Abstract
For optimal activity the catalytic subunit of cAMP-dependent protein kinase requires a phosphate on Thr-197. This phosphate anchors the activation loop in the proper conformation and contributes to catalytic efficiency by enhancing the phosphoryl transfer rate and increasing the affinity for ATP (1). The crystal structure of the catalytic subunit bound to ATP, and the inhibitor peptide, IP20, highlights the contacts made by the Thr-197 phosphate as well as the role adjacent residues play in contacting the substrate peptide. Glu-203 and Tyr-204 interact with arginines in the consensus sequence of PKA substrates at the P-6 and P-2 positions, respectively. To assess the contribution that each residue makes to peptide recognition, the kinetic properties of three mutant proteins (E203A, Y204A, and Y204F) were monitored using multiple peptide substrates. The canonical peptide substrate, Kemptide, as well as a longer 9-residue peptide and corresponding peptides with alanine substitutions at the P-6 and P-2 positions were used. While the effect of Glu-203 is more localized to the P-6 site, Tyr-204 contributes to global peptide recognition. An aromatic hydrophobic residue is essential for optimal peptide recognition and is conserved throughout the protein kinase family.
Highlights
For optimal activity the catalytic subunit of cAMP-dependent protein kinase requires a phosphate on Thr197
A interesting mutation, highlighted in these experiments (Y204A), yielded an enzyme that did not phosphorylate Kemptide, but was able to autophosphorylate when expressed in E. coli
The question of whether Glu-203 would discriminate among substrates as Y204A if there were a P-6 Arg could be determined by using a longer peptide substrate
Summary
For optimal activity the catalytic subunit of cAMP-dependent protein kinase requires a phosphate on Thr197. This phosphate anchors the activation loop in the proper conformation and contributes to catalytic efficiency by enhancing the phosphoryl transfer rate and increasing the affinity for ATP [1]. Residues 188 –192 comprise -sheet 9, the only element of regular secondary structure This segment interacts with the A-helix outside the core as well as the essential phosphate on Thr-197 in the activation loop. The activation loop is a site of regulation for most members of the protein kinase family, where phosphorylation on one or two key Tyr, Thr, or Ser residues is required for optimal activity [7]. These residues serve as an anchor to the large lobe via interaction with Arg280, another conserved residue in the large lobe
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