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 (PDE3A Delta 1) 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 PDE3A Delta 1, 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 PDE3A Delta 1. The K-i of H836A for cGMP was 177-fold higher than that of PDE3A Delta 1. The K-m for E971A was 5-fold higher than PDE3A Delta 1. 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 Mn2+, Mg2+, or Co2+. 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, (Blood, 2000; 95:3380-3386) (C) 2000 by The American Society of Hematology.