Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe

cDNA encoding Schizosaccharomyces pombe alpha -glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Sacc haromyces cerevisiae. The cDNA, 4176 bp in length, included a single ORF co mposed of 2910 bp encoding a polypeptide of 969 amino-acid residues with M- r 106 138. The deduced amino-acid sequence showed a high homology to those of alpha -glucosidases from molds, plants and mammals. Therefore, the enzym e was categorized into the alpha -glucosidase family II. By site-directed m utagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essenti al in the catalytic reaction. The carboxyl group (-COOH) of the Asp647 resi due was for the first time shown to be the most likely proton donor acting as the acid catalyst in the alpha -glucosidase of family II. Studies with t he chemical modifier conduritol B epoxide suggested that the carboxylate gr oup (-COO-) of the Asp481 residue was the catalytic nucleophile, although t he role of the Glu484 residue remains obscure.