Kinetic and binding studies of Mn(II) and fructose 1,6 bisphosphate with rabbit liver hexosebisphosphatase

C. B. Libby, W. A. Frey, J. J. Villafranca, Stephen Benkovic

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The seperate interaction of the substrate fructose 1,6 bisphosphate and a metal ion cofactor Mn2+ with neutral hexosebisphosphatase has been studied under equilibrium conditions at pH 7.5 with gel filtration and electron paramagnetic resonance measurements, respectively. Binding data for both ligands to the enzyme yielded nonlinear Scatchard plots that analyze in terms of four negatively cooperative binding sites per enzyme tetramer. Graphical estimates of the binding constants were refined by a computer searching procedure and nonlinear least squares analysis. These results are qualitatively similar to those obtained from binding studies involving the alkaline enzyme, a modified form of hexosebisphosphatase whose pH optimum is in the alkaline pH region. Both forms of the enzyme enhance the proton relaxation rate of water protons by a factor of 7 to 8 at 24 MHz, demonstrating similar metal ion environments. The activator Co(III) EDTA did not affect Mn2 + binding to the neutral enzyme. In the presence of (α + β)methyl D fructofuranoside 1,6 bisphosphate, however, two sets, each containing four Mn2+ binding sites, were observed per enzyme tetramer with loss of the negatively cooperative interaction. These results are viewed in terms of four noncatalytic and four catalytic Mn2+ binding sites. Parallel kinetic investigations were conducted on the neutral enzyme to determine specific activity as a function of Mn2+ and fructose 1,6 bisphosphate concentration. A pre equilibrium sequential pathway model involving Mn2+ enzyme and the Mn2+ fructose 1,6 bisphosphate complex both as substrate and as an allosteric inhibitor satisfactorily fit the kinetic observations. All possible enzyme species were computed from the determined binding constants and grouped according to the number of moles of Mn2+ fructose 1,6 bisphosphate complex bound to the Mn2+ enzyme, and individual rate constants were calculated. The testing of other models and their failure to describe the kinetic observations are discussed.

Original languageEnglish (US)
Pages (from-to)7564-7573
Number of pages10
JournalJournal of Biological Chemistry
Volume250
Issue number19
StatePublished - 1975

Fingerprint

Liver
Rabbits
Kinetics
Enzymes
Binding Sites
Metal ions
Protons
Metals
fructose-1,6-diphosphate
Ions
Electron Spin Resonance Spectroscopy
Substrates
Least-Squares Analysis
Edetic Acid
Gel Chromatography
Paramagnetic resonance
Rate constants
Gels
Ligands
Water

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Libby, C. B., Frey, W. A., Villafranca, J. J., & Benkovic, S. (1975). Kinetic and binding studies of Mn(II) and fructose 1,6 bisphosphate with rabbit liver hexosebisphosphatase. Journal of Biological Chemistry, 250(19), 7564-7573.
Libby, C. B. ; Frey, W. A. ; Villafranca, J. J. ; Benkovic, Stephen. / Kinetic and binding studies of Mn(II) and fructose 1,6 bisphosphate with rabbit liver hexosebisphosphatase. In: Journal of Biological Chemistry. 1975 ; Vol. 250, No. 19. pp. 7564-7573.
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abstract = "The seperate interaction of the substrate fructose 1,6 bisphosphate and a metal ion cofactor Mn2+ with neutral hexosebisphosphatase has been studied under equilibrium conditions at pH 7.5 with gel filtration and electron paramagnetic resonance measurements, respectively. Binding data for both ligands to the enzyme yielded nonlinear Scatchard plots that analyze in terms of four negatively cooperative binding sites per enzyme tetramer. Graphical estimates of the binding constants were refined by a computer searching procedure and nonlinear least squares analysis. These results are qualitatively similar to those obtained from binding studies involving the alkaline enzyme, a modified form of hexosebisphosphatase whose pH optimum is in the alkaline pH region. Both forms of the enzyme enhance the proton relaxation rate of water protons by a factor of 7 to 8 at 24 MHz, demonstrating similar metal ion environments. The activator Co(III) EDTA did not affect Mn2 + binding to the neutral enzyme. In the presence of (α + β)methyl D fructofuranoside 1,6 bisphosphate, however, two sets, each containing four Mn2+ binding sites, were observed per enzyme tetramer with loss of the negatively cooperative interaction. These results are viewed in terms of four noncatalytic and four catalytic Mn2+ binding sites. Parallel kinetic investigations were conducted on the neutral enzyme to determine specific activity as a function of Mn2+ and fructose 1,6 bisphosphate concentration. A pre equilibrium sequential pathway model involving Mn2+ enzyme and the Mn2+ fructose 1,6 bisphosphate complex both as substrate and as an allosteric inhibitor satisfactorily fit the kinetic observations. All possible enzyme species were computed from the determined binding constants and grouped according to the number of moles of Mn2+ fructose 1,6 bisphosphate complex bound to the Mn2+ enzyme, and individual rate constants were calculated. The testing of other models and their failure to describe the kinetic observations are discussed.",
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Libby, CB, Frey, WA, Villafranca, JJ & Benkovic, S 1975, 'Kinetic and binding studies of Mn(II) and fructose 1,6 bisphosphate with rabbit liver hexosebisphosphatase', Journal of Biological Chemistry, vol. 250, no. 19, pp. 7564-7573.

Kinetic and binding studies of Mn(II) and fructose 1,6 bisphosphate with rabbit liver hexosebisphosphatase. / Libby, C. B.; Frey, W. A.; Villafranca, J. J.; Benkovic, Stephen.

In: Journal of Biological Chemistry, Vol. 250, No. 19, 1975, p. 7564-7573.

Research output: Contribution to journalArticle

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T1 - Kinetic and binding studies of Mn(II) and fructose 1,6 bisphosphate with rabbit liver hexosebisphosphatase

AU - Libby, C. B.

AU - Frey, W. A.

AU - Villafranca, J. J.

AU - Benkovic, Stephen

PY - 1975

Y1 - 1975

N2 - The seperate interaction of the substrate fructose 1,6 bisphosphate and a metal ion cofactor Mn2+ with neutral hexosebisphosphatase has been studied under equilibrium conditions at pH 7.5 with gel filtration and electron paramagnetic resonance measurements, respectively. Binding data for both ligands to the enzyme yielded nonlinear Scatchard plots that analyze in terms of four negatively cooperative binding sites per enzyme tetramer. Graphical estimates of the binding constants were refined by a computer searching procedure and nonlinear least squares analysis. These results are qualitatively similar to those obtained from binding studies involving the alkaline enzyme, a modified form of hexosebisphosphatase whose pH optimum is in the alkaline pH region. Both forms of the enzyme enhance the proton relaxation rate of water protons by a factor of 7 to 8 at 24 MHz, demonstrating similar metal ion environments. The activator Co(III) EDTA did not affect Mn2 + binding to the neutral enzyme. In the presence of (α + β)methyl D fructofuranoside 1,6 bisphosphate, however, two sets, each containing four Mn2+ binding sites, were observed per enzyme tetramer with loss of the negatively cooperative interaction. These results are viewed in terms of four noncatalytic and four catalytic Mn2+ binding sites. Parallel kinetic investigations were conducted on the neutral enzyme to determine specific activity as a function of Mn2+ and fructose 1,6 bisphosphate concentration. A pre equilibrium sequential pathway model involving Mn2+ enzyme and the Mn2+ fructose 1,6 bisphosphate complex both as substrate and as an allosteric inhibitor satisfactorily fit the kinetic observations. All possible enzyme species were computed from the determined binding constants and grouped according to the number of moles of Mn2+ fructose 1,6 bisphosphate complex bound to the Mn2+ enzyme, and individual rate constants were calculated. The testing of other models and their failure to describe the kinetic observations are discussed.

AB - The seperate interaction of the substrate fructose 1,6 bisphosphate and a metal ion cofactor Mn2+ with neutral hexosebisphosphatase has been studied under equilibrium conditions at pH 7.5 with gel filtration and electron paramagnetic resonance measurements, respectively. Binding data for both ligands to the enzyme yielded nonlinear Scatchard plots that analyze in terms of four negatively cooperative binding sites per enzyme tetramer. Graphical estimates of the binding constants were refined by a computer searching procedure and nonlinear least squares analysis. These results are qualitatively similar to those obtained from binding studies involving the alkaline enzyme, a modified form of hexosebisphosphatase whose pH optimum is in the alkaline pH region. Both forms of the enzyme enhance the proton relaxation rate of water protons by a factor of 7 to 8 at 24 MHz, demonstrating similar metal ion environments. The activator Co(III) EDTA did not affect Mn2 + binding to the neutral enzyme. In the presence of (α + β)methyl D fructofuranoside 1,6 bisphosphate, however, two sets, each containing four Mn2+ binding sites, were observed per enzyme tetramer with loss of the negatively cooperative interaction. These results are viewed in terms of four noncatalytic and four catalytic Mn2+ binding sites. Parallel kinetic investigations were conducted on the neutral enzyme to determine specific activity as a function of Mn2+ and fructose 1,6 bisphosphate concentration. A pre equilibrium sequential pathway model involving Mn2+ enzyme and the Mn2+ fructose 1,6 bisphosphate complex both as substrate and as an allosteric inhibitor satisfactorily fit the kinetic observations. All possible enzyme species were computed from the determined binding constants and grouped according to the number of moles of Mn2+ fructose 1,6 bisphosphate complex bound to the Mn2+ enzyme, and individual rate constants were calculated. The testing of other models and their failure to describe the kinetic observations are discussed.

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