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been used to study a site-directed mutant of yeast phosphoglycerate kinase in which the interdomain residue His388 has been replaced by a glutamine residue.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 H-1-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 K(d) 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.