The predominantly nonhydrolytic action of alpha amylases on α-maltosyl fluoride

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Crystalline alpha amylases from a number of sources utilized α-maltosyl fluoride as a glycosyl donor and acceptor at high rates (∼10 to ∼01550 μmol/min/mg of protein, for 30 m M substrate). All enzymes catalyzed conversion of this compound into maltooligosaccharides in preference to causing its hydrolysis. Maltotetraosyl fluoride and maltooligosaccharides of d.p. 3 to 6+ accounted for 75–93% (by weight) of early reaction-products. At a late stage, the yield of maltooligosaccharides was 2–5 times that of maltose, with chains as long as 12 d-glucosyl residues formed by one amylase (from Asp. oryzae), which utilized α-maltosyl fluoride as a donor and as an acceptor at extremely high rates. These results indicate that alpha amylases have a substantial capacity for binding two molecules of this small substrate in a distinctive way, with the CF glycosylic bond of one and the free C-4 hydroxyl group of the other located in theregion of the enzyme's catalytic groups, thereby favoring glycosylation of the suitably positioned acceptor over solvent water. Hydrolysis is assumed to prevail when only a single substrate molecule or segment binds to alpha amylase with a (1→4)-α- d-glucosidic linkage or glycosylic CF bond positioned at the catalytic center. The present demonstration that glycosyl-transfer reactions can be dominantly expressed by alpha amylases, given an appropriate substrate, illustrates the inadequacy of the usual characterization of these enzymes as hydrolases that produce overwhelming hydrolysis of all substrates