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

A beta -glycosidase (M-r 50 000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl P-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 (pK(a1) = 4.9 and pK(a2) = 7.5). Effect of pH on carbodiimide inactivation indicates that the pK(a) 7.5 group is a carboxyl. k(cat) and K-m values were obtained for different p-nitrophenyl beta -glycosides and K-i values were determined for a range of alkyl beta -glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature beta -glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E-187 (proton donor) and E-399 (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. (C) 2001 Elsevier Science B.V. All rights reserved.