title: Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding.
abstract: Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30°C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn(++) or Mg(++) caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, United States. Jackie.corbin@vanderbilt.edu
authors.forename: Jackie D
authors.initials: JD
authors.lastname: Corbin
authors.forename: Sharron H
authors.initials: SH
authors.lastname: Francis
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D002243:Carbolines / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D058986:Phosphodiesterase 5 Inhibitors / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011687:Purines / Q000378:metabolism; D000068677:Sildenafil Citrate; D013379:Substrate Specificity; D013450:Sulfones / Q000378:metabolism*; D000068581:Tadalafil; D014227:Triazines / Q000378:metabolism; D000069058:Vardenafil Dihydrochloride
chemical_list: D002243:Carbolines; D002352:Carrier Proteins; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D058986:Phosphodiesterase 5 Inhibitors; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D014227:Triazines; C022994:cyclic GMP-binding protein; D000069058:Vardenafil Dihydrochloride; D000068581:Tadalafil; D000068677:Sildenafil Citrate; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006152:Cyclic GMP