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cDNA encoding Schizosaccharomyces pombe a-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 M r 106 138. The deduced amino-acid sequence showed a high homology to those of a-glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the a-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 a-glucosidase of family II. Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (-COO 2 ) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure.Keywords: a-glucosidase; proton donor in catalytic reaction; site-directed mutagenesis.a-Glucosidase (EC 3.2.1.20, a-d-glucoside glucohydrolase) catalyzes the liberation of a-glucose from nonreducing terminals of various glucoside substrates. Many a-glucosidases, which show broad substrate specificities [1], have been purified from mammals, plants and microorganisms. They are divided into two groups, a-glucosidase families I and II, based on their amino-acid sequences [2]; families I and II belong to families 13 and 31 of the glycoside hydrolases, respectively [3,4]. Family I, for example, Saccharomyces carlsbergensis (Saccharomyces cerevisiae) a-glucosidase [5] and Bacillus cereus a-glucosidase [6], has the four regions that are conserved among the amino-acid sequences of a-amylases [7]. On the other hand, enzymes such as human lysosomal [8], Aspergillus niger [9,10], and sugar beet [11] a-glucosidases, belong to a-glucosidase family II, and do not have the four conserved regions. There is no similarity in the overall primary structures of a-glucosidase and a-amylase, or of the a-glucosidase families I and II. The threedimensional structure of a-glucosidase was solved only in B. cereus [12], and the catalytic amino-acid residues were deduced from the structure of the catalytic regions conserved in a-amylase [13]. As for the a-glucosidases in family II, however, only one catalytic amino-acid residue was confirmed by chemical modification with conduritol B epoxide (1-d-1,2-anhydro-myo-inositol; CBE). CBE is a known mechanism-based inactivator of a-glucosidase [14±19] that binds specifically to the essential b-carboxyl group of the Asp residue at the active site, which is the catalytic nucleophile (-COO 2 ): the Asp505 and Asp1349 residues of rabbit intestinal sucrase±isomaltase complex [15,16], the Asp518 of human lysosomal a-glucosidase [17], the Asp469 of sugar beet a-glucosidase [18], and the Asp224 of the P2 subunit of A. niger a-glucosidase [19]. The previous kinetic and chemical modification analysis suggested that carboxyl...

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Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an alpha-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey alpha-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.