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 Bac illus circulans (beta -Gal-3 Bcir) are retaining glycosidases that hydrolyz e glycosidic bonds through a double displacement mechanism involving a cova lent glycosyl-enzyme intermediate. The mechanism-based inactivator 2,4-dini trophenyl 2-deoxy-2-fluoro-beta -D-galactopyranoside was shown to inactivat e 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, resp ectively. Peptic digestion of these labeled enzymes and analysis by LC-MS i dentified Glu(260) and Glu(233) as the catalytic nucleophiles involved in t he formation of the glycosyl-enzyme intermediate during catalysis by beta - Gal Xmn and beta -Gal-3 Bcir, respectively. These findings confirm the prev ious prediction of the position of these residues based on primary sequence similarities to other members of the glycoside hydrolase family 35. (C) 20 01 Elsevier Science Ltd. All rights reserved.