CHARACTERIZATION OF CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES WITH CYCLIC-AMP ANALOGS - TOPOLOGY OF THE CATALYTIC SITES AND COMPARISON WITH OTHER CYCLIC AMP-BINDING PROTEINS

To define essential interactions of cAMP with the catalytic sites of c yclic nucleotide phosphodiesterases (PDEs) and to begin to map the top ology of the sites, we have tested a series of cAMP analogs as competi tive inhibitors of the PDEs that hydrolyze cAMP with high efficiency ( PDE1, PDE2, PDE3, and PDE4). Comparisons of IC50 values, relative to c AMP, were used to predict which functional groups on cAMP interact wit h each isozyme. Common to all PDEs tested, except for the calcium/calm odulin-dependent PDE (CaM-PDE, PDE1), is an interaction at the N1-posi tion of cAMP and a distinct lack of binding to the 2'-hydroxyl group o f the ribose moiety. Only the cGMP-stimulated (PDE2) and cAMP-specific (PDE4) PDEs appear to interact strongly at the N7-position. The cGMP- inhibited PDE (cGI-PDE, PDE3) may interact less strongly with this nit rogen. The PDE4 and PDE3 both interact with cAMP through the B-amino g roup, which most likely serves as a hydrogen bond donor. PDE4 and PDE3 appear to be able to bind to the anti-conformer of cAMP, whereas the PDE1 and PDE2 bind the syn-conformer. The CaM-PDE exhibits no apprecia ble specificity for any of the analogs tested, showing little or no in teraction with the 6-amino group or with any of the ring nitrogens. La rge differences exist in the nucleotide-binding requirements for the P DE catalytic sites, compared with the regulatory sites of cAMP-depende nt protein kinase and the catabolite activator protein.