Site-directed mutagenesis of glutamate-190 in the hinge region of yeast 3-phosphoglycerate kinase: implications for the mechanism of domain movement.
In order to evaluate a possible contribution of glutamate-190, situated in the hinge region of yeast 3-phosphoglycerate kinase (PGK), to the mechanism of the substrate- and sulfate-induced domain movement, we have constructed two point mutants, Gln-190 and Asp-190, using oligonucleotide-directed in vitro mutagenesis. The Michaelis constants of the mutants for ATP and 3-phosphoglycerate were not significantly altered, whereas the catalytic activities were decreased, both in the absence and in the presence of sulfate ions. In the absence of sulfate, the Gln-190 and Asp-190 mutants exhibited 26% and 36% of the activity of the native enzyme. In the presence of 30 mM Na2SO4, a concentration at which native PGK exhibits maximum activation, the relative activities of the Gln-190 and Asp-190 mutants were 6% and 9%, respectively. In contrast to the native enzyme, which undergoes activation at low sulfate concentrations and inhibition at high concentrations, both mutants showed a complete loss of the salt activation effect. These results suggest that Glu-190 is not directly involved in the binding of substrates but might be important for conformational flexibility. We have also demonstrated that, similarly to native PGK, both mutants are completely inactivated by the incorporation of 1 mol of glycine ethyl ester/mol of enzyme. Appreciable protection against inactivation is observed in the presence of both substrates, MgATP and 3-phosphoglycerate. Only limited protection is observed in the presence of the individual substrates, suggesting that the modification does not occur at the substrate binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
D003196:Computer Graphics; D005796:Genes; D005800:Genes, Fungal; D005971:Glutamates; D018698:Glutamic Acid; D008958:Models, Molecular; D009154:Mutation; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D005971:Glutamates; D018698:Glutamic Acid; D010735:Phosphoglycerate Kinase