Kinetic analysis of inhibition of cAMP-dependent protein kinase catalytic subunit by the peptide–nucleoside conjugate AdcAhxArg6

Kuznetsov, Aleksei; Uri, Asko; Raidaru, Gerda; Järv, Jaak

doi:10.1016/j.bioorg.2004.05.004

Cited by 11 publications

(5 citation statements)

References 16 publications

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“…Thus H89 revealed at least some properties of bi-substrate inhibitors, which are specially designed to interact simultaneously with the ATP and peptide binding sites, and therefore should affect binding of both substrates. For a bisubstrate inhibitor AdcAhxArg 6 = 12 and = 3 [17]. Consequently, the borderline between the mono-substrate and bisubstrate inhibitors seems to be not very strict, or perhaps even not existing, at least as far as the kinetic mechanism and allosteric cooperativity of PKA is considered.…”

Section: Discussionmentioning

confidence: 99%

“…Linear-free-energy relationship between the negative logarithms of the interaction factor , characterizing allosteric effect of ATP on binding of inhibitory peptides with PKA, upon the binding effectiveness of these peptides with the free enzyme (pK i ). Data for PKA regulatory subunit, PKI and its fragments PKI(5-24) and PKI (14)(15)(16)(17)(18)(19)(20)(21)(22) were compiled from Refs [21][22][23][24][25], data for kemptide binding were taken from Ref (10), data for LRRAALG-NH 2 were taken from Table 1.…”

Section: Discussionmentioning

confidence: 99%

“…This linear-free-energy relationship was extended by including binding data for PKI fragments, namely PKI(5-24), and PKI (14)(15)(16)(17)(18)(19)(20)(21)(22), for which the p( ) and pK i values were calculated from ATP binding data, determined in the presence of these peptide inhibitors by using the acrylodan-modified enzyme [25]. It can be seen in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Initial rate of kemptide phosphorylation was measured at 30°C as described in [16,17]. Briefly, the reaction mixture (final volume 100 l, 50 mM TRIS/HCl, pH 7.5) contained kemptide at various concentrations from 5 to 200 M, -[ 32 P]ATP at various concentrations from 5 to 400 M, 10 mM of MgCl 2 and 0.10-10 μg/ml of the enzyme.…”

Section: Kinetic Measurementsmentioning

confidence: 99%

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Allosteric Cooperativity in Inhibition of Protein Kinase a Catalytic Subunit

Kuznetsov¹,

Järv²

2008

TOEIJ

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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 . This kinetic method was evaluated for differently targeted inhibitors H89 and LRRAALG-NH 2 , 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-NH 2 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 pK i , where p( ) stands for the negative logarithm of the interaction factor and pK i characterizes affinity of the free enzyme for the inhibitory peptide, C=-2.7 and S=0.9, r=0.92.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Kinetic Measurementsmentioning

confidence: 99%

See 2 more Smart Citations

Allosteric Cooperativity in Inhibition of Protein Kinase a Catalytic Subunit

Kuznetsov¹,

Järv²

2008

TOEIJ

Self Cite

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“…The initial rate of peptide phosphorylation was measured at 30 °C as described previously [15,16]. Briefly, the reaction mixture (final volume 100 µL, 50 mM TRIS/HCl, pH 7.5) contained 32 -[ P]ATP γ (concentrations between 2.5 and 150 µM), peptide (concentrations depended on the affinity of protein kinase A for the substrate), 10 mM of MgCl 2 , and 0.015-0.03 µg/mL of the enzyme.…”

Section: Kinetic Measurementsmentioning

confidence: 99%

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

Kuznetsov¹,

Järv

2008

Proc. Estonian Acad. Sci.

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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.

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cAMP-dependent protein kinase

2009

Class 2 Transferases

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Kinetic analysis of inhibition of cAMP-dependent protein kinase catalytic subunit by the peptide–nucleoside conjugate AdcAhxArg6

Cited by 11 publications

References 16 publications

Allosteric Cooperativity in Inhibition of Protein Kinase a Catalytic Subunit

Allosteric Cooperativity in Inhibition of Protein Kinase a Catalytic Subunit

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

cAMP-dependent protein kinase

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