Unequivocal Identification of Asp-214 as the Catalytic Nucleophile of Saccharomyces cerevisiae α-Glucosidase Using 5-Fluoro Glycosyl Fluorides

Abstract

Yeast ␣-glucosidase is a member of a sequence-related family of ␣-glycosidases (Family 13) that includes important digestive ␣-amylases and ␣-glucosidases. These enzymes catalyze the hydrolysis of ␣-linked oligosaccharides by a two-step mechanism involving a glycosylenzyme intermediate. This Glycosyl hydrolases of Family 13 (the ␣-amylase family (1, 2)), which include ␣-amylases and some ␣-glucosidases and oligo-1,6-glucosidases, are important enzymes involved in the digestion of starch and other ␣-linked oligosaccharides in bacteria, plants, and animals. These glycosyl hydrolases cleave the glycosidic bond with net retention of configuration, thus are retaining enzymes that function via a two-step, double displacement mechanism. The first step involves the attack of an enzymic nucleophile at the anomeric center of the sugar, along with general acid catalysis to facilitate loss of the leaving group, leading to the formation of a ␤-D-glycosyl enzyme intermediate. This intermediate can then undergo hydrolysis in a second step, with general base catalysis to facilitate attack of water, resulting in cleavage of the glycosidic bond with net retention of anomeric configuration (see Scheme 2). The active site of these enzymes is known to include a trio of conserved carboxylic acids (3-7). Presumably, one residue functions as the catalytic nucleophile, one as a general acid/base, and the third possibly affords additional stabilization of developing positive charge or modulates the ionization behavior of the other catalytic residues.Considerable progress has been made toward elucidating the roles of these carboxylates in Family 13 enzymes, involving kinetic analyses of site-specific mutants (Ref. 8 and references therein) and x-ray crystallography (3-6). Labeling studies have also been useful. For example, an aspartate that functions as a catalytic nucleophile in a sucrose, ␣-glucan glycosyltransferase, has been identified in Streptococcus ␣-glucosyltransferase by denaturation trapping using radiolabeled sucrose (9). Similarly, in a second group of ␣-glycosyl hydrolases (Family 31), Asp-505 and Asp-1394 have been identified as active site residues in each of the two homologous active sites in the sucraseisomaltase complex by affinity labeling with conduritol B epoxide and have been suggested to be the catalytic nucleophiles (10). The asparagine mutant of the equivalent Asp-518 in human lysosomal ␣-glucosidase exhibits an estimated 6% of wildtype activity, indicating that this residue likely plays an important role in catalysis (11), although this 16-fold reduction in activity is much less than the 10 5 -fold reduction observed upon analysis of nucleophile mutants in mechanistically similar ␤-glycosidases (12-14), leaving some doubt concerning the role of Asp-518. This activity was not, however, based on rigorously purified enzyme, and the assay method used in this study could not distinguish this level of activity from background. Furthermore, lysosomal ␣-glucosidase and sucrase-isomaltase (Family 31) and the ␣-amylases ...

Functional Characterization and Membrane Topology of Escherichia coli WecA, a Sugar-Phosphate Transferase Initiating the Biosynthesis of Enterobacterial Common Antigen and O-Antigen Lipopolysaccharide

“…Replacement of these residues with other amino acids affected the function of WecA, as demonstrated by the lack of in vitro enzymatic activity or reduced in vitro enzymatic activity of the mutated proteins, which could not mediate O-antigen LPS production in vivo (4). Aspartic acids have nucleophilic side chains, which may be involved in binding divalent metal ions (most commonly Mn 2ϩ or Mg 2ϩ ) or in catalysis (23,47,59), as shown previously for other glycosyl and prenyl transferases (10,33,34,50). We hypothesized that Asp90, Asp91, Asp156, and Asp159 are important for the catalytic activity of WecA (4).…”

Identification and Molecular Characterization of the First α-Xylosidase from an Archaeon

“…It has been suggested that the aspartic acid residues identified in sucrase-isomaltase and lysosomal ␣-glucosidase constitute the catalytic nucleophile in this family. However, as already noticed, the assignment of the role played in catalysis by these residues remains equivocal (40). In fact, conduritol epoxide derivatives occasionally have labeled active site residues different from the nucleophile, and the levels of residual enzymatic activity of the human lysosomal ␣-glucosidase mutant Asp-518 3 Asn was incompatible with the essential function proposed (39).…”

Characterization of a New Multigene Family Encoding Isomaltases in the Yeast Saccharomyces cerevisiae, the IMA Family

“…In particular, Ima1p and Ima2p exhibit the same GEMQH consensus, whereas the Ima3p and Ima4p consensus is GEMRH, and the Ima5p consensus diverges from the others with the GEVGI sequence. The Asp residue in region II has been described as the catalytic nucleophile of ␣-glucosidases, whereas Glu (region III) and Asp (region IV) are likely involved in acid/base catalysis (31). The only pertinent difference between the five putative ␣-glucosidases encoded by YGR287c, YIL172c (id.…”