N(β)-methylation changes the recognition pattern of aza-β(3)-amino acid containing peptidomimetic substrates by protein kinase A

The protein kinase A (PKA)-catalyzed phosphorylation of peptide substrate RRASVA analogs, containing N(β)-Me-aza-β(3)-amino acid residues in all subsequent positions, was studied. This work follows along the lines of our previous research of the phosphorylation of aza-β(3)-analogs of RRASVA (the shortest active substrate of PKA) and allows characterizing the influence of N(β)-methylation of aza-β(3)-amino acid residues on substrate recognition by PKA on substrate binding and phosphorylation steps. It was found that the effect of N(β)-methylation was dependent upon the position of the structure alteration. Moreover, the presence of a single N(β)-methylation site in the substrate changed the recognition pattern of this series of peptidomimetics, strongly affecting the phosphorylation step. Structure modeling of aza-β(3)- and N(β)-Me-aza-β(3)-containing substrates revealed that N(β)-methylation of aza-β(3)-moieties changed the peptide bond geometry from trans- to cis-configuration in -CO-NMe- fragments, with an exception for the N-terminally methylated N(β)-Me-aza-β(3)-RRRASVA (with the N-terminal amino group not participating in the peptide bond) and RRAS-N(β)-Me-aza-β(3)-VA. As has been shown in literature, this conformational preference of the backbone has a significant influence on the flexibility of the peptide substrate chain. Following our results, this property seems to have significant influence on the recognition of the amino acid side groups by the enzyme binding site, and in the case of PKA this structural modification was decisive for the phosphate transfer step of the catalytic process.