Allosteric Cooperativity in Inhibition of Protein Kinase a Catalytic Subunit

Abstract

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 ef- fect of cooperativity was characterized in terms of the interaction factor  . This kinetic method was evaluated for differ- ently targeted inhibitors H89 and LRRAALG-NH2, and interaction of these compounds with the free enzyme and the en- zyme-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( ) = C + S pKi, where p( ) 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.