ISOLEUCINE-368 IS INVOLVED IN LOW-AFFINITY BINDING OF N-6-MODIFIED CAMP ANALOGS TO SITE-B OF THE REGULATORY SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE-I

The regulatory (R) subunit of cAMP-dependent protein kinase has a well -defined domain structure including the two in-tandem cAMP-binding sit es that constitute the C-terminus of the protein. The N-terminal bindi ng site (A) has a considerably higher affinity for analogues of cAMP t hat are substituted with bulky and hydrophobic substituents at the 6-a mino group of the adenine ring compared to the affinity observed at th e second site (B). On the basis of the crystal structure of the catabo lite gene activator protein from Escherichia coli, molecular modelling of the binding domains suggested that a tyrosine (Y244) in site A cou ld be involved in a high-affinity hydrophobic interaction, whereas a c orresponding isoleucine (I368) in domain B could lead to steric hindra nce in the binding of bulky N-6-substituted analogues. Site-directed m utagenesis was used to construct mutations in Y244 and I368. Binding d isplacement experiments showed that replacing the tyrosine in site A w ith isoleucine (Y244I) did not affect the interaction of either N-6-su bstituted or otherwise modified analogues with this site. However, rep lacing I368 with tyrosine (I368Y) led to a 3-4-fold increase in affini ty for those N-6-modified analogues that had a hydrophobic group attac hed directly or close to the 6-amino molecule. We conclude that I368 i s involved in the molecular interaction between binding domain B and t he 6-amino group of the adenine moiety of cAMP and that this residue i s partly responsible for the reduced affinity of N-6-substituted cAMP analogues for this site.