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 journalArticle

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

Fingerprint

Glycoside Hydrolases
Glucosides
Catalysis
Hydroxyl Radical
Insects
Carbodiimides
Amino Acids
Spodoptera
Nucleophiles
Sequence Alignment
Enzyme activity
Substrates
Glycosides
Protons
Hydrogen
Catalytic Domain
Hydrogen bonds
Enzymes
Experiments
cellodextrin

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Structural Biology
  • Biophysics

Cite this

Amino acid residues involved in substrate binding and catalysis in an insect digestive β-glycosidase. / Marana, Sandro R.; Jacobs-Lorena, Marcelo; Terra, Walter R.; Ferreira, Clélia.

In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, Vol. 1545, No. 1-2, 09.02.2001, p. 41-52.

Research output: Contribution to journalArticle

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