title: A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the alpha/beta hydrolase fold, acts as a pH-dependent protein-stabilizing element.
abstract: Serine endopeptidases of the chymotrypsin family contain a salt bridge situated centrally within the active site, the acidic component of the salt bridge being adjacent to the catalytically essential serine. Serine carboxypeptidases also contain an acidic residue in this position but it interacts through a short hydrogen bond, probably of low-barrier type, with another acidic residue, hence forming a "glutamic acid bridge." In this study, the residues constituting this structural element in carboxypeptidase Y have been replaced by site-specific mutagenesis. It is demonstrated that the glutamic acid bridge contributes significantly to the stability of the enzyme below pH 6.5 and has an adverse effect at pH 9.5. Carboxypeptidase WII from wheat contains 2 such bridges, and it is more stable than carboxypeptidase Y at acidic pH.
authors.affiliation: Department of Chemistry, Carlsberg Laboratory, Copenhagen, Denmark.
authors.forename: U H
authors.initials: UH
authors.lastname: Mortensen
authors.forename: K
authors.initials: K
authors.lastname: Breddam
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043402:Cathepsin A; D017124:Conserved Sequence; D004271:DNA, Fungal / Q000235:genetics; D004795:Enzyme Stability; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D004271:DNA, Fungal; D005971:Glutamates; D029701:Saccharomyces cerevisiae Proteins; D018698:Glutamic Acid; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase