Restoration of the activity of active-site mutants of the hyperthermophilic beta-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles.
The beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids: Glu387, which acts as the nucleophile of the reaction, and Glu206, which acts as the general acid/base catalyst. The activities of inactive mutants of the catalytic nucleophile Glu387Ala/Gly were restored by externally added nucleophiles. Sodium azide and sodium formate were used as external nucleophiles and the products of their reaction were characterized. Glu387Ala/Gly mutants were reactivated with 2, 4-DNP-beta-Glc substrate and the Glu387Gly mutant showed recovered activity, with the same nucleophiles, also on 2-NP-beta-Glc. The reaction catalyzed by the Glu387Gly mutant proceeded differently depending on the type of externally added nucleophile. Sodium azide restored the catalytic activity of the mutant by attacking the alpha-side of the anomeric carbon of the substrates, thereby yielding an inverting glycosidase. Sodium formate promoted the opposite behavior (retaining) in the mutant, producing 3-O-beta-linked disaccharide derivative of the substrates. A possible role of sodium formate as a biomimicking agent in replacing the natural nucleophile Glu387 is also discussed.
Moracci
M
M
Institute of Protein Biochemistry and Enzymology, Consiglio Nazionale delle Ricerche, Naples, Italy.
Trincone
A
A
Perugino
G
G
Ciaramella
M
M
Rossi
M
M
D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D020134:Catalytic Domain / Q000187:drug effects / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005561:Formates / Q000494:pharmacology; D005959:Glucosidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism / Q000494:pharmacology*; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019810:Sodium Azide / Q000494:pharmacology; D016944:Sulfolobus / Q000201:enzymology*
C116347:2,4-dinitrophenyl-2-deoxy-2-fluoroglucoside; D005561:Formates; D005960:Glucosides; D005973:Glutamine; C030544:formic acid; C070512:2',4'-dinitrophenylglucopyranoside; C018616:2-nitrophenyl-beta-D-glucopyranoside; D018698:Glutamic Acid; D019810:Sodium Azide; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase; D005998:Glycine