CHARACTERIZATION OF CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES WITH CYCLIC-GMP ANALOGS - TOPOLOGY OF THE CATALYTIC DOMAINS

To help define essential interactions of cGMP with the catalytic site, we tested a series of cGMP analogs as competitive inhibitors of each cyclic nucleotide phosphodiesterase (PDE) family known to hydrolyze cG MP (PDE1, PDE2, PDE3, PDE5, and PDE6). IC50 values, relative to cGMP, were used to predict which functional groups of cGMP contribute to bin ding by the catalytic sites of each isozyme. The results indicate that the N1-nitrogen of cGMP contributes to binding at the catalytic site of all PDEs, probably as a hydrogen donor. All PDEs tested, with the e xception of PDE2, also use the 6-oxo group, probably as a hydrogen acc eptor. In contrast to other cGMP-binding enzymes, the 2-amino and 2'-h ydroxyl groups of cGMP are not major requirements for binding to any P DE. The 8-bromo- and 8-p-chlorophenylthio-substituted analogs inhibit PDE1, PDE2, and PDE6 activity with high relative affinities, suggestin g that these PDEs are not sterically hindered with bulky and position substitutions and that they do not preferentially bind the anti-confor mation of cGMP. PDE3 and PDE5 have reduced apparent affinity for these analogs and therefore either are sterically hindered with these subst itutions or bind cGMP in the anti-conformation. Overall, the data show substantial differences in structural requirements for cGMP binding t o the catalytic sites of the different PDE families. Comparisons with published data show different structural requirements for binding to t he catalytic, compared with noncatalytic, binding domains of PDEs. Eve n larger differences are seen between the requirements for binding to PDE catalytic sites and those for the cGMP-dependent protein kinase an d the cGMP-gated cation channel.