Cyclic nucleotide specificity of the activator and catalytic sites of a cGMP‐stimulated cGMP phosphodiesterase from Dictyostelium discoideum

Abstract

The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant-mediated elevations of cGMP levels. The interaction of 20 cGMP derivatives with the activator site and with the catalytic site of the enzyme has been investigated. Binding of cGMP to the activator site is strongly reduced (more than 80-fold) if cGMP is no longer able to form a hydrogen bond at N2Hz or OZ'H. Modifications at N7, C8, 03' and 0'' induce only a small reduction of binding affinity. A cyclic phosphate structure, as well as a negatively charged oxygen atom at phosphorus, are essential to obtain activation of the enzyme. Substitution of the axial exocyclic oxygen atom by sulphur is tolerated; modification of the equatorial oxygen atom reduces the binding activity of cGMP to the activator site by 90-fold.Binding of cGMP to the catalytic site is strongly reduced if cGMP is modified at NIH, C'O, Cs and 03', while modifications at NZHz, N3, N7, 02'H, and 05' have minor effects. Both exocyclic oxygen atoms are important to obtain binding of cGMP to the catalytic site. The results indicate that activation of the enzyme by cGMP and hydrolysis of cGMP occur at different sites of the enzyme. cGMP is recognized at these sites by different types of molecular interaction between cGMP and the protein.cGMP derivatives at concentrations which saturate the activator site do not induce the same degree of activation of the enzyme (activation 2.3 -6.6-fold). The binding affinities of the analogues for the activator site and their maximal activation are not correlated. Our results suggest that the enzyme is activated because cGMP bound to the activator site stabilizes a state of the enzyme which has a higher affinity for cGMP at the catalytic site.Cyclic nucleotides have important functions in the cellular slime mold Dict-yostelium discoideum. This organism lives in the soil where it feeds on bacteria. When the food supply is exhausted the single cells aggregate to form a multicellular slug, which finally differentiates into a fruiting body. Cell aggregation is mediated by chemotaxis to CAMP, which is detected by cell surface receptors. Extracellular cAMP induces a rapid transient accumulation of intracellular cGMP levels, which reach a maximal concentration after about 10 s (for reviews see [l -31).D. discoidewn cells contain two classes of cyclic nucleotide phosphodiesterase activity. One class of enzymes hydrolyzes cAMP and cGMP with similar rates; these enzymes are located extracellularly, intracellularly and on the cell surface [4-61. A second class of enzymes hydrolyzes only cGMP and is localized only intracellularly [7 -91. Non-specific phosphodiesterase is present in large excess over the cCMP-specific enzyme. These enzyme activities can be easily separated by concanavalin-A -Sepharose column chromatography, since all CAMP-hydrolyzing activity binds to the column, while the cGMP-specific enz...

Identification of a Novel Type of cGMP Phosphodiesterase That Is Defective in the ChemotacticstmFMutants

“…The full-length protein was purified by immunoprecipitation with GFP antibodies. The purified PdeD showed cGMP-PDE activity that was stimulated by cGMP and by 8-Br-cGMP, as was previously described for the enzyme lacking in stmF mutants (Kesbeke et al, 1985). In our hands, the stimulation by cGMP was less pronounced than described by Kesbeke et al, which is perhaps a result of the use of different parental strains.…”

“…The cGMP-PDE activity in the PdeD-YFP immunoprecipitate was ϳ5% of the lysate from which it was derived. The immunoprecipitated activity was slightly activated by submicromolar cGMP concentrations and more strongly by 8-Br-cGMP, which is a good activator but a poor substrate for the stmF cGMP-PDE (Kesbeke et al, 1985). cAMP was a very poor competitor for 3 H-cGMP hydrolyzing activity.…”

“…In the original biochemical characterization of the enzyme, stimulation by cGMP was also not noted (Dicou and Brachet, 1980). 8-Br-cGMP induced a more significant stimulation, because it is a poor substrate (Kesbeke et al, 1985). cAMP was no competitor for 3 H-cGMP hydrolysis.…”

“…Studies with cGMP analogues showed that cyclic nucleotide specificity of the activator and catalytic sites of the enzyme are not identical (Kesbeke et al, 1985). Both sites are specific for cGMP, do not bind cAMP, and do not tolerate modification of the exocyclic oxygens.…”

Structural Features of the Noncatalytic cGMP Binding Sites of Frog Photoreceptor Phosphodiesterase Using cGMP Analogs

“…Furthermore, this result shows that substituting sulfur for oxygen at the cyclic phosphate position has a 10-fold greater destabilizing effect than was observed at the C-6 position of the purine ring (1). The small extent of stereospecificity (Ͻ3-fold) exhibited by the R p and S p diastereomers distinguishes the photoreceptor PDE from other cGMP binding sites (22,44). The small difference in binding affinities of the R p and S p analogs of cGMP suggest that either 1) there is a single residue in the binding pocket positioned nearly equidistant from the two exocyclic oxygens, or 2) there are two sites in the binding pocket, each one of which forms a similar interaction with the equatorial and axial exocyclic oxygens.…”