A proton-NMR study of a site-directed mutation (His388----Glu) in the interdomain region of yeast phosphoglycerate kinase. Implications for domain movement.
Proton NMR has been used to study a site-directed mutant of yeast phosphoglycerate kinase in which the interdomain residue His388 has been replaced by a glutamine residue. Using 1H-NMR spectroscopy, it was found that 3-phosphoglycerate binding to the mutant protein induces different conformational effects to those observed for the wild-type enzyme. These differences are not only located at the 3-phosphoglycerate binding site but are also seen as long-range effects at the surface of the protein. Measurements of the Kd for 3-phosphoglycerate from the NMR experiments show that the mutant enzyme has a 30-times reduced affinity for this substrate as compared with the wild-type enzyme. These data are consistent with the suggestion that an aromatic residue at position 388 plays an important role in the proposed hinge-bending mechanism.
Graham
H C
HC
Inorganic Chemistry Laboratory, University of Oxford, UK.
Williams
R J
RJ
Littlechild
J A
JA
Watson
H C
HC
D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D005988:Glyceric Acids / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D005988:Glyceric Acids; D005973:Glutamine; D006639:Histidine; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase