Adenine nucleotides affect the binding of 3-phosphoglycerate to pig muscle 3-phosphoglycerate kinase.
Pig muscle 3-phosphoglycerate kinase was complexed with 1-anilino-8-naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme-dye interaction did not influence the enzymic activity under the experimental conditions used. By measuring the substrate-dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd less than or equal to 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20 degrees C. The dissociation constants obtained for the separate bindings of 3-phosphoglycerate, MgATP, 1,3-bisphosphoglycerate and MgADP were 0.03 +/- 0.01 mM, 0.15 +/- 0.10 mM, 0.00005 +/- 0.00001 mM and 0.15 +/- 0.10 mM respectively. binding of 3-phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3-phosphoglycerate in this ternary complex (0.25 +/- 0.08 mM) is comparable to its Km value (0.38 +/- 0.10 mM). The same weakening can be observed in the non-productive ternary complexes where MgATP is replaced by MgADP (Kd = 0.20 +/- 0.10 mM) or AMP (Kd = 0.12 +/- 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3-phosphoglycerate. In contrast to 3-phosphoglycerate, the binding of the substrate analogue, glycerol 3-phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 +/- 0.10 mM) is comparable to its inhibitory constant (0.70 +/- 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3-phosphate binding (0.40 +/- 0.10 mM) and the Km value of 3-phosphoglycerate (0.38 +/- 0.10 mM) suggest that, during the enzymic reaction, binding of 3-phosphoglycerate occurs probably without involvement of the carboxyl group.
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D005988:Glyceric Acids / Q000378:metabolism*; D005994:Glycerophosphates / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013552:Swine
D000227:Adenine Nucleotides; D000817:Anilino Naphthalenesulfonates; D005988:Glyceric Acids; D005994:Glycerophosphates; C027132:1-anilino-8-naphthalenesulfonate; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; C029620:alpha-glycerophosphoric acid; D010735:Phosphoglycerate Kinase