The identification of the catalytic nucleophiles of two beta-galactosidases from glycoside hydrolase family 35.
The beta-galactosidases from Xanthomonas manihotis (beta-Gal Xmn) and Bacillus circulans (beta-Gal-3 Bcir) are retaining glycosidases that hydrolyze glycosidic bonds through a double displacement mechanism involving a covalent glycosyl-enzyme intermediate. The mechanism-based inactivator 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside was shown to inactivate beta-Gal Xmn and beta-Gal-3 Bcir through the accumulation of 2-deoxy-2-fluorogalactosyl enzyme intermediates with half lives of 40 and 625 h, respectively. Peptic digestion of these labeled enzymes and analysis by LC-MS identified Glu(260) and Glu(233) as the catalytic nucleophiles involved in the formation of the glycosyl-enzyme intermediate during catalysis by beta-Gal Xmn and beta-Gal-3 Bcir, respectively. These findings confirm the previous prediction of the position of these residues based on primary sequence similarities to other members of the glycoside hydrolase family 35.
Blanchard
J E
JE
Department of Chemistry, Protein Engineering Network of Centres of Excellence of Canada, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
Gal
L
L
He
S
S
Foisy
J
J
Warren
R A
RA
Withers
S G
SG
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D014974:Xanthomonas / Q000201:enzymology / Q000235:genetics; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
D017931:DNA Primers; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase