Stability domains, substrate-induced conformational changes, and hinge-bending motions in a psychrophilic phosphoglycerate kinase. A microcalorimetric study.
The cold-active phosphoglycerate kinase from the Antarctic bacterium Pseudomonas sp. TACII18 exhibits two distinct stability domains in the free, open conformation. It is shown that these stability domains do not match the structural N- and C-domains as the heat-stable domain corresponds to about 80 residues of the C-domain, including the nucleotide binding site, whereas the remaining of the protein contributes to the main heat-labile domain. This was demonstrated by spectroscopic and microcalorimetric analyses of the native enzyme, of its mutants, and of the isolated recombinant structural domains. It is proposed that the heat-stable domain provides a compact structure improving the binding affinity of the nucleotide, therefore increasing the catalytic efficiency at low temperatures. Upon substrate binding, the enzyme adopts a uniformly more stable closed conformation. Substrate-induced stability changes suggest that the free energy of ligand binding is converted into an increased conformational stability used to drive the hinge-bending motions and domain closure.
Zecchinon
Laurent
L
Laboratory of Biochemistry, University of Liège, Institute of Chemistry B6a, B-4000 Liège-Sart Tilman, Belgium.
Oriol
Annick
A
Netzel
Ulrike
U
Svennberg
Julie
J
Gerardin-Otthiers
Nicole
N
Feller
Georges
G
D000838:Anions; D001665:Binding Sites; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002412:Cations; D007700:Kinetics; D008968:Molecular Conformation; D016296:Mutagenesis; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D010735:Phosphoglycerate Kinase / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014306:Trioses / Q000737:chemistry; D014466:Ultraviolet Rays
D000838:Anions; D002412:Cations; D009711:Nucleotides; D011994:Recombinant Proteins; D014306:Trioses; D010735:Phosphoglycerate Kinase