Single-subunit allostery in the kinetics of peptide phosphorylation by protein kinase A; pp. 247–254Full article in PDF format | doi: 10.3176/proc.2008.4.07
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 in terms of the interaction factor 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 interrelationship was further formalized in terms of a linear-free-energy relationship pα = C + SpKb, holding between the free energy of the allosteric interaction, quantified by the negative logarithm of the interaction factor α (pα), and the effectiveness of substrate binding quantified by pKb For the peptide phosphorylation reaction C = -1.4 and S = 0.4 were obtained. The negative intercept indicated that the positive cooperativity between the binding sites, characterized by α < 1 at sub-millimolar Kb values, changed into negative cooperativity with α > 1 at millimolar Kb 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.
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