Biologia, Bratislava, 57/Suppl. 11: 171-180, 2002.
ISSN 0006-3088 (Biologia).
Action pattern of a-amylases on modified maltooligosaccharides.
Lili Kandra*, Gyongyi Gyemant & Andras Liptak
Department of Biochemistry, University of Debrecen, Debrecen, P. O. Box 55, H-4010 Hungary; tel.: ++ 36 52 316 666, fax: ++ 36 52 512913, e-mail: firstname.lastname@example.org
* corresponding author
Received: November 26, 2001 / Accepted: February 12, 2002
2-chloro-4-nitrophenyl- (CNP) and 4,6-O-benzylidene-modified 4-nitrophenyl- (Bnl-NP) b-maltooligosaccharides (DP 4-8) were synthesised from cyclodextrins using a chemical procedure. For the preparation of CNP-maltooligosides of longer chain length a new chemoenzymatic procedure was developed using rabbit skeletal muscle glycogen phosphorylase b. These substrates were used for further studies of the action pattern of porcine pancreatic a-amylase (PPA), human salivary a-amylase (HSA) and Bacillus licheniformis a-amylase (BLA). The hydrolysis products and the remaining substrates were separated and quantified by HPLC. Our results suggest at least six subsites in the binding region of HSA; four glycone (-4, -3, -2, -1) and two aglycon binding sites (+1, +2). The binding modes of the benzylidene derivatives indicated a favourable interaction between the Bnl group and subsite (-3) and an unfavourable one with subsite (-4). PPA exhibited a unique pattern of action on CNP-maltooligosaccharides by cleaving maltotriose units from the nonreducing ends and leaving CNP-glycosides, or by cleaving CNP-G2 units from the reducing ends to leave maltooligosaccharides. Modification of the nonreducing end of NP glycosides to give a 4,6-O-benzylidene-D-glucopyranosyl group indicated a favourable interaction between the Bnl group and the subsites (-3) and (-5) but an unfavourable one with subsite (-4), which resulted in a clear shift in the product pattern. The binding region is longer in BLA than in human amylases. Our results suggested the presence of at least eight subsites; five glycone binding sites and three aglycon ones. The binding modes of substrates will be discussed on the basis of the known features of the structures of a-amylases.
Key words: human salivary a-amylase; porcine pancreatic a-amylase; Bacillus licheniformis a-amylase; action patterns; b-maltooligosaccharide glycosides; chemoenzymatic syntheses.