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

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Abstract
CDNA encoding Schizosaccharomyces pombeα‐glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Saccharomyces cerevisiae. The cDNA, 4176 bp in length, included a single ORF composed of 2910 bp encoding a polypeptide of 969 amino‐acid residues with Mr 106 138. The deduced amino‐acid sequence showed a high homology to those of α‐glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the α‐glucosidase family II. By site‐directed mutagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (‐COOH) of the Asp647 residue was for the first time shown to be the most likely proton donor acting as the acid catalyst in the α‐glucosidase of family II. Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (‐COO−) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure.

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