Binding of cGMP to both allosteric sites of cGMP-binding cGMP-specific phosphodiesterase (PDE5) is required for its phosphorylation.
cGMP-binding phosphodiesterases contain two homologous allosteric cGMP-binding sites (sites a and b) that are arranged in tandem; they constitute a superfamily of mammalian cyclic nucleotide receptors distinct from the cyclic nucleotide-dependent protein kinases/cation channels family. The functional role of each of these two sites in the phosphodiesterases is not known. The cGMP-binding sites of one of these phosphodiesterases, the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE, PDE5), have been analysed by using site-directed mutagenesis. Mutations that affect cGMP binding to either one or both allosteric sites do not influence cGMP hydrolysis in the catalytic site under the conditions used. However, compared with wild-type enzyme, the D289A, D478A and D289A/D478A mutants, which are defective in cGMP binding to either site a or site b, or both allosteric sites, require much higher cGMP concentrations for the allosteric stimulation of phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. The cGMP effect is on the cGB-PDE rather than on the catalytic subunit of the protein kinase because the latter enzyme does not require cGMP for activity. The D289N mutant, which has higher binding affinity for cGMP than does the wild-type enzyme, is phosphorylated at lower concentrations of cGMP than is the wild-type enzyme. It is concluded that cGMP binding to the allosteric sites of cGB-PDE does not directly affect catalysis, but binding to both of these sites regulates phosphorylation of this enzyme.
Turko
I V
IV
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615, USA.
Francis
S H
SH
Corbin
J D
JD
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000495:Allosteric Site / Q000235:genetics; D000818:Animals; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity / Q000235:genetics; D014162:Transfection
D011994:Recombinant Proteins; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP