title: Flexibility and folding of phosphoglycerate kinase.
abstract: Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.
authors: Laboratoire d'enzymologie physico-chimique et moléculaire, Unité de Recherche du CNRS, associée à l'Université de Paris-Sud, Orsay, France.
authors: J M
authors: JM
authors: Yon
authors: M
authors: M
authors: Desmadril
authors: J M
authors: JM
authors: Betton
authors: P
authors: P
authors: Minard
authors: N
authors: N
authors: Ballery
authors: D
authors: D
authors: Missiakas
authors: S
authors: S
authors: Gaillard-Miran
authors: D
authors: D
authors: Perahia
authors: L
authors: L
authors: Mouawad
mesh_terms: D000818:Animals; D001665:Binding Sites; D006736:Horses; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D010735:Phosphoglycerate Kinase