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

Abstract

The action pattern of human salivary amylase (HSA) was examined by utilising as model substrates 2-chloro-4-nitrophenyl (CNP) β-glycosides of maltooligosaccharides of dp 4–8 and some 4-nitrophenyl (NP) derivatives modified 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 five 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 six 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 benzylidene derivatives indicated a favourable interaction between the Bnl group and subsite (−3) and an unfavourable one with subsite (−4). Thus, subsite (−4) must be more hydrophylic than hydrophobic. As compared with the action of porcine pancreatic α-amylase (PPA) on the same substrates, the results showed differences in the three-dimensional structure of active sites of HSA and PPA.