title: The slow-refolding step of phosphoglycerate kinase as monitored by pulse proteolysis.
abstract: The kinetics of refolding of yeast phosphoglycerate kinase were studied by following the variation in circular dichroism at 218 nm, the recovery of enzyme activity, and the susceptibility to proteolysis by trypsin and V8-protease. A very rapid phase followed by a slower one was detected by circular dichroism, which revealed the formation of secondary structures. The slower phase, with a macroscopic rate constant of 0.35 min-1, was also detected by the susceptibility of the enzyme to both proteases. It was shown that cleavage sites located in the hinge region, in a part of the C-domain and, to a lesser extent, in a region of the N-domain, which are accessible in the intermediate state, became inaccessible during the slow-refolding step of the molecule. These results demonstrate, on the one hand, the role of domains as folding intermediates, and, on the other hand, the locking of the domain structure and the domain pairing that occurs during the slow-refolding step with a rate constant of 0.35 min-1. The return of the enzyme activity occurred in a slower last step upon conformational readjustments induced by domain interactions.
authors.affiliation: Laboratoire d'Enzymologie Physico-Chimique et Moléculaire, Université de Paris-sud, Orsay, France.
authors.forename: J M
authors.initials: JM
authors.lastname: Betton
authors.forename: D
authors.initials: D
authors.lastname: Missiakas
authors.forename: J M
authors.initials: JM
authors.lastname: Yon
mesh_terms: D002942:Circular Dichroism; D015151:Immunoblotting; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012697:Serine Endopeptidases; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D010735:Phosphoglycerate Kinase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin