Single-subunit allostery in the kinetics of peptide phosphorylation by protein kinase A

Abstract

Allosteric cooperativity between peptide and ATP binding sites on cAMP-dependent protein kinase catalytic subunit was studied kinetically for the reaction of phosphorylation of seven peptide substrates. The allosteric effect was quantified i n terms of the interaction factor α by comparing binding effectiveness of a substrate molecule with the free enzyme and with the enzyme complex with another substrate. It was discovered that the magnitude of the allosteric feedback between these binding sites was governed by the effectiveness of substrate binding, which was varied by using different peptides, and the principle 'better binding: stronger allostery' was formulated. This inte rrelationship was further formalized in terms of a linear-free-en ergy relationship b pC p , S K α =+ holding between the free energy of the allosteric interaction, quantified by the negative logarithm of the interaction factor α (p ) α and the effectiveness of substrate binding quantified by b p. K For the peptide phosphorylation reaction C1 .4 =− and 0.4 S = were obtained. The negative intercept C indicated that the positive cooperativity between the binding sites, characterized by 1 α at millimolar b K values. This means that inversion of the cooperative effect was induced by substrate structure, and allostery was used by this enzyme as an additional mechanism to discriminate between substrates, facilitating phosphorylation of good substrates and providing additional protection against phosphorylation of bad substrates. Some implications of this allosteric mechanism on substrate specificity of protein kinases were discussed.