The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity.

Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide.

Chemical synthesis of a dual branched malto-decaose: a potential substrate for alpha-amylases.

A convergent block strategy for general use in efficient synthesis of complex alpha-(1-->4)- and alpha-(1-->6)-malto-oligosaccharides is demonstrated with the first chemical synthesis of a malto-oligosaccharide, the decasaccharide 6,6''''-bis(alpha-maltosyl)-maltohexaose, with two branch points. Using this chemically defined branched oligosaccharide as a substrate, the cleavage pattern of seven different alpha-amylases were investigated. Alpha-amylases from human saliva, porcine pancreas, barley alpha-amylase 2 and recombinant barley alpha-amylase 1 all hydrolysed the decasaccharide selectively.

Chemoenzymatic synthesis of branched oligo- and polysaccharides as potential substrates for starch active enzymes.

Oligo- and polysaccharides embodying the alpha-maltotriosyl-6(II)-maltotetraosyl structure were readily synthesized by transglycosylation of maltosyl fluoride onto panose and pullulan catalysed by the bacterial transglycosylase cyclodextrin glycosyltransferase (CGTase). The two products obtained proved useful for increasing the knowledge of substrate binding and processing at the active site of barley limit dextrinase that is involved in the metabolism of amylopectin by acting upon its branch points.

Selection of a high-energy bioactive conformation of a sulfonium-ion glycosidase inhibitor by the enzyme glucoamylase G2.

Transferred nuclear Overhauser effect and rotating-frame Overhauser enhancement NMR spectroscopies are used to probe the conformation of a bicyclic sulfonium ion, which is an analogue of the naturally occurring glycosidase inhibitor castanospermine, bound to the enzyme glucoamylase G2. Enzyme inhibition assays indicate that the bicyclic sulfonium ion is a slightly better inhibitor (K(i) = 1.32 mM) of glucoamylase G2 than the naturally occurring sulfonium-ion glycosidase inhibitor, salacinol, with a K(i) value of 1.

Chemoenzymatic syntheses of linear and branched hemithiomaltodextrins as potential inhibitors for starch-debranching enzymes.

Oligosaccharides embodying the S-maltosyl-6-thiomaltosyl structure have been readily synthesised by using convergent chemoenzymatic approaches. The key steps for the preparation of these molecules involved: 1) transglycosylation reactions of maltosyl fluorides onto suitable acceptors catalysed by the bacterial transglycosylase, cyclodextrin glycosyltransferase (CGTase), and 2) the SN2-type displacement of a 6-halide from acetylated acceptors by activated 1-thioglycoses. The target molecules, which were obtained in good overall yields, proved to be useful for investigating substrate binding in the active sites of several enzymes that act upon the alpha-1,6-linkage of pullulan and/or amylopectin.

Synthesis of selenium analogues of the naturally occurring glycosidase inhibitor salacinol and their evaluation as glycosidase inhibitors.

The syntheses of two selenium analogues (10 and 11) of the naturally occurring sulfonium ion, salacinol (3), are described. Salacinol is one of the active principles in the aqueous extracts of Salacia reticulata that are traditionally used in Sri Lanka and India for the treatment of diabetes. The synthetic strategy relies on the nucleophilic attack of a 2,3,5-tri-O-benzyl-1,4-anhydro-4-seleno-D-arabinitol at the least hindered carbon of benzyl- or benzylidene-protected D- or L-erythritol-1,3-cyclic sulfate.