Amino acid substitution reveals the role of V-shape helix on construction of yeast carboxypeptidase Y

Carboxypeptidase Y (CPY) is a vacuolar serine-type protease of yeast Saccharomyces cerevisiae to release wide variety of L-amino acid including proline from C termini of peptides and proteins. According to three-dimensional structure of mature CPY determined crystallographically, there is a characteristic region constructed with two adjacent α-helices, called ‘V-shape helix’, which lies over the active site cavity just like a door. In order to clarify the role of V-shape helix, we attempted to immobilize the V-shape helix by introducing amino acid substitution to create additional disulfide bridge between the V-shape helix and the main body of CPY. Resulting mutant enzymes presented with misfolding or hydrolase activity loss, suggesting that replacement of the residues at or near V-shape helix would influence on the protein folding and enzyme activity. The results of this report demonstrated that the insertion domain including V-shape helix in α/β hydrolasefold serine carboxypeptidases plays a role in the construction of functional enzyme.