An Essential Aspartic Acid at Each of Two Allosteric cGMP-binding Sites of a cGMP-specific Phosphodiesterase

Abstract

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...

Characterization of the Tandem GAF Domain of Human Phosphodiesterase 5 Using a Cyanobacterial Adenylyl Cyclase as a Reporter Enzyme

“…within 5 min after removal of cGMP by an 840-fold dilution, because activities were identical when assayed at 5 or 250 min after dilution (8 data points assayed). Those fast k on and k off values for hPDE5 GAF-cyaB1 AC are at variance with results for cGMP binding to PDE5, where on and off kinetics were about 15 min for the on-reaction (11) and up to 7 h for the off-reaction (10,24). Similarly, binding assays with the isolated PDE5 GAF domains have demonstrated that cGMP binding and release are slow (15,17,18,24).…”

“…In mPDE5 cGMP-mediated stimulation predominantly involves the first of the two GAF domains, i.e. GAF A (10,11,15,24), whereas in cyaB1 AC, GAF B mediates cAMP signaling (6). Therefore, we were surprised that swapping the tandem GAF domain of cyaB1 with that of hPDE5 was successful.…”

“…The tandem GAF domains in mammalian PDEs 2, 5, and 6, which bind cGMP, have been analyzed intensively (2,(7)(8)(9)(10)(11)(12). The studies have been hampered by the fact that cGMP concurrently serves as a substrate and as an allosteric regulator creating an unsolvable kinetic conundrum.…”

Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: Modes of ligand binding and dimerization

“…In a chimera containing PDE6 GAF-A͞B and PDE5 catalytic domain, an Asn-to-Ala mutation in either GAF domain abolished noncatalytic cGMP binding (22). The Asp to Ala mutation in PDE5 in GAF-A or -B reduced affinity for cGMP (23). Asn, Lys, and Asp mutations to Ala in GAF-A did likewise, except for Glu to Ala, which had no effect (19).…”

Interaction of caspase‐3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1)

“…Indeed, we have shown that in the presence of the inhibitory protein (PDEc) of the rod photoreceptor PDE6, PDE5A1 is a substrate for a low activity preparation of purified caspase-3 [8]. Sitedirected mutagenesis studies have defined the position of the GAF domains [9][10][11] and key amino acid residues involved in the metal ion coordination and catalytic activity [12][13][14][15]. These are shown in Fig.…”