Identification of overlapping but distinct cAMP and cGMP interaction siteswith cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B

Cyclic nucleotide phosphodiesterase 3A (PDE3A) hydrolyzes cAMP to AMP, but is competitively inhibited by cGMP due to a low k(cat) despite a tight K-m. Cyclic AMP elevation is known to inhibit all pathways of platelet activati on, and thus regulation of PDE3 activity is significant. Although cGMP elev ation will inhibit platelet function, the major action of cGMP in platelets is to elevate cAMP by inhibiting PDE3A. To investigate the molecular detai ls of how cGMP, a similar but not identical molecule to cAMP, behaves as an inhibitor of PDE3A. we constructed a molecular model of the catalytic doma in of PDE3A based on homology to the recently determined X-ray crystal stru cture of PDE4B. Based on the excellent fit of this model structure, we muta ted nine amino acids in the putative catalytic cleft of PDE3A to alanine us ing site-directed mutagenesis. Six of the nine mutants (Y751A, H840A, D950A , F972A, Q975A, and F1004A) significantly decreased catalytic efficiency, a nd had k(cat)/K-m less than 10% of the wild-type PDE3A using cAMP as substr ate. Mutants N845A, F972A, and F1004A showed a 3- to 12-fold increase of K- m for cAMP. Four mutants (Y751A, H840A, D950A, and F1004A) had a 9- to 200- fold increase of K-i for cGMP in comparison to the wild-type PDE3A. Studies of these mutants and our previous study identified two groups of amino aci ds: E866 and F1004 contribute commonly to both cAMP and cGMP interactions w hile N845, E971, and F972 residues are unique for cAMP and the residues Y75 1, H836, H840, and D950 interact with cGMP. Therefore, our results provide biochemical evidence that cGMP interacts with the active site residues diff erently from cAMP.