Conformational Difference between PDE4 Apoenzyme and Holoenzyme

Abstract

The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that PDE4 exists in two conformations that bind the inhibitor rolipram with affinities differing by more than 100-fold. Here we report that these two conformations are the consequence of PDE4 binding to its metal cofactor such as Mg(2+). Using a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay, we identified that L-791,760, a fluorescent inhibitor, binds to the apoenzyme (free enzyme) and the holoenzyme (enzyme bound to Mg(2+)) with comparable affinities (K(d) approximately 30 nM). By measuring the displacement of the bound L-791,760, we have also identified that other inhibitors bind differentially with the apoenzyme and the holoenzyme depending upon their structure. CDP-840, SB-207499, and RP-73401 bind preferentially to the holoenzyme. The conformational-sensitive inhibitor (R)-rolipram binds to the holoenzyme and apoenzyme with affinities (K(d)) of 5 and 300 nM, respectively. In contrast to its high affinity (K(d) approximately 2 microM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K(d) approximately 170 microM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.

Deletion of phosphodiesterase 4D in mice shortens α2-adrenoceptor–mediated anesthesia, a behavioral correlate of emesis

“…Four representative human enzymes, PDE4A 248 (HSPDE4A4B ) and its PDE4B, PDE4C, and PDE4D equivalents, with the divergent N-termini of the splice variants truncated at the conserved GlnThr dipeptide upstream of the catalytic domain for each PDE4 subtype, were expressed as GST fusion proteins and purified to homogeneity from Sf9 cells according to the published procedure (11). IC 50 values (mean ± SD, n ≥ 3) of inhibitors were determined from an 11-point dose-response curve performed in duplicate at 0.1 µM 3 H-cAMP in 20 mM HEPES (pH 7.5), 10 mM MgCl 2 , 1 mM EDTA, and 100 mM KCl at 30°C (12).…”

“…(24) Table 1. The biochemical characterization of PMNPQ was previously published under the incorrect name of RS14203 following a mistake in the identification of the compound (11,26). The following drugs and test compounds were also used: erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA; BIOMOL Research Laboratories Inc., Plymouth Meeting, Pennsylvania, USA), milrinone and dipyridamole (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada), xylazine (Rompun; Bayer, Etobicoke, Ontario, Canada), ketamine (Ketaset; Ayerst, Montreal, Quebec, Canada), polyethylene glycol (PEG; molecular weight 200; Sigma, Milwaukee, Wisconsin, USA), vinpocetine and dipyridamole (Tocris Cookson Inc., Ballwin, Missouri, USA), sodium pentobarbital (Somnotol; MTC Pharmaceuticals, Cambridge, Ontario, Canada), and MK-912 (synthesized at Merck Research Laboratories, Rahway, New Jersey, USA).…”

“…The reversible binding of the cation cofactors (e.g., Mg 2+ ) results in the presence of two coexisting conformers that bind inhibitors differently: the holoenzyme (enzyme bound with Mg 2+ ) and the apoenzyme (free enzyme) (11,12). In the past, it was observed that the potency of some inhibitors (e.g., rolipram) on PDE activity deviated from their affinity at the high-affinity rolipram binding site (HARBS); this led to the proposal that inhibitors with a reduced potency on the HARBS may have an improved therapeutic index over that of firstgeneration compounds (13-15).…”

“…In the past, it was observed that the potency of some inhibitors (e.g., rolipram) on PDE activity deviated from their affinity at the high-affinity rolipram binding site (HARBS); this led to the proposal that inhibitors with a reduced potency on the HARBS may have an improved therapeutic index over that of firstgeneration compounds (13-15). It has now been clarified that the HARBS corresponds to the holoenzyme conformer responsible for PDE4 catalysis (11,12).…”

“…In the past, it was observed that the potency of some inhibitors (e.g., rolipram) on PDE activity deviated from their affinity at the high-affinity rolipram binding site (HARBS); this led to the proposal that inhibitors with a reduced potency on the HARBS may have an improved therapeutic index over that of firstgeneration compounds (13-15). It has now been clarified that the HARBS corresponds to the holoenzyme conformer responsible for PDE4 catalysis (11,12).The PDE4 family is composed of four subtypes (PDE4A-D) and multiple splice variants (16). If it were possible to identify the subtype(s) responsible for the beneficial and the side effects associated with PDE4 inhibition, then subtype-selective inhibitors devoid of the tendency to induce nausea and vomiting could be developed.…”