AN ESSENTIAL ASPARTIC-ACID AT EACH OF 2 ALLOSTERIC CGMP-BINDING SITESOF A CGMP-SPECIFIC PHOSPHODIESTERASE

The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP -binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE ( cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repea ts a and b of all known cGMP-binding PDEs was changed to alanine by si te directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB- PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (nati ve) and WT cGB-PDE displayed identical cGMP-binding kinetics, with sim ilar to 1.8 mu M cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (K-d > 10 mu M) and the D478 A mutant showed increased affinity for cGMP (K-d approximate to 0.5 mu M) as compared to WT and native cGB-PDE. WT and native cGB-PDE dis pl ayed an identical curvilinear profile of cGMP dissociation which was c onsistent with the presence of distinct slowly dissociating (k(off) = 0.26 h(-1)) and rapidly dissociating (k(off) = 1.00 h(-1)) sites of cG MP binding. In contrast, the D289A mutant displayed a single k(off) = 1.24 h(-1) which was similar to the calculated k(off) for the fast sit e of WT and native cGB-PDE, and the D478A mutant displayed a single k( off) = 0.29 h(-1), which was similar to that calculated for the slow s ite of WT and native cGB-PDE. These results were consistent with the l oss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PD E, and the loss of a fast site in repeat b of the D478A mutant, sugges ting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for i nteraction with cGMP at each site.