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  • Title: High affinity binding of the heat-stable protein kinase inhibitor to the catalytic subunit of cAMP-dependent protein kinase is selectively abolished by mutation of Arg133.
    Author: Wen W, Taylor SS.
    Journal: J Biol Chem; 1994 Mar 18; 269(11):8423-30. PubMed ID: 8132568.
    Abstract:
    The two classes of physiological inhibitors of the catalytic subunit of cAMP-dependent protein kinase are the regulatory subunits and the heat-stable protein kinase inhibitors (PKIs), and both share a common mechanism of inhibition. Each has a similar inhibitor site that resembles a peptide substrate, and this occupies the P-3 to P+1 portion of the peptide recognition site. However, in addition to this consensus site, each inhibitor requires a peripheral binding site to achieve high affinity binding. Arg134 and Arg133 lie on the surface of the catalytic subunit with Arg133 coming close to the amphipathic helix of PKI(5-24) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N.-h., Taylor, S. S., and Sowadski, J. M. (1991) Science 253, 414-420). Replacement of Arg134 and Arg133 with Ala selectively abolishes the high affinity binding of PKI. Replacement of Arg133 alone is sufficient to give the same phenotype. In the presence of MgATP, the Kd,app, is increased from < 0.2 to 105 nM and, in the absence of ATP, the Kd is too large to be reliably measured. Based on the crystal structure, Arg133 hydrogen bonds to the P-7 backbone carbonyl of PKI(5-24). However, more importantly, it also contributes to the hydrophobicity of the P-11 binding site in the C.PKI(5-24) complex. We predict that it is the perturbation of this hydrophobic pocket that accounts for the effects of this mutation. In the absence of peptide, Arg133 may help to stabilize Glu230, a buried carboxylate that binds to the P-2 Arg in the crystal structure of C.PKI(5-24). Replacement of Arg133 and Arg134 with Ala has little effect on catalysis using a heptapeptide substrate and has no effect on the inhibition of the catalytic subunit by the regulatory subunit. The results thus demonstrate that these two inhibitor proteins that both bind to the catalytic subunit with a high affinity utilize different sites on the enzyme to achieve tight binding. The gamma isoform of the catalytic subunit is insensitive to inhibition by PKI and in this isoform Arg133 is replaced with Gln. We predict that this change accounts for the altered inhibitor properties of C gamma.
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