The identification of the catalytic nucleophiles of two β-galactosidases from glycoside hydrolase family 35 Original Research Article

The β-galactosidases from Xanthomonas manihotis (β-Gal Xmn) and Bacillus circulans (β-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-β-d-galactopyranoside was shown to inactivate β-Gal Xmn and β-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 Glu260 and Glu233 as the catalytic nucleophiles involved in the formation of the glycosyl–enzyme intermediate during catalysis by β-Gal Xmn and β-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.