Thermodynamic linkages in rabbit muscle pyruvate kinase

Kinetic, equilibrium, and structural studies

Robert W. Oberfelder, Lucy L Y Lee, James Lee

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23°C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23°C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].

Original languageEnglish (US)
Pages (from-to)3813-3821
Number of pages9
JournalBiochemistry
Volume23
Issue number17
StatePublished - 1984
Externally publishedYes

Fingerprint

Pyruvate Kinase
Phenylalanine
Thermodynamics
Muscle
Rabbits
Muscles
Kinetics
Phosphoenolpyruvate
Sedimentation
Allosteric Regulation
Enzymes
Hydrodynamics
Binding Sites
Databases
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Thermodynamic linkages in rabbit muscle pyruvate kinase : Kinetic, equilibrium, and structural studies. / Oberfelder, Robert W.; Lee, Lucy L Y; Lee, James.

In: Biochemistry, Vol. 23, No. 17, 1984, p. 3813-3821.

Research output: Contribution to journalArticle

Oberfelder, RW, Lee, LLY & Lee, J 1984, 'Thermodynamic linkages in rabbit muscle pyruvate kinase: Kinetic, equilibrium, and structural studies', Biochemistry, vol. 23, no. 17, pp. 3813-3821.
Oberfelder, Robert W. ; Lee, Lucy L Y ; Lee, James. / Thermodynamic linkages in rabbit muscle pyruvate kinase : Kinetic, equilibrium, and structural studies. In: Biochemistry. 1984 ; Vol. 23, No. 17. pp. 3813-3821.
@article{c0a49491739e424dba76073c647affa4,
title = "Thermodynamic linkages in rabbit muscle pyruvate kinase: Kinetic, equilibrium, and structural studies",
abstract = "The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23°C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23°C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90{\%} complete when only 25{\%} of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].",
author = "Oberfelder, {Robert W.} and Lee, {Lucy L Y} and James Lee",
year = "1984",
language = "English (US)",
volume = "23",
pages = "3813--3821",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",

}

TY - JOUR

T1 - Thermodynamic linkages in rabbit muscle pyruvate kinase

T2 - Kinetic, equilibrium, and structural studies

AU - Oberfelder, Robert W.

AU - Lee, Lucy L Y

AU - Lee, James

PY - 1984

Y1 - 1984

N2 - The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23°C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23°C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].

AB - The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23°C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23°C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].

UR - http://www.scopus.com/inward/record.url?scp=0021762383&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021762383&partnerID=8YFLogxK

M3 - Article

VL - 23

SP - 3813

EP - 3821

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 17

ER -