Matthiesen Karina, Nielsen Jacob
Department of Molecular Neurobiology, H. Lundbeck A/S, Ottiliavej 9, 2500 Copenhagen-Valby, Denmark.
Biochem J. 2009 Oct 12;423(3):401-9. doi: 10.1042/BJ20090982.
To date eleven human PDE (3',5'-cyclic nucleotide phosphodiesterase) families have been identified. Of these, five families contain non-catalytic tandem GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domains, GAFa and GAFb, in the N-terminal part of the enzyme. For PDE2A, PDE5A and PDE6 the GAF domains have been shown to bind cGMP with high affinity. For PDE2A and PDE5A this ligand binding has been shown to stimulate the catalytic activity of the enzyme. PDE10A and PDE11A are the two most recently described PDEs and it has been suggested that their GAF domains bind to cAMP and cGMP respectively. We have developed a scintillation proximity-based assay to directly measure cyclic nucleotide binding to the PDE2A, PDE10A and PDE11A GAF domains, and in the present study we demonstrate binding of cyclic nucleotides to the PDE10A and PDE11A GAF domains. We show that these non-catalytic sites bind cAMP and cGMP respectively with much higher affinity than has previously been suggested using indirect assessment of the interaction. The GAFb domain of PDE10A binds cAMP with a Kd of 48 nM and the GAFa domain of PDE11A binds cGMP with a Kd of 110 nM. The effect of cyclic nucleotides binding to the GAF domains on the enzyme activity was investigated through the use of modified cyclic nucleotides. In contrast with other GAF domain-containing PDEs, and with what has previously been predicted, ligand binding to the GAF domains of PDE10A and PDE11A does not stimulate catalytic activity.
迄今为止，已鉴定出11个人类磷酸二酯酶（3',5'-环核苷酸磷酸二酯酶）家族。其中，五个家族的酶在N端含有非催化性串联GAF（cGMP特异性和受刺激的磷酸二酯酶、鱼腥藻腺苷酸环化酶和大肠杆菌FhlA）结构域，即GAFa和GAFb。对于PDE2A、PDE5A和PDE6，已证明GAF结构域能以高亲和力结合cGMP。对于PDE2A和PDE5A，已证明这种配体结合能刺激酶的催化活性。PDE10A和PDE11A是最近描述的两种磷酸二酯酶，有人提出它们的GAF结构域分别与cAMP和cGMP结合。我们开发了一种基于闪烁邻近的测定法，以直接测量环核苷酸与PDE2A、PDE10A和PDE11A GAF结构域的结合，在本研究中，我们证明了环核苷酸与PDE10A和PDE11A GAF结构域的结合。我们表明，这些非催化位点分别结合cAMP和cGMP的亲和力比以前使用相互作用的间接评估所表明的要高得多。PDE10A的GAFb结构域以48 nM的解离常数结合cAMP，PDE11A的GAFa结构域以110 nM的解离常数结合cGMP。通过使用修饰的环核苷酸，研究了环核苷酸与GAF结构域结合对酶活性的影响。与其他含GAF结构域的磷酸二酯酶以及先前的预测相反，配体与PDE10A和PDE11A的GAF结构域结合不会刺激催化活性。
