title: Limited proteolysis of branching enzyme from Escherichia coli.
abstract: Branching enzyme is involved in determining the structure of starch and glycogen. It catalyzes the formation of branch points by cleavage and transfer of alpha-1,4-glucan chains to alpha-1,6 branch points. Branching enzyme belongs to the amylolytic family of enzymes containing four conserved regions in a central (alpha/beta)8-barrel. Limited proteolysis of the branching enzyme from Escherichia coli (84 kDa) by proteinase K produced a truncated protein of 70-kDa, which still retained 40-60% of branching activity, depending on the type of assay used. Amino acid sequencing showed that the 70-kDa protein lacked 111 or 113 residues at the amino terminal, whereas the carboxy terminal was still intact. We purified this truncated enzyme to homogeneity and analyzed its properties. The enzyme had a three- to fourfold lower catalytic efficiency than the native enzyme, whereas the substrate specificity was unaltered. Furthermore, a branching enzyme with 112 residues deleted at the amino terminal was constructed by recombinant technology and found to have properties identical to those of the proteolyzed enzyme.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
authors.forename: K
authors.initials: K
authors.lastname: Binderup
authors.forename: R
authors.initials: R
authors.lastname: Mikkelsen
authors.forename: J
authors.initials: J
authors.lastname: Preiss
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016296:Mutagenesis; D010957:Plasmids; D013997:Time Factors
chemical_list: D015061:1,4-alpha-Glucan Branching Enzyme; D019286:Endopeptidase K