Limited proteolysis of branching enzyme from Escherichia coli

Branching enzyme is involved in determining the structure of starch and gly cogen. 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 be longs 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, depen ding on the type of assay used. Amino acid sequencing showed that the 70-kD a protein lacked 111 or 113 residues at the amino terminal, whereas the car boxy terminal was still intact. We purified this truncated enzyme to homoge neity and analyzed its properties. The enzyme had a three- to fourfold lowe r catalytic efficiency than the native enzyme, whereas the substrate specif icity was unaltered. Furthermore, a branching enzyme with 112 residues dele ted at the amino terminal was constructed by recombinant technology and fou nd to have properties identical to those of the proteolyzed enzyme. (C) 200 0 Academic Press.