases and other enzymes. (11, )... 

Host 3-Glucan Hydrolase Induction. A further response to microbial invasion of host tissues involves an increase in activity of certain host enzymes, especially the glycan hydrolases which are potentially able to depolymerise microbial polysaccharides. 

Extra- and intracellular glycan hydrolases were isolated from Aspergillus flavus and partially characterized both preparations exhibited /3-D-galactanase activity (see p. 499). Gel chromatography of the extracellular enzyme preparation revealed one protein fraction containing jS-D-galactanase activity and a second one exhibiting mainly (3-D-xylanase activity. 

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