title: Role of the C-terminal helix in the folding and stability of yeast phosphoglycerate kinase.
abstract: In order to determine the role of the C-terminal helix in the folding and stability of yeast phosphoglycerate kinase, a mutant deleted of the 12 C-terminal residues (PGK delta 404-415) was constructed. This mutant folds in a conformation very similar to that of the wild-type protein, but exhibits a very low activity (0.1% of that of the wild-type enzyme). The main structural effect of the deletion of the C-terminal helix is an increase in flexibility of the whole protein and a decrease in stability by about 5 kcal/mol. The structural properties of the truncated protein are very similar, at least qualitatively, to those in the isolated domains. The accessibility of the thiol group of Cys 97 is identical to that in the isolated N-domain. The large solvent effect on the tryptophan fluorescence in the native protein at very low concentration of denaturant reveals an increase of flexibility of the C-domain, similar to that observed on the isolated C-domain. NMR measurements show that the pH dependence of His C2H and C4H chemical shifts in the truncated protein perfectly matches those of the isolated domains. The addition of the missing peptide provokes a 40-fold increase in enzyme activity at saturation. A dissociation constant of 80 microM was determined. This peptide, which displays a random structure in solution, folds in a helical structure in the region 405-410 as assessed by TRNOESY. All these results show that the C-terminal part of yeast phosphoglycerate kinase is not necessary for most of the initial folding steps but acts to lock the C-domain on the N-domain, thus ensuring the expression of full enzyme activity. Without this sequence, the protein has the sum of the properties of the two isolated domains.
authors: Laboratoire d'Enzymologie Physicochimique et Moléculaire, Université de Paris-Sud, Orsay, France.
authors: M
authors: M
authors: Ritco-Vonsovici
authors: B
authors: B
authors: Mouratou
authors: P
authors: P
authors: Minard
authors: M
authors: M
authors: Desmadril
authors: J M
authors: JM
authors: Yon
authors: M
authors: M
authors: Andrieux
authors: E
authors: E
authors: Leroy
authors: E
authors: E
authors: Guittet
mesh_terms: D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000648:ultrastructure; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D006146:Guanidines; D010446:Peptide Fragments; D010735:Phosphoglycerate Kinase; D019791:Guanidine; D003545:Cysteine