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 in 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 interrelationship was further formalized in terms of a linear-free-energy relationship b p C p , S K α < at sub-millimolar b K values, changed into negative cooperativity with 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.Abbreviations: Ala-kemptide -peptide inhibitor LRRAALG; AMPPNP -, -imidoadenosine β γ 5′-triphosphate; kemptidepeptide substrate LRRASLG; protein kinase A -catalytic subunit of cAMP dependent protein kinase.Enzyme: cAMP dependent protein kinase -EC 2.7.11.1.

Ligand structure controlled allostery in cAMP-dependent protein kinase catalytic subunit

“…Our recent kinetic studies have revealed that the catalytic activity of protein kinase A was indeed governed by interaction between binding sites of two different substrates which can be said to be allosteric [10,11]. Moreover, allosterically regulated ligand binding properties have been observed for this enzyme also in several ligand binding studies, as summarized below.…”

“…4, as initially mentioned in our previous paper [10]. These data were characterized by the intercept value C b = -1.4 ± 0.1 and by the slope value S b = 0.43 ± 0.03.…”

“…This trend had been previously observed, and the principle "better binding: stronger allostery" was formulated [10,11]. This qualitative pattern was formalized in terms of linear freeenergy relationships, as described below.…”

“…These data were compiled from our recent study, where kinetics of phosphorylation of seven different peptides was measured and both pK b and pα values were calculated from the steady-state kinetic data [10]. As substrates of various structure and binding affinity were used in this work, clear dependence of the interaction factor upon substrate structure was revealed.…”

“…This principle has been formalized in terms of linear free-energy relationships between pα and pK b values [10] and pγ and pK i values [11], using the following correlation equations:…”