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

A β-glycosidase (M_r 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 (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 β-glycosides and K_i 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.