Zhang W, Colman R W
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Blood. 2000 Jun 1;95(11):3380-6.
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.
磷酸二酯酶（PDE）3A的活性需要二价阳离子。在两个高度保守的金属结合基序HXXXH(X)(25)E处预计存在假定的金属结合位点。一种包含催化结构域的功能性截短重组PDE3A（PDE3AΔ1）和突变蛋白在杆状病毒/Sf9细胞系统中表达。所有突变蛋白的催化效率（k(cat)/K(m)）均降低。与催化效率为1.86 s⁻¹的PDE3AΔ1相比，突变体H752A、H756A和E825A的k(cat)小于0.0008 s⁻¹至0.0475 s⁻¹，而K(m)不变。尽管E866A的k(cat)为0.235 s⁻¹，但其对环磷酸腺苷（cAMP）的K(m)增加了11倍，对环磷酸鸟苷（cGMP）的K(i)比PDE3AΔ1高27倍。H836A对cGMP的K(i)比PDE3AΔ1高177倍。E971A的K(m)比PDE3AΔ1高5倍。这些结果表明，cAMP和cGMP结合位点相互重叠但不相同，涉及共同和不同的氨基酸。突变体E825A、H836A和E866A在无金属离子的测定中显示出低活性；然而，在含有Mn²⁺、Mg²⁺或Co²⁺的缓冲液中，它们的酶活性增加了4至10倍。这一观察结果表明，第二个金属结合基序中的保守氨基酸可能不参与二价阳离子的结合，但可能在PDE3A中发挥其他功能，如底物或抑制剂结合。
