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Mg2+ affects the binding of ADP but not ATP to 3-phosphoglycerate kinase. Correlation between equilibrium dialysis
Abstract:
The role of Mg2+ in the binding of ADP and ATP to pig muscle and yeast 3-phosphoglycerate kinases has been studied by equilibrium dialysis. Whereas the Kd of ATP binding varies between 0.17 and 0.23 mM (S.E.M. 0.03 mM) for both enzymes, independently of the presence of Mg2+, the Kd values for ADP and MgADP binding are in the range 0.18-0.27 mM (S.E.M. 0.04 mM) and 0.05-0.06 mM (S.E.M. 0.01 mM) respectively. Thus Mg2+ exclusively tightens the interaction of ADP, but not of ATP, with the protein molecule. Although the equilibrium dialysis data are consistent with a model possessing a single site for nucleotides, the existence of a much weaker secondary site (with a Kd value at least two orders of magnitude larger) cannot be excluded. The binding of AMP and adenosine to pig muscle 3-phosphoglycerate kinase is weaker than binding of MgATP; the respective Kd values are 0.36 +/- 0.05 mM and 0.65 +/- 0.05 mM. Thus, in addition to the interaction of the alpha-phosphate that is detectable by crystallography [Banks, Blake, Evans, Haser, Rice, Hardy, Merrett and Phillips (1979) Nature (London) 279, 773-777], the beta- and/or gamma-phosphate(s) of MgATP may also interact with the enzyme molecule. The fact that MgADP binds more tightly than ADP is consistent with its stronger inhibition of the reaction catalysed by the enzyme between 3-phosphoglycerate and MgATP. MgADP is a product of this reaction, and inhibits it competitively with both substrates; as an inhibitor its KI is comparable with the Kd found in binding studies. At the same time, the Km value for MgADP in the reverse reaction (0.18 +/- 0.05 mM; mean +/- S.E.M.) is higher than these constants; this may be due either to a different kinetic mechanism in this direction of the enzymic reaction, or to different binding modes of MgADP as inhibitor and as substrate. The reason why inhibition by MgADP is competitive with 3-phosphoglycerate may be that its binding prevents the specific change in conformation that the enzyme undergoes [Harlos, Vas and Blake (1992) Proteins 12, 133-144] when it binds 3-phosphoglycerate.
Insights
Magnesium ions (Mg2+) significantly enhance ADP binding to 3-phosphoglycerate kinase enzymes, but do not affect ATP binding. This differential effect of Mg2+ on nucleotide interactions is crucial for enzyme regulation.
Area of Science:
- Biochemistry
- Enzymology
- Protein-ligand interactions
Background:
- 3-phosphoglycerate kinase (PGK) is a key enzyme in glycolysis.
- Understanding nucleotide binding is essential for elucidating enzyme mechanisms and regulation.
Purpose of the Study:
- To investigate the role of Mg2+ in the binding of ADP and ATP to pig muscle and yeast 3-phosphoglycerate kinases.
- To characterize the binding affinities (Kd values) of various nucleotides and their magnesium complexes.
Main Methods:
- Equilibrium dialysis was employed to measure the binding constants.
- Kinetic studies were performed to assess inhibition constants (KI) and Michaelis constants (Km).
Main Results:
- Mg2+ significantly tightens the binding of ADP to PGK (lower Kd), but does not alter ATP binding affinity.
- MgADP exhibits tighter binding and stronger inhibition compared to ADP.
- The binding of AMP and adenosine is weaker than that of MgATP.
Conclusions:
- Mg2+ plays a specific role in modulating ADP interaction with PGK, impacting enzyme regulation.
- The binding characteristics of MgADP are consistent with its role as a product inhibitor.
- Potential for secondary nucleotide binding sites exists, though not definitively proven by these studies.