MG.ATP2--DEPENDENT INTERACTION OF THE INHIBITOR PROTEIN OF THE CAMP-DEPENDENT PROTEIN-KINASE WITH THE CATALYTIC SUBUNIT

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  • 1 January 1983
    • journal article
    • research article
    • Vol. 258  (6) , 3682-3692
Abstract
The interaction between the inhibitor protein and the catalytic subunit of the cAMP-dependent protein kinase was investigated by steady state kinetics and by an assessment of the requirement of this interaction for ATP. By analysis for tightly bound inhibitors, inhibition by the inhibitor protein was competitive vs. peptide substrate and uncompetitive vs. Mg.cntdot.ATP2-. This, together with the observations of Gronot et al. and those given in the accompanying paper, would indicate that the probable reaction mechanism of the protein kinase is ordered with the nucleotide binding first and that the inhibitor protein blocks catalysis by interaction with the catalytic subunit-Mg.cntdot.ATP complex. The Ki for this interaction at saturating Mg.cntdot.ATP and zero peptide substrate is 0.49 nM. Multiple inhibition analysis in the presence of 5''-adenylimidodiphosphate (AMP.cntdot.PNP) indicates that the inhibitor protein does not interact with a catalytic subunit-AMP.cntdot.PNP complex. The requirement for ATP for the inhibitor protein-catalytic subunit interaction has also been demonstrated by direct binding measurements and by the observation that the efficiency of the inhibitor protein is increased by preincubation of the inhibitor protein, catalytic subunit and ATP in the absence of peptide substrate. By either measurement, the catalytic subunit in the presence of the inhibitor protein, was shown to exhibit an apparent Kd of 20 .apprx. 60 nM for ATP; this value is 2 orders of magnitude higher than the affinity for ATP by the catalytic subunit alone. This high apparent affinity of the catalytic subunit for ATP (in the presence of the inhibitor) does not require that there be a specific binding site on the inhibitor protein for some moiety of the ATP, but may simply be a reflection of the formation of a catalytic subunit-Mg.cntdot.ATP.cntdot.inhibitor protein complex with resultant displacement of the equilibrium of ATP binding to the protein kinase.