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The Allosteric Effect of Fructose Bisphosphate on Muscle Pyruvate Kinase Studied by Infrared Spectroscopy
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2011 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 115, no 39, 11501-11505 p.Article in journal (Refereed) Published
Abstract [en]

Pyruvate kinase exhibits allosteric properties. The allosteric effect of fructose 1,6-bisphosphate (FBP) on phosphoenolpyruvate (PEP) binding to rabbit muscle pyruvate kinase (PK) in the presence of various ions (mg(2+), mn(2+), Na(+)) was studied by attenuated total reflection infrared spectroscopy in combination with a dialysis accessory. The experiments indicated that FBP binding causes conformational changes of PK that are of the same order of magnitude as those of PEP binding. The conformational change of PEP binding to PK/Mg(2+)/K(+) in the presence of FBP was about twice as large as in its absence, which is tentatively ascribed to a higher occupancy the closed state. The affinity for PEP increased in the presence of Mg(2+) and K(+). No such effects were observed with the other ion combinations Mn(2+)/K(+) and Mg(2+)/Na(+) or in D(2)O (with Mg(2+)/K(+)), and therefore we did not detect an allosteric effect on PEP binding under these conditions.

Place, publisher, year, edition, pages
2011. Vol. 115, no 39, 11501-11505 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-69877DOI: 10.1021/jp206272xISI: 000295245400022OAI: oai:DiVA.org:su-69877DiVA: diva2:479814
Note
authorCount :2Available from: 2012-01-18 Created: 2012-01-15 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Infrared studies: Method development and binding of ligands to pyruvate kinase
Open this publication in new window or tab >>Infrared studies: Method development and binding of ligands to pyruvate kinase
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Infrared spectroscopy is a valuable technique for the study of ligand induce change in biomolecules. Our development of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. We use this method to understand the binding of phosphoenolpyruvate (PEP) and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. To investigate the effect of the protein environment on the bound PEP, we used labeled PEP, which helped assign and evaluate the infrared absorption bands. The effects of different divalent and monovalent ions on PEP binding to PK were also studied. We could demonstrate that the β-sheets were perturbed differently with Na+ as compared to the other monovalent ions. The pattern of structural changes does not correlate with the activity profiles of the monovalent ions. Thus, it seems unlikely that the ion effects on activity are due to the ion effects on the structure of the PEP:PK complex. Comparing different divalent ions, a particularly large conformational change and a more homogeneous binding mode was observed with Mn2+ and attributed to a more closed conformation of the complex. The allosteric effect of fructose 1, 6 bisphosphate (FBP) on PEP binding to PK in presence of various ions (Mg2+, Mn2+, K+, Na+) was studied. The experiments indicated that the conformational change of PEP binding to PK:Mg2+:K+ in the presence of FBP was about twice as large as in its absence, which is tentatively ascribed to a higher occupancy of the closed state of PK. The affinity for PEP increased in presence of Mg2+ and K+. No allosteric effects were observed with the other ion combinations Mn2+/K+ and Mg2+/Na+. A method of ligand binding by observing a change in water absorption was developed and established with four different proteins. The results suggest that the decrease of water absorption is due to the release of bound water into the bulk during the ligand binding process, which can be a used as label-free indicator of ligand-protein binding.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 7 p.
National Category
Natural Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-56754 (URN)978-91-7447-297-4 (ISBN)
Public defence
2011-05-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Accepted. Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2011-05-09 Created: 2011-04-26 Last updated: 2012-01-18Bibliographically approved

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