Examination of the active sites of human salivary alpha-amylase (HSA)

The action pattern of human salivary amylase (HSA) was examined by utilisin g as model substrates 2-chloro-4-nitrophenyl (CNP) beta -glycosides of malt ooligosaccharides of dp 4-8 and some 4-nitrophenyl (NP) derivatives modifie d at the nonreducing end with a 4,6-O-benzylidene (Bnl) group. The product pattern and cleavage frequency were investigated by product analysis using HPLC. The results revealed that the binding region in HSA is longer than fi ve subsites usually considered in the literature and suggested the presence of at least six subsites; four glycone binding sites (- 4, - 3, - 2, - 1) and two aglycone binding sites (+ 1, + 2). In the ideal arrangement, the si x subsites are filled by a glucosyl unit and the release of maltotetraose ( G(4)) from the nonreducing end is dominant. The benzylidene group was also recognisable by subsites (- 3) and ( - 4). The binding modes of the benzyli dene derivatives indicated a favourable interaction between the Bnl group a nd subsite (- 3) and an unfavourable one with subsite (- 4). Thus, subsite (- 4) must be more hydrophylic than hydrophobic. As compared with the actio n of porcine pancreatic alpha -amylase (PPA) on the same substrates, the re sults showed differences in the three-dimensional structure of active sites of HSA and PPA. (C) 2000 Elsevier Science Ltd. All rights reserved.