Thermodynamic analysis of protein kinase A Iα activation

Thermodynamic analysis of protein kinase A (PKA) Iα 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 (iso-density polarizable continuum model) computations (ɛ = 4.0). It is demonstrated that thermodynamically favorable agonist-induced PKA Iα 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 (ΔG_R^o = −23.9 ± 8.2 kJ/mol). Second, 3′:5′-AMP affinity to the regulatory subunit conformation observed after agonist-induced PKA Iα activation is higher than that to inactive holoenzyme complex (ΔG_{3′:5′−AMP}^o = −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 Iα holoenzyme were found to be 60 and 57 μM 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 μM, are proved to be direct measures of 3′:5′-AMP-PKA Iα binding affinity. Their values are in a reasonable agreement with the changes in 3′:5′-AMP concentration in the cell (2-55 μM) and account for PKA Iα activation in response to adequate stimuli.