Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase

Electron paramagnetic resonance studies with oxygen-17-labeled ligands

Thomas S. Leyh, Paula J. Goodhart, Ann C. Nguyen, George L. Kenyon, George H. Reed

Research output: Contribution to journalArticle

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Abstract

Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II) ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by Chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture. Spectra for samples of the equilibrium mixture set up initially with creatine and either [α-17O]ATP, [β-17O]ATP, or [γ-17O]ATP show equivalent inhomogeneous broadening of signals from both central complexes irrespective of the position of the 17O label. Similar measurements with the nonreactive complex of 1-(carboxymethyl)-2-iminoimidazolidin-4-one and the 17O-labeled forms of ATP show that Mn(II) is coordinated to all three phosphate groups of ATP in this complex. These results show that an α,β,γ-tridentate complex of Mn(II)ATP is the substrate in the forward direction and Mn(II) remains coordinated to the α- and β-phosphates of ADP and to phosphocreatine in the product complex. A definitive model for the involvement of the divalent metal ion in the entire reaction sequence emerges from these results.

Original languageEnglish (US)
Pages (from-to)308-316
Number of pages9
JournalBiochemistry
Volume24
Issue number2
StatePublished - 1985
Externally publishedYes

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Phosphocreatine
Electron Spin Resonance Spectroscopy
Creatine Kinase
Manganese
Adenosine Diphosphate
Paramagnetic resonance
Adenosine Triphosphate
Oxygen
Ligands
Creatine
Enzymes
Phosphates
Substrates
Equilibrium constants
Complex Mixtures
Metal ions
Conformations
Labels
Quenching
Assays

ASJC Scopus subject areas

  • Biochemistry

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Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase : Electron paramagnetic resonance studies with oxygen-17-labeled ligands. / Leyh, Thomas S.; Goodhart, Paula J.; Nguyen, Ann C.; Kenyon, George L.; Reed, George H.

In: Biochemistry, Vol. 24, No. 2, 1985, p. 308-316.

Research output: Contribution to journalArticle

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title = "Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase: Electron paramagnetic resonance studies with oxygen-17-labeled ligands",
abstract = "Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II) ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by Chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture. Spectra for samples of the equilibrium mixture set up initially with creatine and either [α-17O]ATP, [β-17O]ATP, or [γ-17O]ATP show equivalent inhomogeneous broadening of signals from both central complexes irrespective of the position of the 17O label. Similar measurements with the nonreactive complex of 1-(carboxymethyl)-2-iminoimidazolidin-4-one and the 17O-labeled forms of ATP show that Mn(II) is coordinated to all three phosphate groups of ATP in this complex. These results show that an α,β,γ-tridentate complex of Mn(II)ATP is the substrate in the forward direction and Mn(II) remains coordinated to the α- and β-phosphates of ADP and to phosphocreatine in the product complex. A definitive model for the involvement of the divalent metal ion in the entire reaction sequence emerges from these results.",
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T1 - Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase

T2 - Electron paramagnetic resonance studies with oxygen-17-labeled ligands

AU - Leyh, Thomas S.

AU - Goodhart, Paula J.

AU - Nguyen, Ann C.

AU - Kenyon, George L.

AU - Reed, George H.

PY - 1985

Y1 - 1985

N2 - Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II) ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by Chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture. Spectra for samples of the equilibrium mixture set up initially with creatine and either [α-17O]ATP, [β-17O]ATP, or [γ-17O]ATP show equivalent inhomogeneous broadening of signals from both central complexes irrespective of the position of the 17O label. Similar measurements with the nonreactive complex of 1-(carboxymethyl)-2-iminoimidazolidin-4-one and the 17O-labeled forms of ATP show that Mn(II) is coordinated to all three phosphate groups of ATP in this complex. These results show that an α,β,γ-tridentate complex of Mn(II)ATP is the substrate in the forward direction and Mn(II) remains coordinated to the α- and β-phosphates of ADP and to phosphocreatine in the product complex. A definitive model for the involvement of the divalent metal ion in the entire reaction sequence emerges from these results.

AB - Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II) ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by Chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture. Spectra for samples of the equilibrium mixture set up initially with creatine and either [α-17O]ATP, [β-17O]ATP, or [γ-17O]ATP show equivalent inhomogeneous broadening of signals from both central complexes irrespective of the position of the 17O label. Similar measurements with the nonreactive complex of 1-(carboxymethyl)-2-iminoimidazolidin-4-one and the 17O-labeled forms of ATP show that Mn(II) is coordinated to all three phosphate groups of ATP in this complex. These results show that an α,β,γ-tridentate complex of Mn(II)ATP is the substrate in the forward direction and Mn(II) remains coordinated to the α- and β-phosphates of ADP and to phosphocreatine in the product complex. A definitive model for the involvement of the divalent metal ion in the entire reaction sequence emerges from these results.

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