Amino acid residues involved in substrate binding and catalysis in an insect digestive β-glycosidase

Sandro R. Marana, Marcelo Jacobs-Lorena, Walter R. Terra, Clélia Ferreira

Research output: Contribution to journalArticlepeer-review

Abstract

A β-glycosidase (Mr 50 000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl β-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pKa1=4.9 and pKa2=7.5). Effect of pH on carbodiimide inactivation indicates that the pKa 7.5 group is a carboxyl. kcat and Km values were obtained for different p-nitrophenyl β-glycosides and Ki values were determined for a range of alkyl β-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature β-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E187 (proton donor) and E399 (nucleophile); (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite.

Original languageEnglish (US)
Pages (from-to)41-52
Number of pages12
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1545
Issue number1-2
DOIs
StatePublished - Feb 9 2001
Externally publishedYes

Keywords

  • Enzyme specificity
  • Glycoside hydrolase
  • Insect
  • β-Glucosidase
  • β-Glycosidase

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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