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J. Biol. Chem., Vol. 281, Issue 39, 29236-29244, September 29, 2006

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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^*

Su-Hwi Hung¹, Wei Zhang¹, Robin A. Pixley, Bradford A. Jameson, Yu Chu Huang^¶, Roberta F. Colman^¶2, and Robert W. Colman²³

From the Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, the Department of Biochemistry, Drexel University College of Medicine, Philadelphia, Pennsylvania 19104, and the ^¶Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716

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 K_m but no change in k_cat. 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 K_m and k_cat 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 k_cat but not K_m, 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.

Received for publication, July 10, 2006

^* This work was supported by Pennsylvania/Delaware Affiliated American Heart Association Grant 026439U (to S.-H. H.), National Institutes of Health Grant HL-64943 (to R. W. C.), and National Science Foundation Grant MCB97-28202 (to R. F. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² These authors should both be considered senior authors.

³ To whom correspondence should be addressed: Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad St., Philadelphia, PA 19140. Tel.: 215-707-4665; Fax: 215-707-4855; E-mail: colmanr{at}temple.edu.

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