Conformational changes in yeast phosphoglycerate kinase upon ligand binding: fluorescence of a linked probe and chemical reactivity of genetically introduced cysteinyl residues.
The effects of ligands on the conformation of yeast phosphoglycerate kinase were explored by introducing cysteinyl residues at different positions in the molecule by site-directed mutagenesis. Thus several mutants were constructed, each containing a unique cysteinyl residue. Neither the conformation nor the enzyme activity was affected by the substitutions. The reactivity of the thiol groups and the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine covalently linked to these thiols were used to monitor the conformational changes induced upon ligand binding. It was found that the observed changes mainly involve the part of the protein located in the cleft, particularly the environment of residues 35 and 183. No alteration was observed on the external side of the protein. Only 3-Phosphoglycerate induced these conformational changes. However, when the fluorescent probe was attached to residue 377, the binding of the two substrates was required to induce a modification in the fluorescence of the probe. These results indicate that the substrates separately or together induce discrete molecular motions in phosphoglycerate kinase.
Desmadril
M
M
Laboratorie d'Enzymologie physico-chimique et moléculaire, Groupe de Recherche du Centre National de la Recherche Scientifique associé à l'Université de Paris-Sud, Orsay, France.
Minard
P
P
Ballery
N
N
Gaillard-Miran
S
S
Hall
L
L
Yon
J M
JM
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes; D005988:Glyceric Acids / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013439:Sulfhydryl Reagents
D005456:Fluorescent Dyes; D005988:Glyceric Acids; D008024:Ligands; D009282:Naphthalenesulfonates; D013439:Sulfhydryl Reagents; C048876:1,5-AEDANS; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; D003545:Cysteine