Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3 0 5 0 -Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits Rait Kivi 1,2 • Karina Solovjova 1,3 • To ˜iv Haljasorg 1 • Piret Arukuusk 2 • Jaak Ja ¨rv 1 Published online: 15 November 2016 Ó Springer Science+Business Media New York 2016 Abstract The allosteric influence of adenosine triphos- phate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3 0 5 0 -cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme- ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denatu- ration assay, respectively It was found that the principle ‘‘better binding—stronger allostery’’ holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand–pro- tein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allos- teric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme’s overall intrinsic dynamics. Keywords cAMP-dependent protein kinase catalytic subunit  Allosteric regulation mechanism  ATP binding  Peptide binding  Ligand structure effect Abbreviations ATP Adenosine triphosphate cAMP 3 0 ,5 0 -Cyclic adenosine monophosphate 6-Cl-HOBt 6-Chloro-1-hydroxybenzotriazole DIPEA Diisopropylethylamine Fmoc Fluorenylmethyloxycarbonyl group HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate HPLC High-performance liquid chromatography MOPS 3-(N-morpholino)propanesulfonic acid PKAc 3 0 5 0 -Cyclic adenosine monophosphate dependent protein kinase catalytic subunit PKI 76-Amino acid natural protein, efficient inhibitor of 3 0 5 0 -cyclic adenosine monophosphate dependent protein kinase PKI(5-25) Peptide inhibitor TTYADFIASGRTGRRNAI, fragment of the protein kinase inhibitor protein TFA Trifluoroacetic acid TIS Triisopropylsilane 1 Introduction Protein kinases comprise an important group of enzymes known to regulate the majority of cellular pathways in humans. Since these enzymes have profound effects within the cell, they are attractive drug targets, and the estab- lishment of interactions between their distinct binding sites is an important and challenging topic for drug design [1]. As protein kinases are bisubstrate enzymes, where & Jaak Ja ¨rv jaak.jarv@ut.ee 1 Institute of Chemistry, University of Tartu, Tartu, Estonia 2 Institute of Technology, University of Tartu, Tartu, Estonia 3 Present Address: CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic 123 Protein J (2016) 35:459–466 DOI 10.1007/s10930-016-9691-9