Abstract
Yeast carboxypeptidase Y (CPY) is a serine protease with broad substrate specificity. Structurally, CPY belongs to the α/β hydrolase fold family and contains characteristic large helices, termed the V‐shape helix, above the active site cavity. Four intramolecular disulfide bonds are located in and around the V‐shape helix. In this study, mutant CPYs were constructed in which one of these disulfide bonds was disrupted. Mutants lacking the C193–C207 bond located at the beginning of the V‐shape helix aggregated easily, while mutants lacking the C262–C268 bond located at the end of the V‐shape helix displayed decreased hydrolytic activity. The results indicate that the V‐shape helix is involved in CPY catalysis and in maintenance of its conformation.
Highlights
Yeast carboxypeptidase Y (CPY) is a serine protease with broad substrate specificity
The results indicate that the disruption of C262–C268 affects the catalytic activity of CPY, where anilidase activity is more susceptible than peptidase activity
The insertion domain in lipases and acetylcholinesterases, which is present in most members of the a/b hydrolase fold family, has a unique catalytic role [7–10]
Summary
Yeast carboxypeptidase Y (CPY) is a serine protease with broad substrate specificity. CPY belongs to the a/b hydrolase fold family and contains characteristic large helices, termed the V-shape helix, above the active site cavity. Analysis of the structural anatomy of CPY using computer graphics revealed that mCPY has a typical a/b hydrolase fold pattern with two domains: a core domain (1–179 and 318–421) and an ‘insertion’ domain (180–317; Fig. 1A). The insertion domain contains helices that are attached to the conserved core folding topology (Fig. 1B). This helical insertion topology is often found in the a/b hydrolase fold family. Examination of the crystal structure of mCPY has revealed that the V-shape helix juts out from the core domain, reminiscent of a bird wing.
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