title: Glutamate-459 is important for Escherichia coli branching enzyme activity.
abstract: The branching enzyme belongs to the amylolytic family, a group of enzymes that cleave and/or transfer chains of glucan. The amylolytic enzymes are homologous and all contain four conserved regions, proposed to contain the active site. By primary structure analysis, a conserved position unique to branching enzymes has been identified. This residue, which is either Asp or Glu, depending on the species, is located immediately after the putative catalytic Glu-458 (Escherichia coli numbering). Branching enzymes differ from other amylolytic enzymes in having this acid pair, and we asked if this motif could be essential for branching enzyme action. We used site-directed mutagenesis of the Glu-459 residue in the E. coli branching enzyme in order to determine the significance of the conserved Asp/Glu in branching enzymes. A substitution of Glu-459 to Asp resulted in increased specific activity compared to wild-type, suggesting that the mutation had created a more efficient enzyme. Changing Glu-459 to Ala, Lys, or Gln lowered the specific activities and altered the preferred substrate from amylose to amylopectin.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
authors.forename: K
authors.initials: K
authors.lastname: Binderup
authors.forename: J
authors.initials: J
authors.lastname: Preiss
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics; D006003:Glycogen / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D010940:Plant Proteins; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D006003:Glycogen; D015061:1,4-alpha-Glucan Branching Enzyme; D008239:Lysine; D000409:Alanine