Amino acid residues involved in substrate binding and catalysis in an insect digestive beta-glycosidase.
A beta-glycosidase (M(r) 50000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl beta-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.
Marana
S R
SR
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil.
Jacobs-Lorena
M
M
Terra
W R
WR
Ferreira
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002482:Cellulose / Q000031:analogs & derivatives* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003912:Dextrins / Q000378:metabolism; D017344:Genes, Insect; D006027:Glycosides / Q000378:metabolism; D006868:Hydrolysis; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D003912:Dextrins; D006027:Glycosides; D019476:Insect Proteins; D002482:Cellulose; C068994:cellodextrin; D001617:beta-Glucosidase