New Insights from the Structure-Function Analysis of the Catalytic Region of Human Platelet Phosphodiesterase 3A A ROLE FOR THE UNIQUE 44-AMINO ACID INSERT

Abstract Human phosphodiesterase 3A (PDE3A) degrades cAMP, the major inhibitor of platelet function, thus potentiating platelet function. Of the 11 human PDEs, only PDE3A and 3B have 44-amino acid inserts in the catalytic domain. Their function is not clear. Incubating Sp-adenosine-3′,5′-cyclic-S-(4-bromo-2,3-di-oxobutyl) monophosphorothioate (Sp-cAMPS-BDB) with PDE3A irreversibly inactivates the enzyme. High pressure liquid chromatography (HPLC) analysis of a tryptic digest yielded an octapeptide within the insert of PDE3A ((K)T806YNVTDDK813), suggesting that a substrate-binding site exists within the insert. Because Sp-cAMPS-BDB reacts with nucleophilic residues, mutants Y807A, D811A, and D812A were produced. Sp-cAMPS-BDB inactivates D811A and D812A but not Y807A. A docking model showed that Tyr807 is 3.3 angstroms from the reactive carbon, whereas Asp811 and Asp812 are >15 angstroms away from Sp-cAMPS-BDB. Y807A has an altered Km but no change in kcat. Activity of wild type but not Y807A is inhibited by an anti-insert antibody. These data suggest that Tyr807 is modified by Sp-cAMPS-BDB and involved in substrate binding. Because the homologous amino acid in PDE3B is Cys792, we prepared the mutant Y807C and found that its Km and kcat were similar to the wild type. Moreover, Sp-cAMPS-BDB irreversibly inactivates Y807C with similar kinetics to wild type, suggesting that the tyrosine may, like the cysteine, serve as a H donor. Kinetic analyses of nine additional insert mutants reveal that H782A, T810A, Y814A, and C816S exhibit an altered kcat but not Km, indicating that catalysis is modulated. We document a new functional role for the insert in which substrate binding may produce a conformational change. This change would allow the substrate to bind to Tyr807 and other amino acids in the insert to interact with residues important for catalysis in the active site cleft.