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≤ 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20°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 ± 0.1 0mM, 0.00005 ± 0.00001 mM and 0.15 ± 0.10mM 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 ± 0.10mM). 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‐hosphoglycerate. 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 ± 0.10mM) and the Km value of 3‐phosphoglycerate (0.38 ± 0.10mM) suggest that, during the enzymic reaction, binding of 3‐phosphoglycerate occurs probably without involvement of the carboxyl group.
|Number of pages||9|
|Journal||European Journal of Biochemistry|
|Publication status||Published - Feb 1984|
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