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 cGMP (PDE1, PDE2, PDE3, PDE5, and PDE6). IC50 values, relative to cGMP, were used to predict which functional groups of cGMP contribute to binding 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 exception of PDE2, also use the 6-oxo group, probably as a hydrogen acceptor. In contrast to other cGMP-binding enzymes, the 2-amino and 2'-hydroxyl groups of cGMP are not major requirements for binding to any PDE. The 8-bromo- and 8-p-chlorophenylthio-substituted analogs inhibit PDE1, PDE2, and PDE6 activity with high relative affinities, suggesting that these PDEs are not sterically hindered with bulky 8-position substitutions and that they do not preferentially bind the anti-conformation of cGMP. PDE3 and PDE5 have reduced apparent affinity for these analogs and therefore either are sterically hindered with these substitutions or bind cGMP in the anti-conformation. Overall, the data show substantial differences in structural requirements for cGMP binding to the catalytic sites of the different PDE families. Comparisons with published data show different structural requirements for binding to the catalytic, compared with noncatalytic, binding domains of PDEs. Even larger differences are seen between the requirements for binding to PDE catalytic sites and those for the cGMP-dependent protein kinase and the cGMP-gated cation channel.
Beltman
J
J
Department of Pharmacology, University of Washington, Seattle 98195.
Becker
D E
DE
Butt
E
E
Jensen
G S
GS
Rybalkin
S D
SD
Jastorff
B
B
Beavo
J A
JA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D018411:Spodoptera
D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP