No abstract

The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dicfyostelium discoideum has been investigated by measuring the apparent K, and maximal velocity (V) of 24 derivatives of adenosine 3',5'-nionophosphate (CAMP). Several analogs have increased K,,, values, but unaltered V values if compared to CAMP; also the contrary (unaltered X, and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of CAMP.cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme.The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.cAMP is a key regulator of metabolism, function and growth of many cell types [l]. In mammalian cells CAMP is the second messenger of many hormones; it achieves its function via binding to an intracellular receptor, which is a protein kinase. In the cellular slime mold Dicfyostelium discoideum cAMP acts as the first messenger [2]; it is excreted by the cells and achieves its function via binding to a cell surface receptor. In D. discoideum cAMP induces chemotaxis and cell aggregation (for reviews see [3,4]).The level of cAMP is controlled by the rate of synthesis catalyzed by adenylate cyclase, the rate of excretion, and the rate of degradation by cyclic nucleotide phosphodiesterase.In mammalian tissue at least three types of phosphodiesterase have been demonstrated : a calmodulin-dependent enzyme, a CAMP-specific enzyme, and a third form characterized by cGMP activation (for reviews see [5,6]). In the cellular slime molds at least two enzymes have been demonstrated: one enzyme is localized on the cell surface and in the extracellular medium [7,8] and hydrolyzes cAMP and cGMP with about equal rates, and one cGMP-specific enzyme is localized intracellularly [9-111. cAMP and c...

Cyclic nucleotide derivatives have been used as a tool to investigate the existence of distinctive activating and hydrolytic sites 011 the phosphodiesterase from rat liver activated by cGMP (guanosine 3',5'-monophosphate). This positively cooperative enzyme was stimulated up to 30-fold by 3 pM cGMP when 3 pM CAMP (adenosine 3',5'-monophosphate) was used as substrate. All analogues were less potent activators than cGMP. Most CAMP derivatives were inactive, with two exceptions : 7-deazaadenosine 3',5'-monophosphate and 3'-amino-3'-deoxyadenosine 3',5'-monophosphate. Benzimidazole ribonucleoside 3',5'-monophosphate, where the two atoms of nitrogen 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 analogue was always the more potent activator suggesting that the activating site is sensitive to a guaninetype cyclic nucleotide structure. Degradation of the derivatives by the enzyme was measured by highperformance liquid chromatography : no relation could be established between hydrolysis and effectiveness of activation. In addition, there was no parallelism between inhibitory and activating potency for ten cyclic nucleotide derivatives. Since the chemical interactions between the analogues at the activating site on the one hand and at the catalytic sitc on the other, are different, it is proposed that the sites are distinct. Consequently, it is suggested that the enzyme operates in steps. In the first activating step, cGMP is fixed by at least two hydrogen 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 second hydrolytic step, several purine-related cyclic nucleotides are converted to the corresponding 5' nucleotides.

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...

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.