Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3380-3386
Conserved amino acids in metal-binding motifs of PDE3A are
involved in substrate and inhibitor binding
Wei Zhang and
Robert W. Colman
From the Sol Sherry Thrombosis Research Center, Temple University
School of Medicine, Philadelphia, PA
The activity of phosphodiesterase (PDE)3A requires divalent cations.
Putative metal-binding sites are expected at 2 highly conserved
metal-binding motifs, HXXXH(X)25E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3A
1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All
the mutant proteins had decreased catalytic efficiency
(kcat/Km). Mutants H752A, H756A, and E825A had
kcat of less than 0.0008 s
1 to 0.0475 s1 compared to PDE3A1, with 1.86 second1, with unchanged Km. Although E866A
had a kcat of 0.235 s1, the Km
for cyclic adenosine monophosphate (cAMP) was increased 11-fold and the
Ki for cyclic guanosine monophosphate (cGMP) was 27-fold
higher than PDE3A1. The Ki of H836A for cGMP was
177-fold higher than that of PDE3A1. The Km for E971A
was 5-fold higher than PDE3A1. 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.
