The catalytic mechanism of protein kinase A involves direct transfer of the 7-phosphate group of ATP to the phosphorylatable residue of peptide substrate [9], and occurs via formation of the ternary complex including the enzyme (E), ATP (A), and the phosphorylatable protein/peptide substrate (B).
Various indications can be found in earlier papers about the significant role of the allosteric cooperativity in ligand binding with protein kinase A. Most of these data have been still discussed as the influence of ATP (more precisely the ATP-Mg complex) on the binding effectiveness of peptide or protein inhibitors of this enzyme, like the regulatory subunit of protein kinase A and the heat-stable protein inhibitor [I I], but also short peptide inhibitors like peptide inhibitor LRRAALG (Ala-kemptide) [12].
In the present study allostery was revealed in the protein kinase A catalysed reaction of peptide phosphorylation, and the effect was quantified in terms of the interaction factor [alpha] for seven substrates.
The catalytic subunit [C.sub.alpha] of mouse cAMP-dependent protein kinase (protein kinase A), recombinantly expressed in E.
Briefly, the reaction mixture (final volume 100 [micro]L, 50 mM TRIS/HCI, pH 7.5) contained y-[[sup.32]P]ATP (concentrations between 2.5 and 1501yM), peptide (concentrations depended on the affinity of protein kinase A for the substrate), 10 MM of Mg[Cl.sub.2], and 0.015-0.03 [micro]g/mL of the enzyme.
The catalytic subunit of protein kinase A was expressed using plasmid pRSET B based on the T7 promoter expression system (from Invitrogen).
The catalytic subunit of protein kinase A was purified on the P-11 phosphocellulose column using isocratic elution.
Peptide phosphorylation by protein kinase A was carried out at 30[degrees]C in 100 [micro]L reaction mixture composed as follows: peptide substrate at concentrations from 5 to 200 [micro]M (the stock solutions were prepared in 50 mM TRIS/HCl, pH 7.5); [gamma]-[[sup.32]P]ATP at concentrations from 5 to 1000 [micro]M (the stock solutions had specific radioactivity 60-200 cpm/pmol); 10 [micro]L of 100 mM Mg[Cl.sub.2] solution; 15 [micro]L of the enzyme solution in buffer containing 50 mM TRIS/HCl (pH 7.5); and 1 mg/mL BSA.
This ternary complex is formed reversibly and there are indications that the formation of both enzyme-peptide and enzyme--ATP complexes follows the random mechanism in the case of protein kinase A [6].
Steady-state kinetic analysis was used to characterize the interaction of peptide substrate and ATP with protein kinase A. This analysis is based on the application of second-order rate constants, which do not reflect the kinetic details of the catalytic steps as well as the accompanying conformational and diffusion-controlled phenomena.