Effect of two simultaneous aza-β3-amino acid substitutions on recognition of peptide substrates by cAMP dependent protein kinase catalytic subunit

► Peptidomimetics containing two aza-β3-amino acids were phosphorylated by protein kinase A. ► Effect of amino acid replacement by aza-β3 analogs was dependent on position of this replacement. ► Influence of two aza-β3-amino acids was bigger than sum of effects caused by single replacements. ► Substrate backbone specificity clusters can be defined by using peptidomimetics.