BINDING OF CGMP TO BOTH ALLOSTERIC SITES OF CGMP-BINDING CGMP-SPECIFIC PHOSPHODIESTERASE (PDE5) IS REQUIRED FOR ITS PHOSPHORYLATION

cGMP-binding phosphodiesterases contain two homologous allosteric cGMP -binding sites (sites (a) under bar and (b) under bar) that are arrang ed in tandem; they constitute a superfamily of mammalian cyclic nucleo tide receptors distinct from the cyclic nucleotide-dependent protein k inases/cation channels family. The functional role of each of these tw o sites in the phosphodiesterases is not known. The cGMP-binding sites of one of these phosphodiesterases, the cGMP-binding cGMP-specific ph osphodiesterase (cGB-PDE, PDES), have been analysed by using site-dire cted mutagenesis. Mutations that affect cGMP binding to either one or both allosteric sites do not influence cGMP hydrolysis in the catalyti c site under the conditions used. However, compared with wildtype enzy me, the D289A, D478A and D289A/D478A mutants, which are defective in c GMP binding to either site (a) under bar or site (b) under bar, or bot h allosteric sites, require much higher cGMP concentrations for the al losteric stimulation of phosphorylation by the catalytic subunit of cA MP-dependent protein kinase. The cGMP effect is on the cGB-PDE rather than on the catalytic subunit of the protein kinase because the latter enzyme does not require cGMP for activity. The D289N mutant, which ha s higher binding affinity for cGMP than does the wild-type enzyme, is phosphorylated at lower concentrations of cGMP than is the wildtype en zyme. It is concluded that cGMP binding to the allosteric sites of cGB -PDE does not directly affect catalysis, but binding to both of these sites regulates phosphorylation of this enzyme.