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 ARG(133)

The two classes of physiological inhibitors of the catalytic subunit o f cAMP-dependent protein kinase are the regulatory subunits and the he at-stable protein kinase inhibitors (PKIs), and both share a common me chanism of inhibition. Each has a similar inhibitor site that resemble s 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 af finity binding. Arg(134) and Arg(133) lie on the surface of the cataly tic subunit with Arg(133) coming close to the amphipathic helix of PKI (5-24) (Knighton, D. R., Zheng, J., Ten Eyck, L. F., Xuong, N. h., Tay lor, S. S., and Sowadski, J. M. (1991) Science 253, 414-420). Replacem ent of Arg(134) and Arg(133) with Ala selectively abolishes the high a ffinity binding of PKI. Replacement of Arg(133) alone is sufficient to give the same phenotype. In the presence of MgATP, the K-d,K-app,K- i s increased from <0.2 to 105 nM and, in the absence of ATP, the K-d is too large to be reliably measured. Based on the crystal structure, Ar g(133) hydrogen bonds to the P-7 backbone carbonyl of PKI(5-24). Howev er, 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 t he perturbation of this hydrophobic pocket that accounts for the effec ts of this mutation. In the absence of peptide, Arg(133) may help to s tabilize Glu(230), a buried carboxylate that binds to the P-2 Arg in t he crystal structure of C.PKI(5-24). Replacement of Arg(133) and Arg(1 34) With Ala has little effect on catalysis using a heptapeptide subst rate and has no effect on the inhibition of the catalytic subunit by t he regulatory subunit. The results thus demonstrate that these two inh ibitor proteins that both bind to the catalytic subunit with a high af finity utilize different sites on the enzyme to achieve tight binding. The gamma isoform of the catalytic subunit is insensitive to inhibiti on by PKI and in this isoform Arg(133) is replaced with Gln. We predic t that this change accounts for the altered inhibitor properties of C gamma.