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Phosphoenolpyruvate and Mg2+ binding to pyruvate kinase monitored by infrared spectroscopy
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.ORCID-id: 0000-0001-5784-7673
2010 (Engelska)Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 98, nr 9, s. 1931-1940Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg2+ binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg2+ binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-C-13(2)-labeled PEP. We obtained the following assignments: 1589 cm(-1) (antisymmetric carboxylate stretching vibration); 1415 cm(-1) (symmetric carboxylate stretching vibration); 1214 cm(-1) (C-O stretching vibration); 1124 and 1110 cm(-1) (asymmetric PO32- stretching vibrations); and 967 cm(-1) (symmetric PO32- stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm-1. The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na+ exhibited the same band positions as in the presence of K+, indicating very similar interaction strengths between ligand and protein in both cases.

Ort, förlag, år, upplaga, sidor
2010. Vol. 98, nr 9, s. 1931-1940
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:su:diva-49234DOI: 10.1016/j.bpj.2009.12.4335ISI: 000277377300027OAI: oai:DiVA.org:su-49234DiVA, id: diva2:376758
Anmärkning
authorCount :2Tillgänglig från: 2010-12-13 Skapad: 2010-12-13 Senast uppdaterad: 2022-02-24Bibliografiskt granskad
Ingår i avhandling
1. Infrared studies: Method development and binding of ligands to pyruvate kinase
Öppna denna publikation i ny flik eller fönster >>Infrared studies: Method development and binding of ligands to pyruvate kinase
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. s. 7
Nationell ämneskategori
Naturvetenskap
Forskningsämne
biofysik
Identifikatorer
urn:nbn:se:su:diva-56754 (URN)978-91-7447-297-4 (ISBN)
Disputation
2011-05-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
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.Tillgänglig från: 2011-05-09 Skapad: 2011-04-26 Senast uppdaterad: 2022-02-24Bibliografiskt granskad

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Kumar, SarojBarth, Andreas

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