Specificity of Cyclic GMP Activation of a Multi-substrate Cyclic Nucleotide Phosphodiesterase from Rat Liver

Abstract
Cyclic nucleotide derivatives were used to investigate the existence of distinctive activating and hydrolytic sites on the phosphodiesterase from rat liver activated by cGMP. This positively cooperative enzyme was stimulated up to 30-fold by 3 .mu.M cGMP when 3 .mu.M cAMP was used as substrate. All analogs were less potent activators than cGMP. Most cAMP derivatives were inactive, with 2 exceptions: 7-deazaadenosine 3'',5''-monophosphate and 3''-amino-3''-deoxy-adenosine 3'',5''-monophosphate. Benzimidazole ribonucleoside 3'',5''-monophosphate, where the 3 atoms of N of the pyrimidine ring are missing was a better stimulator than the intact purine-related cyclic derivative. When cAMP and cGMP with identical chemical ligands substituted at the same position were compared, the cGMP analog was always the more potent activator suggesting that the activating site is sensitive to a guanine-type cyclic nucleotide structure. Degradation of the derivatives by the enzyme was measured by high-performance liquid chromatography; no relation could be established between hydrolysis and effectiveness of activation. There was no parallelism between inhibitory and activating potency for 10 cyclic nucleotide derivatives. Since the chemical interactions between the analogs at the activating site and at the catalytic site are different, it is proposed that the sites are distinct. It is suggested that the enzyme operates in steps. In the 1st activating step cGMP is fixed by at least 2 H bonds at a specific binding site of the enzyme. This is followed by a conformational change of the protein and subsequently a change of the kinetic parameters. In a rather unspecific process and in a 2nd hydrolytic step, several purine-related cyclic nucleotides are converted to the corresponding 5'' nucleotides.

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