Cyclic nucleotide derivatives as probes of phosphodiesterase catalytic and regulatory sites.
The present data are consistent with the following mechanism of activation of the cGMP-stimulated PDE: cGMP is first bound to an allosteric site of the enzyme; this is followed by a conformational change in protein structure, a shift of kinetic behavior, and sequential activation of cAMP PDE hydrolysis (18). Evidence for the existence of distinct activating and catalytic sites is obtained from the use of several cyclic nucleotide derivatives to elucidate the essential molecular interactions at both of these sites (4). Since the liver PDE under study exhibits positive homotropic cooperativity by cAMP, the stimulatory effect of low concentrations of MIX is consistent with its binding at the substrate-active sites. As shown previously, this enzymatic mechanism is reproduced by an analog of the substrate, c6clPMP, but not by cGMP. Since the order of potency of a series of competitive inhibitors of two PDE (i.e., the cyclic GMP-sensitive enzyme and a calmodulin-sensitive enzyme), is not parallel, it is suggested that the active sites of these enzymes are distinct. The interaction of MIX at the active site of two PDE studied here could reflect a general binding mechanism of the xanthine due to similar chemical forces. Since we propose that the allosteric-activation site is specific for cGMP, the xanthine does not bind to that site. This is also suggested from substrate-velocity relationships measured in the presence of MIX and cGMP (Table 3). Complete characterizations of the cGMP-activating site and the xanthine-sensitive catalytic site are required in order to elucidate the exact chemical interactions at both sites on the cGMP-stimulated PDE.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009712:Nucleotides, Cyclic / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats
D009712:Nucleotides, Cyclic; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases