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

Narayanan Ramasubbu, Chandran Ragunath, Prasunkumar J. Mishra, Leonard M. Thomas, G. Gyémánt, L. Kandra

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

The nonreducing end of the substrate-binding site of human salivary α-amylase contains two residues Trp58 and Trp59, which belong to β2-α2 loop of the catalytic (β/α)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 Km 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 α-amylase.

Original languageEnglish
Pages (from-to)2517-2529
Number of pages13
JournalEuropean Journal of Biochemistry
Volume271
Issue number12
DOIs
Publication statusPublished - Jun 2004

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Enzyme activity
Amylases
Oligosaccharides
Substrates
Enzymes
Starch
Acarbose
Glucose
Maltose
Human Activities
Cats
Hydrolysis
Binding Sites
Electrons
Mutation
Carrier concentration
Kinetics

Keywords

  • Crystal structure
  • Oligosaccharide hydrolysis
  • Salivary α-amylase
  • Site-directed mutagenesis
  • Subsite engineering

ASJC Scopus subject areas

  • Biochemistry

Cite this

Human salivary α-amylase Trp58 situated at subsite -2 is critical for enzyme activity. / Ramasubbu, Narayanan; Ragunath, Chandran; Mishra, Prasunkumar J.; Thomas, Leonard M.; Gyémánt, G.; Kandra, L.

In: European Journal of Biochemistry, Vol. 271, No. 12, 06.2004, p. 2517-2529.

Research output: Contribution to journalArticle

Ramasubbu, Narayanan ; Ragunath, Chandran ; Mishra, Prasunkumar J. ; Thomas, Leonard M. ; Gyémánt, G. ; Kandra, L. / Human salivary α-amylase Trp58 situated at subsite -2 is critical for enzyme activity. In: European Journal of Biochemistry. 2004 ; Vol. 271, No. 12. pp. 2517-2529.
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abstract = "The nonreducing end of the substrate-binding site of human salivary α-amylase contains two residues Trp58 and Trp59, which belong to β2-α2 loop of the catalytic (β/α)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 Km 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 α-amylase.",
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