New Insights from the Structure Function of the Catalytic Region of Human Platelet PDE3A: The Role of the Unique 44 Amino Acid Insert.

Abstract

Human platelet PDE3A degrades cAMP, the major intracellular inhibitor of platelet function, and thus potentiates platelet activation. PDE3A is irreversibly inactivated by the affinity label Sp-cAMPS-BDB. The inactivation is prevented by Sp-cAMPS indicating that the affinity label is targeted at the cAMP binding site. We now use Sp-cAMPS-BDB with the aim of identifying nonconserved amino acids in substrate binding. After incubating Sp-cAMPS-BDB with PDE3A followed by reduction with [3H]NaBH4, the incorporation was 1.1 mol/mol. HPLC analysis of the tryptic digest yielded a radioactive octapeptide T806YNVTDDK813 in the 44-amino acid insert of PDE3A. Molecular modeling of PDE3A based on the PDE3B structure suggests the insert is a flexible loop. Incorporation of Sp-cAMPS-BDB indicates loop interaction with the substrate. Since Sp-cAMPS-BDB reacts with the nucleophilic residues, Y807, D811 and D812 were each mutated to alanine. Sp-cAMPS-BDB inactivates D811A and D812A but not Y807A, suggesting Y807 is the residue modified by Sp-cAMPS-BDB. Y807A affects the Km but not kcat, suggesting its involvement in cAMP binding. Kinetic analyses of 11 loop mutants reveal that H782A, T810A, Y814A and C816S each affects the kcat but not Km, indicating that catalysis is modulated. We conclude that binding of cAMP to the flexible loop of platelet PDE3A induces a conformational change which allows interaction with essential catalytic residues. These findings provide a new strategy for developing antiplatelet agents to treat patients with reocclusion of coronary arteries who are resistant to aspirin or whose chronic congestive heart failure prevents utilizing cilostazol.