Mg2+ affects the binding of ADP but not ATP to 3-phosphoglycerate kinase. Correlation between equilibrium dialysis binding and enzyme kinetic data

M. Molnar, M. Vas

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24 Citations (Scopus)

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 K(d) 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 K(d) values for ADP and MgADP binding are in the range 0.18-0.27mM (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 K(d) 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 K(d) values are 0.36 ± 0.05 mM and 0.65 ± 0.05 mM. Thus, in addition to the interaction of the α-phosphate that is detectable by crystallography the β- and/or γ-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 K(I) is comparable with the K(d) found in binding studies. At the same time, the K(m) 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.

Original languageEnglish
Pages (from-to)595-599
Number of pages5
JournalBiochemical Journal
Volume293
Issue number2
Publication statusPublished - 1993

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Phosphoglycerate Kinase
Enzyme kinetics
Dialysis
Adenosine Diphosphate
Adenosine Triphosphate
Enzymes
Scanning electron microscopy
Muscle
Swine
Phosphates
3-phosphoglycerate
Muscles
Crystallography
Molecules
Substrates
Adenosine Monophosphate
Yeast
Adenosine
Conformations
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Mg2+ affects the binding of ADP but not ATP to 3-phosphoglycerate kinase. Correlation between equilibrium dialysis binding and enzyme kinetic data",
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 K(d) 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 K(d) values for ADP and MgADP binding are in the range 0.18-0.27mM (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 K(d) 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 K(d) values are 0.36 ± 0.05 mM and 0.65 ± 0.05 mM. Thus, in addition to the interaction of the α-phosphate that is detectable by crystallography the β- and/or γ-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 K(I) is comparable with the K(d) found in binding studies. At the same time, the K(m) 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.",
author = "M. Molnar and M. Vas",
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journal = "Biochemical Journal",
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T1 - Mg2+ affects the binding of ADP but not ATP to 3-phosphoglycerate kinase. Correlation between equilibrium dialysis binding and enzyme kinetic data

AU - Molnar, M.

AU - Vas, M.

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N2 - 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 K(d) 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 K(d) values for ADP and MgADP binding are in the range 0.18-0.27mM (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 K(d) 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 K(d) values are 0.36 ± 0.05 mM and 0.65 ± 0.05 mM. Thus, in addition to the interaction of the α-phosphate that is detectable by crystallography the β- and/or γ-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 K(I) is comparable with the K(d) found in binding studies. At the same time, the K(m) 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.

AB - 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 K(d) 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 K(d) values for ADP and MgADP binding are in the range 0.18-0.27mM (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 K(d) 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 K(d) values are 0.36 ± 0.05 mM and 0.65 ± 0.05 mM. Thus, in addition to the interaction of the α-phosphate that is detectable by crystallography the β- and/or γ-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 K(I) is comparable with the K(d) found in binding studies. At the same time, the K(m) 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.

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