Structure-function relationships in human salivary α-amylase: Role of aromatic residues

The active site of human salivary α-amylase consists of six subsites (-4 through +2). The aromatic residues, Trp58, Trp59, Tyr151 and Phe256 are prominently located at the active site (immediately next to the subsites -1 and +1 where cleavage occurs). We focused on determining the role of these aromatic residues in the structure-function relationships of salivary α-amylase. While previous studies had showed that glucose production was significant when residues Trp58 and Trp59 were mutated, results presented here show that at low concentrations of substrates, glucose production by Trp58 mutants was even higher. Unlike the wild-type enzyme, these Trp mutants acquired the ability to hydrolyze maltose and maltotriose, suggesting that impaired binding at subsites -2 and -3 might have increased the productive binding modes for the smaller oligosaccharides. The increase in transglycosylation activity by the mutant Y151M was further studied by analyzing the crystal structure of a complex between acarbose and Y151M. The mutant Y151M also modified acarbose in the crystal; however, only glucose.acarviosine was fitted at the active site (subsites -2, -1, +1). The aromatic residues, Phe256 and Phe295 prominent at the chloride-binding site (about 5Å away from the subsites -1 and +1) did not exhibit any significant effect in chloride binding; however, Phe256 helps orient a water chain that may be involved in the starch hydrolysis. From these studies, we conclude that aromatic residues in the vicinity of the active site of human salivary α-amylase play a crucial role in substrate binding, enzyme activity and catalysis.