Using site specific mutagenesis to change residues in the substrate binding clef

Question

Using site specific mutagenesis to change residues in the substrate binding cleft of PKA (not residues involved in catalytic roles), how would you alter PKA’s substrate specifity? By this, I mean how would you specifically make mutations in the PKA enzyme amino acid sequence (not the substrate sequence) that would alter amino acid preferences for particular residues on a substrate peptide that you might want to design for this enzyme. Please think carefully about what this means. It does not mean changing ATP binding or altering the active site base or Mg binding site, as such mutations would only decrease overall catalytic activity of the phosphotransfer reaction. Please provide three detailed examples of how you could try to modify the substrate specificity of PKA (assuming that protein sequences exist in the cell that would match the newly designed substrate specificity of the enzyme).

Explanation / Answer

PKA(protein kinase A)enzyme is cyclic AMP dependent enzyme I.e it get activated only in presence of cAMP,activated PKA further activate other enzymes that are involved in various cellular processes. The catalytic site of PKA consists serine and threonine amino acid residues that catalyze the substrate reaction. Substrate specificity of pka can be changed by bringing site specific mutations in its catalytic subunit using PCR based oligo nucleotide primer technique. As you said changing one or two amino acids residues just may reduce catalytic activity but donot completely change substrate specificity but changing all the nucleotide sequence in the catalytic subunit in the PKA enzyme coding cDNA using PCR based site specific mutation techniques it is possible to change substrate specificity of PKA enzyme by mimicking the catalytic site nucleotide sequence of any other known enzyme. Later cloning the same cDNA in a suitable specific host.

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