Action of magnesium ion on diphosphopyridine nucleotide linked isocitrate dehydrogenase from bovine heart. Characterization of the forms of the substrate and the modifier of the reaction

G. W E Plaut, Vern L. Schramm, T. Aogaichi

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Abstract

The activity of DPN linked isocitrate dehydrogenase is dependent on the concentration of magnesium D isocitrate (MgI-) under conditions where the concentrations of free Mg2+ and isocitrate approach zero and saturation, repectively. These results can be interpreted in terms of magnesium isocitrate as the active substrate, however the present data cannot rule out the possibility of free Mg2+ and isocitrate as the substrates if the reaction mechanism is rapid equilibrium ordered with free Mg2+ being the first reactant. In the absence of ADP and in presence of 0.33 mM DPN+, S(0.5) of magnesium DL isocitrate has been found to be 0.2, 0.4, 0.5, and 1.0 mM at pH 6.65, 7.2, 7.4, and 7.75, respectively, with values of Hill slopes (n) near 2. Excess free Mg2+ causes apparent inhibition which is competitive with respect to magnesium isocitrate, the proposed substrate of the reaction. The inhibition by Mg2+ is much more severe at pH 6.65 than at pH 7.2 and above, e.g. the inhibition constant for Mg2+ is 40 to 80 fold larger at pH 7.2 than at pH 6.65. In contrast to the results which are consistent with magnesium isocitrate as the substrate, free ADP3- is the modifier of the enzyme and MgADP- is inactive. At pH 7.2 and 7.4, increasing concentrations of ADP3- lead to decreasing values of S(0.5) for magnesium isocitrate accompanied by a decline of Hill slopes from n = 2 to near unity; these changes are less pronounced at pH 6.65. The values of V(max) are the same in the absence and presence of ADP at each pH tested; however, V(max) at pH 6.65 is about one half of that at pH 7.2 and above. There is an interdependence in the interaction of the enzyme with substrate and modifier. Thus, at pH 7.2 and 7.4, the values of K(m) for ADP3- decline with increasing concentrations of magnesium isocitrate; however, the Hill slopes for ADP3- are not influenced by substrate concentration and remain constant at m = 1 between pH 6.65 and 7.75. A reaction model has been proposed in which initial binding of substrate to the enzyme at one site leads to a conformational change and subsequent binding of magnesium isocitrate at an additional site which is catalytically active.

Original languageEnglish (US)
Pages (from-to)1848-1856
Number of pages9
JournalJournal of Biological Chemistry
Volume249
Issue number6
StatePublished - 1974
Externally publishedYes

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Isocitrate Dehydrogenase
NAD
Magnesium
Ions
Substrates
Adenosine Diphosphate
Enzymes
isocitric acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{6f9ac03ab4934647b74f244164ab6e89,
title = "Action of magnesium ion on diphosphopyridine nucleotide linked isocitrate dehydrogenase from bovine heart. Characterization of the forms of the substrate and the modifier of the reaction",
abstract = "The activity of DPN linked isocitrate dehydrogenase is dependent on the concentration of magnesium D isocitrate (MgI-) under conditions where the concentrations of free Mg2+ and isocitrate approach zero and saturation, repectively. These results can be interpreted in terms of magnesium isocitrate as the active substrate, however the present data cannot rule out the possibility of free Mg2+ and isocitrate as the substrates if the reaction mechanism is rapid equilibrium ordered with free Mg2+ being the first reactant. In the absence of ADP and in presence of 0.33 mM DPN+, S(0.5) of magnesium DL isocitrate has been found to be 0.2, 0.4, 0.5, and 1.0 mM at pH 6.65, 7.2, 7.4, and 7.75, respectively, with values of Hill slopes (n) near 2. Excess free Mg2+ causes apparent inhibition which is competitive with respect to magnesium isocitrate, the proposed substrate of the reaction. The inhibition by Mg2+ is much more severe at pH 6.65 than at pH 7.2 and above, e.g. the inhibition constant for Mg2+ is 40 to 80 fold larger at pH 7.2 than at pH 6.65. In contrast to the results which are consistent with magnesium isocitrate as the substrate, free ADP3- is the modifier of the enzyme and MgADP- is inactive. At pH 7.2 and 7.4, increasing concentrations of ADP3- lead to decreasing values of S(0.5) for magnesium isocitrate accompanied by a decline of Hill slopes from n = 2 to near unity; these changes are less pronounced at pH 6.65. The values of V(max) are the same in the absence and presence of ADP at each pH tested; however, V(max) at pH 6.65 is about one half of that at pH 7.2 and above. There is an interdependence in the interaction of the enzyme with substrate and modifier. Thus, at pH 7.2 and 7.4, the values of K(m) for ADP3- decline with increasing concentrations of magnesium isocitrate; however, the Hill slopes for ADP3- are not influenced by substrate concentration and remain constant at m = 1 between pH 6.65 and 7.75. A reaction model has been proposed in which initial binding of substrate to the enzyme at one site leads to a conformational change and subsequent binding of magnesium isocitrate at an additional site which is catalytically active.",
author = "Plaut, {G. W E} and Schramm, {Vern L.} and T. Aogaichi",
year = "1974",
language = "English (US)",
volume = "249",
pages = "1848--1856",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "6",

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TY - JOUR

T1 - Action of magnesium ion on diphosphopyridine nucleotide linked isocitrate dehydrogenase from bovine heart. Characterization of the forms of the substrate and the modifier of the reaction

AU - Plaut, G. W E

AU - Schramm, Vern L.

AU - Aogaichi, T.

PY - 1974

Y1 - 1974

N2 - The activity of DPN linked isocitrate dehydrogenase is dependent on the concentration of magnesium D isocitrate (MgI-) under conditions where the concentrations of free Mg2+ and isocitrate approach zero and saturation, repectively. These results can be interpreted in terms of magnesium isocitrate as the active substrate, however the present data cannot rule out the possibility of free Mg2+ and isocitrate as the substrates if the reaction mechanism is rapid equilibrium ordered with free Mg2+ being the first reactant. In the absence of ADP and in presence of 0.33 mM DPN+, S(0.5) of magnesium DL isocitrate has been found to be 0.2, 0.4, 0.5, and 1.0 mM at pH 6.65, 7.2, 7.4, and 7.75, respectively, with values of Hill slopes (n) near 2. Excess free Mg2+ causes apparent inhibition which is competitive with respect to magnesium isocitrate, the proposed substrate of the reaction. The inhibition by Mg2+ is much more severe at pH 6.65 than at pH 7.2 and above, e.g. the inhibition constant for Mg2+ is 40 to 80 fold larger at pH 7.2 than at pH 6.65. In contrast to the results which are consistent with magnesium isocitrate as the substrate, free ADP3- is the modifier of the enzyme and MgADP- is inactive. At pH 7.2 and 7.4, increasing concentrations of ADP3- lead to decreasing values of S(0.5) for magnesium isocitrate accompanied by a decline of Hill slopes from n = 2 to near unity; these changes are less pronounced at pH 6.65. The values of V(max) are the same in the absence and presence of ADP at each pH tested; however, V(max) at pH 6.65 is about one half of that at pH 7.2 and above. There is an interdependence in the interaction of the enzyme with substrate and modifier. Thus, at pH 7.2 and 7.4, the values of K(m) for ADP3- decline with increasing concentrations of magnesium isocitrate; however, the Hill slopes for ADP3- are not influenced by substrate concentration and remain constant at m = 1 between pH 6.65 and 7.75. A reaction model has been proposed in which initial binding of substrate to the enzyme at one site leads to a conformational change and subsequent binding of magnesium isocitrate at an additional site which is catalytically active.

AB - The activity of DPN linked isocitrate dehydrogenase is dependent on the concentration of magnesium D isocitrate (MgI-) under conditions where the concentrations of free Mg2+ and isocitrate approach zero and saturation, repectively. These results can be interpreted in terms of magnesium isocitrate as the active substrate, however the present data cannot rule out the possibility of free Mg2+ and isocitrate as the substrates if the reaction mechanism is rapid equilibrium ordered with free Mg2+ being the first reactant. In the absence of ADP and in presence of 0.33 mM DPN+, S(0.5) of magnesium DL isocitrate has been found to be 0.2, 0.4, 0.5, and 1.0 mM at pH 6.65, 7.2, 7.4, and 7.75, respectively, with values of Hill slopes (n) near 2. Excess free Mg2+ causes apparent inhibition which is competitive with respect to magnesium isocitrate, the proposed substrate of the reaction. The inhibition by Mg2+ is much more severe at pH 6.65 than at pH 7.2 and above, e.g. the inhibition constant for Mg2+ is 40 to 80 fold larger at pH 7.2 than at pH 6.65. In contrast to the results which are consistent with magnesium isocitrate as the substrate, free ADP3- is the modifier of the enzyme and MgADP- is inactive. At pH 7.2 and 7.4, increasing concentrations of ADP3- lead to decreasing values of S(0.5) for magnesium isocitrate accompanied by a decline of Hill slopes from n = 2 to near unity; these changes are less pronounced at pH 6.65. The values of V(max) are the same in the absence and presence of ADP at each pH tested; however, V(max) at pH 6.65 is about one half of that at pH 7.2 and above. There is an interdependence in the interaction of the enzyme with substrate and modifier. Thus, at pH 7.2 and 7.4, the values of K(m) for ADP3- decline with increasing concentrations of magnesium isocitrate; however, the Hill slopes for ADP3- are not influenced by substrate concentration and remain constant at m = 1 between pH 6.65 and 7.75. A reaction model has been proposed in which initial binding of substrate to the enzyme at one site leads to a conformational change and subsequent binding of magnesium isocitrate at an additional site which is catalytically active.

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