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 cGMPbinding 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 repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with ϳ1.8 M cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (K d > 10 M) and the D478A mutant showed increased affinity for cGMP (K d Ϸ 0.5 M) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (k off ‫؍‬ 0.26 h ؊1 ) and rapidly dissociating (k off ‫؍‬ 1.00 h ؊1 ) sites of cGMP 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 site 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 site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site. Cyclic nucleotide phosphodiesterases (PDEs)1 constitute a complex family of enzymes which catalyze the hydrolysis of 3Ј:5Ј-cyclic nucleotides to the corresponding nucleoside 5Ј-monophosphates. The multiple PDEs differ in their substrate specificities, sensitivities to inhibitors, modes of regulation, and tissue distributions. Most PDEs are chimeric multidomain proteins, possessing distinct catalytic and regulatory domains (1). A 250-amino acid segment of sequence, which is conserved among all mammalian PDEs and is located in the more carboxyl-terminal portions of the PDE molecules, contains the catalytic site of these enzymes (1-4). Domains of the PDEs which interact with allosteric/regulatory factors are thought to be located within the more amino-terminal regions (1, 5, 6).The cGMP-binding PDEs comprise a heterogeneous subgroup of PDEs, all of which exhibit allosteric cGMP-binding sites that are distinct from the sites of cyclic nucleotide hydrolysis. This group consists of at least three classes of PDEs: the cGMP-stimulated PDEs (cGS-PDEs, or PDE2s 2 ) (7), the photoreceptor PDEs (rod outer segment PDE (ROS-PDE; PDE6A/B) (8) and cone PDE (PDE6C) (9)), and the cGMP-binding, cGMPspecific PDE (cGB-PDE; PDE5A) (10). The s...

, and Asp 289 in cGMP binding. These residues could be presented as a putative NKX n D motif, and their functions were predicted based on analogy with the canonical NKXD motif in GTP-binding proteins. No marked differences in catalytic functions such as specific activity, K m for cGMP, and IC 50 for zaprinast or 3-isobutyl-1-methylxanthine were found among wild-type and mutant cGB-PDEs. This suggested that cGMP binding to site a does not influence the catalytic properties of cGB-PDE.3Ј:5Ј-Cyclic nucleotide phosphodiesterases (PDEs) 1 catalyze the hydrolysis of 3Ј:5Ј-cyclic nucleotides to the corresponding nucleoside 5Ј-monophosphates. On the basis of their regulatory features and substrate specificities, the multiple PDEs can be classified into at least seven major families, three of which exhibit cGMP-binding sites that are distinct from sites of cyclic nucleotide hydrolysis (1). Comparison of their amino acid sequences reveals a conserved segment located toward the N terminus, which has been proposed to constitute an allosteric cGMP-binding region. This region consists of two similar blocks of conserved residues: block a ϭ NKX 5 or 7 FX 3 DE and block b ϭ N(K/R)X 4 or 10 FX 3 DE. These internally homologous repeats were postulated to be contained in two cGMP-binding sites, a and b, which have different affinities for cGMP (2).Recently, studies of site-directed mutagenesis of the cGMPbinding, cGMP-specific PDE (cGB-PDE; PDE 5A) (3) provided evidence for the importance of Asp 289 in block a and Asp 478 in block b for cGMP binding. These two residues are the first amino acid residues to be identified as critical for cGMP binding in allosteric sites of cGMP-binding PDEs. [ 32 P]cGMP dissociation kinetics of each of the mutant cGB-PDEs identified site a and site b as the high affinity site and low affinity site, respectively.Since the discovery of cyclic nucleotides, the characterization of cAMP and cGMP binding to their receptors has been the subject of intense investigations. As a result, the cyclic nucleotide-binding sites of catabolite gene activator protein (CAP) (4), cAMP-and cGMP-dependent protein kinases (5-7), and the cGMP-gated cation channels (8) have been described in detail. The major interactions for all of these proteins are similar: glutamic acid bonds with the ribose 2Ј-OH, while arginine forms an ion pair with the charged phosphate oxygen. The cGMP-binding sites of cGMP-binding PDEs do not exhibit any sequence similarity with CAP-related proteins (9), and the cyclic nucleotide-binding characteristics between these two families of proteins are quite different. In order to probe the structural elements that contribute to cGMP binding and to develop a better understanding of the functions of these elements in the cGMP-binding sites of PDEs, five mutants in the invariant N 276 KX 7 FX 3 DE 290 sequence of site a of cGB-PDE were generated. N276A, K277A, K277R, D289A, and E290A mutants were expressed in insect High Five cells, and then purified. These mutations were selected in part because of the res...

Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6␣) in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6␣/PDE5 enzyme containing the PDE6␣ noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/ High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6␣. The inhibitory Photoreceptor phosphodiesterases (PDEs) 1 serve as effector enzymes in the G protein-mediated visual transduction cascade (1-3). During transduction of the visual signal in vertebrate photoreceptor rod and cone cells, the activated G protein (transducin) ␣ subunit stimulates PDE catalytic activity by relieving the inhibitory constraint imposed by two identical inhibitory P␥ subunits. A recently adopted classification of cyclic nucleotide PDEs recognizes seven different families based on primary sequence and regulation (4). PDEs within each of the families have 60% or more homology while similarities between different families are 40% or less. According to this nomenclature, photoreceptor rod and cone PDEs comprise the PDE6 family (4). Rod photoreceptor PDE is composed of two large homologous catalytic ␣ and ␤ subunits of nearly identical size (molecular masses of 99.2 and 98.3 kDa) and two copies of an inhibitory ␥ subunit (molecular mass 9.7 kDa) (5-8). Cone PDE is composed of two identical ␣Ј subunits (molecular masses of 98.7 kDa) (9, 10), which share Ͼ60% homology with PDE6␣ and PDE6␤ (11). An inhibitory cone P␥ subunit that is highly homologous to rod P␥ and specific for a subset of cone photoreceptors has been identified (12). Recently, a rod-specific

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The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors of the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photoaffinity labeling was progressively blocked by cGMP at concentrations higher than 10 microM, whereas cAMP or 5'-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, cIMP, and beta-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 microM, which compared favorably with an IC50 of 5 microM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.