Adenine nucleotides affect the binding of 3‐phosphoglycerate to pig muscle 3‐phosphoglycerate kinase

Abstract

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 .ltoreq. 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20.degree. C. The Kd 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 Kd 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 analog, glycerol 3-phosphate is practically not affected by the presence of MgATP: the Kd 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 Kd of glycerol 3-phosphate binding (0.40 .+-. 0.01 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.