title: Refolding of a carboxypeptidase Y folding intermediate in vitro by low-affinity binding of the proregion.
abstract: Efficient folding of carboxypeptidase Y is dependent on the presence of the proregion. Thus, denatured procarboxypeptidase Y, in contrast to the mature enzyme, refolds efficiently in vitro in low ionic strength buffers. Under these conditions denatured mature carboxypeptidase Y forms an inactive, soluble folding intermediate, which has been characterized in the present study. The inactive intermediate can be folded into the active enzyme at a low efficiency (5-10%) by the addition of 0.9 M ammonium sulfate. The refolding is accompanied by pronounced structural changes. As seen for other protease zymogens the isolated proregion from carboxypeptidase Y was found to stimulate refolding without covalent linkage to the mature part. However, the added proregion does not form a stable complex with the native enzyme and requires the presence of 0.9 M ammonium sulfate to exhibit its function. The proregion increases the yield of correctly folded enzyme, and kinetic analysis suggests that this is due to a reduction of the rate of nonproductive folding or aggregation. In addition, the proregion stabilizes carboxypeptidase Y toward thermoinactivation.
authors.affiliation: Department of Yeast Genetics, Carlsberg Laboratory, Copenhagen, Denmark.
authors.forename: J R
authors.initials: JR
authors.lastname: Winther
authors.forename: P
authors.initials: P
authors.lastname: Sørensen
authors.forename: M C
authors.initials: MC
authors.lastname: Kielland-Brandt
mesh_terms: D002268:Carboxypeptidases / Q000737:chemistry* / Q000378:metabolism*; D043402:Cathepsin A; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D011485:Protein Binding; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase