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

Abstract

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.

Carboxypeptidase Y activity and maintenance is modulated by a large helical structure

“…Indeed, some amino acid residues at the V-shape helix comprise part of those substrate-binding sites. Our previous findings support the idea that the V-shape helix participates in the catalysis because the replacement of the residues at or near the V-shape helix influences the hydrolase activity of CPY [14]. To further elucidate this function of the V-shape helix, dynamic analyses, such as molecular dynamic simulations or kinetic analyses, would be required.…”

“…Sequence data of CPY, prc1/YMR297W (SGD ID: SGD: S000004912), were obtained from the Saccharomyces Genome Database (http://www.yeastgenome.org/). The recombinant wild-type CPY expression vector was prepared as described [14]: The prc1 fragment encoding full-length wildtype CPY was prepared from yeast genomic DNA extracted from yeast wild-type strain using PCR amplification with primers containing a SmaI or XhoI restriction site for the 5 0 -and 3 0 -termini, respectively. This fragment was inserted into the pGEM Ò -T Easy Vector (Promega, Madison, WI, USA) to construct the cloning vector, which was digested with SmaI and XhoI to generate an insert fragment.…”