Open AccessCyclic nucleotide specificity of the activator and catalytic sites of a cGMP‐stimulated cGMP phosphodiesterase from Dictyostelium discoideum
Author(s) -
KESBEKE Fanja,
BARANIAK Janina,
BULGAKOV Roman,
JASTORFF Bernd,
MORR Michael,
PETRIDIS Georg,
STEC Wojciech J.,
SEELA Frank,
HAASTERT Peter J. M.
Publication year - 1985
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1985.tb09083.x
Subject(s) - activator (genetics) , pde10a , dictyostelium discoideum , phosphodiesterase , phosphodiesterase 3 , chemistry , binding site , enzyme activator , enzyme , active site , biochemistry , stereochemistry , receptor , gene
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 N 2 H 2 or O 2 H. Modifications at N 7 , C 8 , O 3′ and O 5′ 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 N 1 H, C 6 O, C 8 and O 3′ , while modifications at N 2 H 2 , N 3 , N 7 , O 2′ H, and O 5′ 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.