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 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 alpha-glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the alpha-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 alpha-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.
Okuyama
M
M
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
Okuno
A
A
Shimizu
N
N
Mori
H
H
Kimura
A
A
Chiba
S
S
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011522:Protons; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000378:metabolism
D018076:DNA, Complementary; D011522:Protons; D012333:RNA, Messenger; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D000520:alpha-Glucosidases