Thermodynamic analysis of protein kinase A Ialpha activation

Thermodynamic analysis of protein kinase A (PKA) Ialpha activation was performed using Quantum 3.3.0 docking software and a Gaussian 03W quantum mechanical computational package. Expected stacking interactions between adenine of 3':5'-AMP and aromatic moieties of amino acids were taken into account by means of MP2/6-31G(d) IPCM (isodensity polarizable continuum model) computations (epsilon = 4.0). It is demonstrated that thermodynamically favorable agonist-induced PKA Ialpha activation is mediated by two processes. First, 3':5'-AMP binding is accompanied by structural changes leading to a thermodynamically favorable regulatory subunit conformation, which is hardly realized in the absence of the ligand (DeltaG degrees (R) = -23.9 +/- 8.2 kJ/mol). Second, 3':5'-AMP affinity to the regulatory subunit conformation observed after agonist-induced PKA Ialpha activation is higher than that to inactive holoenzyme complex (DeltaG degrees (3':5'-AMP) = -28.1 +/- 9.7 kJ/mol). ATP is capable of docking into the 3':5'-AMP-binding site B of the regulatory subunit complexed with the catalytic one, resulting in inhibition of kinase activation. True constants of 3':5'-AMP binding to PKA Ialpha holoenzyme were found to be 60 and 57 microM for the regulatory subunit domains A and B, respectively. These constants, unlike the binding equilibrium constant determined using established experimental techniques and ranging from 15 nM to 2.9 microM, are proved to be direct measures of 3':5'-AMP-PKA Ialpha binding affinity. Their values are in a reasonable agreement with the changes in 3':5'-AMP concentration in the cell (2-55 microM) and account for PKA Ialpha activation in response to adequate stimuli.