Thermodynamic Aspects of cAMP Dependent Protein Kinase Catalytic Subunit Allostery
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Kinetics of thermal inactivation of acrylodan-labeled cAMP dependent protein kinase catalytic subunit, its binary complexes with ATP and peptide inhibitor PKI[5–24], respectively, and the ternary complex involving both of these ligands were studied at different temperatures (5–50 °C). The thermodynamic parameters ΔH and ΔS for ligand binding equilibria as well as for the allosteric interaction between the binding sites of these ligands were obtained by using the Van’t Hoff analysis. The results indicated that more inter- and intra-molecular non-covalent bonds were involved in ATP binding with the protein when compared to the peptide binding. Similarly, nucleotide and peptide binding steps were accompanied with different entropy effects, while almost no entropy change accompanied PKI[5–24] binding, suggesting that the protein flexibility was not affected in this case. Differently from the binary complex formation the ternary complex formation was accompanied by a significant entropy change and with intensive formation of new non-covalent interactions (ΔH). At the same time both ligand binding steps as well as the allosteric interaction between ligand binding sites could be described by a common entropy–enthalpy compensation plot, pointing to a similar mechanism of these phenomena. It was concluded that numerous weak interactions govern the allostery of cAMP dependent protein kinase catalytic subunit.
KeywordscAMP-Dependent protein kinase catalytic subunit Protein denaturation Fluorescence spectroscopy Acrylodan Ligand binding Protein stabilization Thermodynamics Allostery
cAMP-Dependent protein kinase A catalytic subunit from Mus musculus
This work was financially supported by the Estonian Ministry of Education and Research, Grants SF0180064s08 and IUT14-20, by Graduate School “Functional materials and technologies” receiving funding from the European Social Fund under Project 1.2.0401.09-0079 in the University of Tartu, and Kristjan Jaak and DoRa T6 Travel Grants to RK.
- 2.Kuznetsov A, Järv J (2009) Ligand structure controlled allostery in cAMP-dependent protein kinase catalytic subunit. Cent Eur J Biol 4:131–141Google Scholar
- 4.Segel IH (1975) Enzyme kinetics. Wiley-Interscience, New YorkGoogle Scholar
- 7.Kuznetsov A, Väärtnõu-Järv H, Järv J (2003) Kinetic model for protein kinase simultaneous interaction with peptide, ATP, and bi-functional inhibitor. Proc Estonian Acad Sci 52:178–187Google Scholar
- 11.Prendergast FG, Meyer M, Carlson GL, Iida S, Potter JD (1983) Synthesis, spectral properties, and use of 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan). A thiol-selective, polarity-sensitive fluorescent probe. J Biol Chem 258:7541–7544Google Scholar
- 14.Laidler KJ, Bunting PS (1973) Chemical kinetics of enzyme action. Oxford University Press, OxfordGoogle Scholar
- 18.Braxton BL, Tlapak-Simmons VL, Reinhart GD (1994) Temperature-induced inversion of allosteric phenomena. J Biol Chem 269:47–50Google Scholar
- 22.Eller M, Järv J, Toomik R, Ragnarsson U, Ekman P, Engstrom L (1993) Substrate specificity of protein kinase c studied with peptides containing d-amino acid residues. J Biochem 114:177–180Google Scholar