Human salivary α‐amylase Trp58 situated at subsite −2 is critical for enzyme activity

Abstract

The nonreducing end of the substrate-binding site of human salivary a-amylase contains two residues Trp58 and Trp59, which belong to b2-a2 loop of the catalytic (b/a) 8 barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k cat , an increase in K m and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites )2, )3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites )2 and )3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary a-amylase.Keywords: salivary a-amylase; site-directed mutagenesis; subsite engineering; oligosaccharide hydrolysis; crystal structure.a-Amylases (a-1,4-D-glucan glucanohydrolases, EC 3.2.1.1) are endoglucanases, widely distributed in all three domains of life (Bacteria, Archaea and Eucarya), and catalyze reactions such as hydrolysis and transglycosylation of polysaccharides [1,2]. These enzymes, belonging to the glycoside hydrolase family 13 [3], possess very low overall sequence similarity among the various members; nonetheless, in four small regions around the active site, the members exhibit a strong sequence similarity [4][5][6] and harbor the (b/a) 8 barrel topology [7]. This small number of conserved but critical short regions whose residues are lined up along the surface of a deep cleft carries out substrate binding and catalysis in a-amylases [2].In humans, a-amylase is present in both salivary and pancreatic secretions; the overall primary sequences of the pancreatic and salivary a-amylases are highly homologous, and exhibit a high level of structural similarity [8,9]. Human salivary a-amylase (HSAmy) is monomeric, calcium binding protein with a single polypeptide chain of 496 amino acids [9]. The structure of HSAmy consists of three domains: domain A (residues 1-99, 170-404), domain B (residues 100-169) and domain C (residues 405-496). The domain A adopts a (b/a) 8 barrel structure bearing three catalytic residues Asp197, Glu233 an...

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“…The active site of human saliva amylase consists of seven subsites ( -4, -3, -2, -1, +1, +2, +3) and the catalytic site is located between subsites ( -1) and (+1) [31]. The nonreducing end of the substrate-binding site of human salivary aamylase contains two residues Trp58 and Trp59, which belong to b2-a2 loop of the catalytic (b/a)(8) barrel [32]. Trp58 and Trp59 are both located near the subsites ( -3)/ (-2) [31].…”

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