Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives.
Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002147:Calmodulin / Q000494:pharmacology; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D006152:Cyclic GMP / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009712:Nucleotides, Cyclic / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats
D002147:Calmodulin; D009712:Nucleotides, Cyclic; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP