The effects of ligands on the conformation of phosphoglycerate kinase: fluorescence anisotropy decay and theoretical interpretation.
Horse muscle phosphoglycerate kinase (PGK) is a monomer folded into two widely distant domains. In the glycolytic pathway, this enzyme catalyzes the first reaction that produces ATP. It was suggested, by analogy with yeast hexokinase, that a hinge-bending motion may be induced by the binding of specific substrates to the protein. To analyze such a motion, or any structural changes induced by ligand binding, fluorescence anisotropy decay of tryptophan residues in free and liganded PGK was studied. At 293 K, for the free protein and the binary complex with 3-phosphoglycerate, a single correlation time of 26 ns was observed, corresponding to the rotation of the overall protein, whereas upon addition of MgADP, this correlation time decreased to 10 ns. Such a decrease cannot be merely due to a change of the protein's shape and volume. To explain this, it was suggested that the fluorescence anisotropy decay of the PGK-MgADP complex corresponded to the rotation of the only buried tryptophan (Trp 335). The rotational paths of this tryptophan, in the presence and absence of the nucleotide, were established by potential energy minimization calculations. The results indicated that MgADP induces a displacement of helix alpha-13 that decreases the rotational energy barrier of Trp 335 from 16 kcal/mol in the free protein to 8 kcal/mol in the complex.
Mouawad
L
L
Laboratoire d'Enzymologie Physico-chimique et Moléculaire, Groupe de Recherche CNRS associé à l'Université de Paris-Sud, France.
Desmadril
M
M
Perahia
D
D
Yon
J M
JM
Brochon
J C
JC
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001718:Birefringence; D005454:Fluorescence Polarization; D005988:Glyceric Acids / Q000378:metabolism; D006019:Glycolysis; D006736:Horses; D007536:Isomerism; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism
D005988:Glyceric Acids; D008024:Ligands; D046911:Macromolecular Substances; D000244:Adenosine Diphosphate; C005156:3-phosphoglycerate; D014364:Tryptophan; D010735:Phosphoglycerate Kinase