title: Investigating interdomain region mutants Phe194----Leu and Phe194----Trp of yeast phosphoglycerate kinase by 1H-NMR spectroscopy.
abstract: Site-directed mutagenesis has been used to produce two mutant forms of yeast phosphoglycerate kinase in which the interdomain residue Phe194 has been replaced by a leucine or tryptophan residue. Using 1H-NMR spectroscopy, it was found that the mutations at position 194 induce both local and long-range conformational changes in the protein. It was also found that 3-phosphoglycerate binding to the mutant proteins induces somewhat different conformational effects to those observed for wild-type phosphoglycerate kinase. The affinity of mutant Phe194----Trp for 3-phosphoglycerate was found by NMR studies to be unaffected, while the affinity of Phe194----Leu mutant is reduced by about threefold relative to the wild-type enzyme. The binding of ATP at the electrostatic site of the mutant proteins is also seen to be about three times weaker for the Phe194----Leu mutant when compared to wild-type or Phe194----Leu mutant. These results are discussed in the light of the kinetic studies on the mutants which show that for Phe194----Leu mutant the Km values for both 3-phosphoglycerate and ATP, as well as the Vmax, are decreased relative to the wild-type enzyme, while for mutant Phe194----Trp, the Km values for 3-phosphoglycerate and ATP are unaffected and the Vmax is decreased when compared to wild-type enzyme. Kinetic studies in the presence of sulphate reveal that the anion activation is greater for mutant Phe194----Trp and less for mutant Phe194----Leu, relative to that observed for wild-type phosphoglycerate kinase. The NMR data, taken together with the kinetic data, are consistent with the on and off rates of 3-phosphoglycerate being affected by the mutations at position 194. It is suggested that Phe194 is important for the mobility of the interdomain region and the relative movement of the 3-phosphoglycerate binding site which allows the optimum conformation for catalysis to be attained. Apparently Trp194 reduces the mobility of the interdomain region of the protein, while Leu194 increases it.
authors.affiliation: Inorganic Chemistry Laboratory, University of Oxford, England.
authors.forename: H C
authors.initials: HC
authors.lastname: João
authors.forename: N
authors.initials: N
authors.lastname: Taddei
authors.forename: R J
authors.initials: RJ
authors.lastname: Williams
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002413:Cations, Divalent; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014364:Tryptophan / Q000235:genetics
chemical_list: D002413:Cations, Divalent; D010649:Phenylalanine; D014364:Tryptophan; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; D007930:Leucine; D008274:Magnesium