Conserved amino acids in metal-binding motifs of PDE3A are involved in substrate and inhibitor binding.
The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)(25)E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3Atriangle up1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (k(cat)/K(m)). Mutants H752A, H756A, and E825A had k(cat) of less than 0.0008 s(-1) to 0.0475 s(-1) compared to PDE3Atriangle up1, with 1.86 second(-1), with unchanged K(m). Although E866A had a k(cat) of 0.235 s(-1), the K(m) for cyclic adenosine monophosphate (cAMP) was increased 11-fold and the K(i) for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3Atriangle up1. The K(i) of H836A for cGMP was 177-fold higher than that of PDE3Atriangle up1. The K(m) for E971A was 5-fold higher than PDE3Atriangle up1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn(2+), Mg(2+), or Co(2+). This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.
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Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Colman
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D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology*; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D014162:Transfection
D011994:Recombinant Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human