Allosteric Cooperativity in Inhibition of Protein Kinase a Catalytic Subunit

Allosteric cooperativity in inhibition of protein kinase A was studied for the first time kinetically, by using the second-order rate constants of kemptide phosphorylation, measured in the absence and presence of inhibitors, and the effect of cooperativity was characterized in terms of the interaction factor γ. This kinetic method was evaluated for differently targeted inhibitors H89 and LRRAALG-NH2, and interaction of these compounds with the free enzyme and the enzyme- substrate complexes was quantified. The inhibitory effect of these compounds was asymmetric relatively ATP and kemptide, and allosteric enhancement of LRRAALG-NH2 binding in the presence of ATP was revealed. This cooperative effect was compared with results of ligand binding studies and the principle better binding - stronger allostery was formulated and formalized in terms of a linear-free-energy relationship p(γ) = C + S pKi, where p(γ) stands for the negative logarithm of the interaction factor and pKi characterizes affinity of the free enzyme for the inhibitory peptide, C=-2.7 and S=0.9, r=0.92.