I. Huq et al., 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, Biochemical journal, 316, 1996, pp. 337-343
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.