Binding of substrates and other anions to yeast phosphoglycerate kinase.
1. The binding of all four substrates to yeast phosphoglycerate kinase has been studied using a gel filtration technique. The binding of phosphate and sulphate anions has also been investigated. 2. Two sites for each adenine nucleotide were found, one site being weaker than the other by between 30 and 50-fold. Only one binding site for the phosphoglycerate substrates was found. 3. 1,3-Bisphosphoglycerate (1,3-P2-glycerate) bound to the enzyme approximately 1000 times tighter than the other three substrates, its dissociation constant being 0.06 micrometer at ionic strength 0.15 M. 4. Sulphate and phosphate were mutually competitive and sulphate competed with the binding of all substrates except MgADP. MgADP bound to the enzyme more weakly in the presence of sulphate. The dissociation constant for sulphate binding was 1.6 mM at ionic strength of 0.15 M, and 0.05 mM at ionic strength 0.015 M. 5. These results are consistent with sulphate acting as a competitive inhibitor, as found by kinetic studies at high sulphate concentrations. The activatory effect of sulphate at lower concentrations and the substrate activation phenomea displayed by this enzyme, are interpreted in terms of a two-step dissociation of 1, 3-P2-glycerate. The presence of moderate concentrations of MgATP, 3-phosphoglycerate or sulphate causes acceleration of the rate of dissociation of the product, 1, 3-P2-glycerate, this being the rate-limiting step in the overall enzyme reaction.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000838:Anions; D001665:Binding Sites; D001667:Binding, Competitive; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D005988:Glyceric Acids / Q000378:metabolism; D007700:Kinetics; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000838:Anions; D004163:Diphosphoglyceric Acids; D005988:Glyceric Acids; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase