Phosphorylation increases affinity of the phosphodiesterase-5 catalytic site for tadalafil.

Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotide-dependent protein kinases. To test for a direct effect of phosphorylation on the PDE5 catalytic site, independent of cGMP binding to the allosteric sites of the enzyme, binding of the catalytic site-specific substrate analog [(3)H]tadalafil to PDE5 was measured. Phosphorylation increased [(3)H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [(3)H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [(3)H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic site-specific cGMP analog, 2'-O-anthraniloyl-cGMP, by approximately 3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independently of cGMP binding to the allosteric sites. The results suggested that phosphorylation acts in concert with allosteric cGMP binding to stimulate the PDE5 catalytic site, which should promote negative feedback regulation of the cGMP pathway in intact cells. By increasing the affinity of the catalytic site, phosphorylation should also consequently increase the potency and duration of PDE5 inhibitor action.

Conversion of phosphodiesterase-5 (PDE5) catalytic site to higher affinity by PDE5 inhibitors.

Phosphodiesterase-5 (PDE5) specifically hydrolyzes cGMP, thereby contributing to modulation of intracellular levels of this nucleotide. In the present study, preincubation with cGMP increased PDE5 catalytic activity for cGMP degradation, and it converted the PDE5 catalytic site to a form that was more potently inhibited by each of the three PDE5 catalytic site-specific inhibitors: sildenafil, vardenafil, and tadalafil. These results implied that elevated cGMP initiates a physiological negative feedback on the cGMP pathway by increasing the affinity of the PDE5 catalytic site for cGMP. This increase in catalytic site activity or affinity for inhibitors could be caused by binding of cGMP to either the PDE5 allosteric sites, catalytic site, or both. Whether occupation of the catalytic site alone could mediate the effect was examined using radiolabeled PDE5 inhibitors in the absence of cGMP. Exchange-dissociation of [(3)H]sildenafil (Viagra), [(3)H]vardenafil (Levitra), or [(3)H]tadalafil (Cialis) from full-length PDE5 or isolated catalytic domain revealed two kinetic components (slow and fast). Extended preincubation of full-length PDE5, but not isolated catalytic domain, with (3)H inhibitors converted the biphasic pattern to a single slow (high-affinity) component. Studies of amino-terminally truncated PDE5 established that full-length mammalian GAF-B (cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlA) subdomain conjoined with the catalytic domain was sufficient for this conversion. In conclusion, binding of substrate or substrate analogs such as PDE5 inhibitors to the catalytic site converts a fast (low-affinity) inhibitor dissociation component of the PDE5 catalytic site to a slow (high-affinity) inhibitor dissociation component. This effect is predicted to improve the substrate affinity or inhibitory potencies of these compounds in intact cells.

Phosphorylation of phosphodiesterase-5 is promoted by a conformational change induced by sildenafil, vardenafil, or tadalafil.

Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, vardenafil, or tadalafil) or phosphorylation by cyclic nucleotide-dependent protein kinase causes an apparent conformational change in PDE5, as indicated by a shift in migration on non-denaturing PAGE gels and an altered pattern of tryptic digestion. Combination of cGMP and a PDE5 inhibitor or phosphorylation does not cause a further gel shift or change in tryptic digest. Phosphorylation of PDE5 is stimulated by inhibitors, and combination of cGMP and inhibitor does not cause further phosphorylation. Dephosphorylation of PDE5 by either purified phosphoprotein phosphatase-1 or -2A catalytic subunit or by a crude phosphatase mixture is not affected by cGMP or inhibitors, suggesting that phosphorylation itself maintains conformational exposure of the phosphorylation site. The combined results imply that cGMP binding to the catalytic site initiates negative feedback control of many cellular cGMP signaling pathways by directly stimulating phosphorylation and activation of PDE5; by exploiting this molecular mechanism, PDE5 inhibitors stimulate their own potencies.

A 46-amino acid segment in phosphodiesterase-5 GAF-B domain provides for high vardenafil potency over sildenafil and tadalafil and is involved in phosphodiesterase-5 dimerization.

Phosphodiesterase-5 (PDE5) contains a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that contains two mammalian cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlAs (GAFs) (A and B) and a phosphorylation site for cyclic nucleotide-dependent protein kinases (cNPKs). Binding of cGMP to GAF-A increases cNPK phosphorylation of PDE5 and improves catalytic site affinity for cGMP or inhibitors. GAF-B contributes to dimerization of PDE5, inhibition of cGMP binding to GAF-A, and sequestration of the phosphorylation site. To probe potential PDE5 R domain effects on catalytic site affinity for certain inhibitors, four N-terminal truncation mutants were generated: PDE5Delta1-321 contained GAF-B domain, C domain, and the sequence between GAF-A and -B; PDE5Delta1-419 contained GAF-B and C domain; PDE5Delta1-465 contained the C domain and the C-terminal portion of GAF-B; and PDE5Delta1-534 contained only C domain. Truncated proteins with a complete GAF-B were dimers, but those lacking the N-terminal 46 amino acids of GAF-B were monomers, indicating that these residues are vital for GAF-B-mediated PDE5 dimerization. K(m) values of the mutants for cGMP were similar to that of full-length PDE5. All PDE5 constructs had similar affinities for 3-isobutyl-1-methylxanthine, sildenafil, tadalafil, and UK-122764, but mutants containing a complete GAF-B had 7- to 18-fold higher affinity for vardenafil-based compounds compared with those lacking a complete GAF-B. This indicated that the N-terminal 46 amino acids in GAF-B are required for high vardenafil potency. This is the first evidence that PDE5 R domain, and GAF-B in particular, influences affinity and selectivity of the catalytic site for certain classes of inhibitors.

Correlation of seasonal acclimatization in metabolic enzyme activity with preferred body temperature in the Eastern red spotted newt (Notophthalmus viridescens viridescens).

Eastern red spotted newts, as aquatic adults, are active year round. They are small and easy to handle, and thus lent themselves to a laboratory study of seasonal changes in preferred body temperature and biochemical acclimatization. We collected newts in summer (n=20), late fall (n=10) and winter (n=5). Ten each of the summer and late fall newts were subjected to an aquatic thermal gradient. Summer newts maintained higher cloacal temperatures than late fall newts (26.8+/-0.5 degrees C and 17.2+/-0.4 degrees C, respectively). In addition, the activity of three muscle metabolic enzymes (cytochrome c oxidase (CCO), citrate synthase (CS) and lactate dehydrogenase (LDH)) was studied in all newts collected. Newts compensated for lower late fall and winter temperatures by increasing the activity of CCO during those seasons over that in summer newts at all assay temperatures (8, 16 and 26 degrees C). The activity of CS was greater in winter over summer newts at 8 and 16 degrees C. No seasonal differences in LDH activity were demonstrated. These data in newts indicate that this amphibian modifies some muscle metabolic enzymes in relation to seasonal changes and can modify its behavioral in a way that correlates with those biochemical changes.

Structural and functional features in human PDE5A1 regulatory domain that provide for allosteric cGMP binding, dimerization, and regulation.

The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K(D) < 30 nM) and between GAF b modules (K(D) = 1-20 pM), and the sequence between the GAFs (Thr322-Asp403) contributes to dimer stability; 2) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(D) < 40 nM) than does the isolated R domain (K(D) = 110 nM) or holoenzyme (K(D) = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu321) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser102 phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.

Binding of tritiated sildenafil, tadalafil, or vardenafil to the phosphodiesterase-5 catalytic site displays potency, specificity, heterogeneity, and cGMP stimulation.

Sildenafil, tadalafil, and vardenafil each competitively inhibit cGMP hydrolysis by phosphodiesterase-5 (PDE5), thereby fostering cGMP accumulation and relaxation of vascular smooth muscle. Biochemical potencies (affinities) of these compounds for PDE5 determined by IC(50), K(D) (isotherm), K(D) (dissociation rate), and K(D) ((1/2) EC(50)), respectively, were the following: sildenafil (3.7 +/- 1.4, 4.8 +/- 0.80, 3.7 +/- 0.29, and 11.7 +/- 0.70 nM), tadalafil (1.8 +/- 0.40, 2.4 +/- 0.60, 1.9 +/- 0.37, and 2.7 +/- 0.25 nM); and vardenafil (0.091 +/- 0.031, 0.38 +/- 0.07, 0.27 +/- 0.01, and 0.42 +/- 0.10 nM). Thus, absolute potency values were similar for each inhibitor, and relative potencies were vardenafil >> tadalafil > sildenafil. Binding of each (3)H inhibitor to PDE5 was specific as determined by effects of unlabeled compounds. (3)H Inhibitors did not bind to isolated PDE5 regulatory domain. Close correlation of EC(50) values using all three (3)H inhibitors competing against one another indicated that each occupies the same site on PDE5. Studies of sildenafil and vardenafil analogs demonstrated that higher potency of vardenafil is caused by differences in its double ring. Exchange-dissociation studies revealed two binding components for each inhibitor. Excess unlabeled inhibitor did not significantly affect (3)H inhibitor dissociation after infinite dilution, suggesting the absence of subunit-subunit cooperativity. cGMP addition increased binding affinity of [(3)H]tadalafil or [(3)H]vardenafil, an effect presumably mediated by cGMP binding to PDE5 allosteric sites, implying that either inhibitor potentiates its own binding to PDE5 in intact cells by elevating cGMP. Without inhibitor present, cGMP accumulation would stimulate cGMP degradation, but with inhibitor present, this negative feedback process would be blocked.

Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity.

Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain). The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate. A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied. The rate, cGMP dependence, and stoichiometry of phosphorylation of the PDE5 R domain by the catalytic subunit of cAMP-dependent protein kinase are comparable with that of the holoenzyme. Migration in native polyacrylamide gels suggests that either cGMP binding or phosphorylation produces distinct conformers of the R domain. Phosphorylation of the R domain increases affinity for cGMP approximately 10-fold (K(D) values 97.8 +/- 17 and 10.0 +/- 0.5 nm for unphospho- and phospho-R domains, respectively). [(3)H]cGMP dissociates from the phospho-R domain with a single rate (t(12) = 339 +/- 30 min) compared with the biphasic pattern of the unphospho-R domain (t(12) = 39.0 +/- 4.8 and 265 +/- 28 min, for the fast and slow components, respectively). Thus, cGMP-directed regulation of PDE5 phosphorylation and the resulting increase in cGMP binding affinity occur largely within the R domain. Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis.

A conserved serine juxtaposed to the pseudosubstrate site of type I cGMP-dependent protein kinase contributes strongly to autoinhibition and lower cGMP affinity.

Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type Ialpha and type Ibeta (PKG-Ialpha and PKG-Ibeta), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-Ialpha (S64A, S64T, S64D, and S64N) and PKG-Ibeta (S79A). In wild-type PKG-Ialpha, basal kinase activity ratio (-cGMP/+cGMP) is 0.11, autophosphorylation increases this ratio 3-fold, and the K(a) and K(D) values for cGMP are 127 and 36 nm, respectively. S64A PKG-Ialpha basal kinase activity ratio increases 2-fold, cGMP binding affinity increases approximately 10-fold in both K(a) and K(D), and activation by autophosphorylation is slight. S64D and S64N mutants are nearly constitutively active in the absence of cGMP, cGMP binding affinity in each increases 18-fold, and autophosphorylation does not affect the kinase activity of these mutants. Mutation of the homologous site in PKG-Ibeta (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-Ibeta. The combined results demonstrate that a conserved serine juxtaposed to the pseudosubstrate site in type I PKGs contributes importantly to enzyme function by increasing autoinhibition and decreasing cGMP binding affinity.