Thermodynamic linkages in rabbit muscle pyruvate kinase: analysis of experimental data by a two-state model

Rw, Oberfelder; Bg, Barisas; Jc, Lee

doi:10.1021/bi00312a005

Cited by 35 publications

(48 citation statements)

References 5 publications

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“…Concentration of RMPK was adjusted using absorptivity of 0.54 mg ml −1 cm −1 at 280 nm (12) and molecular weight of 220 kDa (8). A single batch of protein was used for all experiments.…”

Section: Methodsmentioning

confidence: 99%

Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 1. Calorimetric Study

Heřman

Lee

2009

Biochemistry

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Rabbit muscle pyruvate kinase (RMPK) is an important allosteric enzyme of the glycolytic pathway catalyzing a transfer of the phosphate from phosphoenolpyruvate (PEP) to ADP. The energetic landscape of the allosteric regulatory mechanism of RMPK was characterized by isothermal titration calorimetry (ITC) in the temperature range from 4°C to 45°C. ITC data for RMPK binding to substrates PEP and ADP, for the allosteric inhibitor Phe, as well as for combination of ADP and Phe were globally analyzed. The thermodynamic parameters characterizing the linked-multiple- equilibria system were extracted. Four novel insights were uncovered 1. The binding preference of ADP for either the T- or R-state is temperature dependent; namely, more favorably to the T- and R-state at high and low temperature, respectively. This cross over of affinity towards R and T-state implies that ADP plays a complex role in modulating the allosteric behavior of RMPK. Depending on the temperature, binding of ADP can regulate RMPK activity by favoring the enzyme to either the R- or T-state. 2. The binding of Phe is negatively coupled to that of ADP i.e. Phe and ADP prefer not to bind to the same subunit of RMPK. 3. The release or absorption of protons linked to the various equilibria is specific to the particular reaction. As a consequence, pH will exert a complex effect on these linked equilibria resulting in proton being an allosteric regulatory ligand of RMPK. 4. The R↔T equilibrium is accompanied by a significant ΔCp rendering RMPK most sensitive to temperature under physiological conditions. During muscle activity, both pH and temperature fluctuations are known to happen; thus, results of this study are physiologically relevant.

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Section: Methodsmentioning

confidence: 99%

Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 1. Calorimetric Study

Heřman

Lee

2009

Biochemistry

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“…Our model is based on a two-state model for cooperative binding of ligands to macromolecules that has been published by Monod et al (28) and adapted by Oberfelder et al (2) for the tetrameric RMPK. Description and application of this model for analysis of calorimetric RMPK data can be found in the previous paper (1).…”

Section: Methodsmentioning

confidence: 99%

“…Based on a simple two-state model for the cooperative binding of ligands by macromolecules earlier described by Monod and customized for the tertameric RMPK by Oberfelder et al (2), four novel insights on the mechanism of allosteric regulation of RMPK were uncovered: 1. A temperature dependent cross over of ADP affinity towards R and T-state; more favorably to the R- and T-state at high and low temperature, respectively.…”

mentioning

confidence: 99%

Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 2. Fluorescence Study

Heřman

Lee

2009

Biochemistry

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The energetic landscape of the allosteric regulatory mechanism of rabbit muscle pyruvate kinase (RMPK) was characterized by isothermal titration calorimetry (ITC). Four novel insights were uncovered 1. ADP exhibits a dual property. Depending on the temperature, ADP can regulate RMPK activity by switching the enzyme to either the R-or T-state. 2. The assumption that ligand binding to RMPK is state dependent is only correct for PEP but not Phe/ADP. 3. The pH effect on the regulatory behavior of RMPK is partly due to the complex pattern of proton release or absorption linked to the multiple linked equilibria which govern the activity of the enzyme. 4. The R↔T equilibrium is accompanied by a significant ΔC p rendering RMPK most sensitive to temperature under physiological conditions. In order to rigorously test the validity of conclusions derived from the ITC data, in this study a fluorescence approach, albeit indirect, that tracks continuous structural perturbations was employed. Intrinsic Trp fluorescence of RMPK in the absence and in the presence of substrates phosphoenolpyruvate (PEP) and ADP, and the allosteric inhibitor Phe was measured in the temperature range between 4°C and 45°C. For data analysis the fluorescence data were complemented by ITC experiments to obtain extended data set allowing more complete characterization of the RMPK regulatory mechanism. Twenty-one thermodynamic parameters were derived to define the network of linked interactions involved in regulating the allosteric behavior of RMPK through global analysis of the ITC and fluorescent data sets. In this study 27 independent curves with more than 1600 experimental points were globally analyzed. Consequently, the consensus results not only substantiate the conclusions derived from the ITC data but also structural information characterizing the transition between the active and the inactive state of RMPK and the antagonism between ADP and Phe binding. The latter observation reveals a novel role for ADP in the allosteric regulation of RMPK. KeywordsTwo state model; global analysis; allosterism; fluorescence; calorimetry In the previous article of this series isothermal titration calorimetry, ITC, was employed to define the energy landscape of allosteric regulation of rabbit muscle pyruvate kinase, RMPK (1). Based on a simple two-state model for the cooperative binding of ligands by macromolecules earlier described by Monod and customized for the tertameric RMPK by Oberfelder et al. (2), four novel insights on the mechanism of allosteric regulation of RMPK *TO WHOM CORRESPONDENCE SHOULD BE ADDRESSED: For J.C.L., tel. (409) 772-2281, fax (409) 772-4298, jclee@utmb.edu. For P.H., tel. +420-221911461, fax +420-224922797, herman@karlov.mff.cuni.cz. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 October 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript were uncovered: 1. A temperature dependent cross over of ADP affinity towards R and T-state; more favorably to the R-and ...

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“…In that regard it is of interest to note that pyruvate kinase is an allosteric enzyme (Oberfelder et al, 1984a, b) for which preexistence of the isomerization equilibrium has been established (Harris and Winzor, 1988b). Because this isomerization is displaced heavily toward the kinetically active isomer (Oberfelder et al, 1984b;Harris and Winzor, 1988b), the experimental data of Fig. 5a are examined in the light that only the inactive isomer of pyruvate kinase exhibits affinity for myofibrillar matrix sites.…”

Section: Interaction Of Pyruvate Kinase With Myofibrilsmentioning

confidence: 99%

“…(12)-(14) to the pyruvate kinase data with values of 4 for f and 10 for Y (Oberfelder et al, 1984b;Harris and Winzor, 1988b) and a total concentration of acceptor sites (2.34/xM) based on a myofibrillar capacity of 78nmol/g (Harris and Winzor, 1989) for glycolytic enzymes. Analysis of the results on this basis gives rise to a multivalent Scatchard plot (Fig.…”

Section: Interaction Of Pyruvate Kinase With Myofibrilsmentioning

confidence: 99%

Effects of heterogeneity and cooperativity on the forms of binding curves for multivalent ligands

Harris

Jackson²,

Winzor

1995

J Protein Chem

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An exploratory investigation is made of the binding behavior that is likely to be encountered with multivalent ligands under circumstances where a single intrinsic binding constant does not suffice to describe all acceptor-ligand interactions. Numerical simulations of theoretical binding behavior have established that current criteria for recognizing heterogeneity and cooperativity of acceptor sites on the basis of the deviation of the binding curve from rectangular hyperbolic form for univalent ligands also apply to the interpretation of the corresponding binding curves for multivalent ligands. However, for systems in which the source of the departure from equivalence and independence of binding sites resides in the ligand, these criteria are reversed. On the basis of these observations a case is then made for attributing results of an experimental binding study of the interaction between pyruvate kinase and muscle myofibrils to positive cooperativity of enzyme sites rather than to heterogeneity or negative cooperativity of the myofibrillar sites.

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Thermodynamic linkages in rabbit muscle pyruvate kinase: analysis of experimental data by a two-state model

Cited by 35 publications

References 5 publications

Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 1. Calorimetric Study

Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 1. Calorimetric Study

Functional Energetic Landscape in the Allosteric Regulation of Muscle Pyruvate Kinase. 2. Fluorescence Study

Effects of heterogeneity and cooperativity on the forms of binding curves for multivalent ligands

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