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• W2015996622 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. 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. The anti-platelet drugs aspirin and clopidogrel have proven efficacy in secondary prevention of stroke, myocardial infarction, and peripheral vascular reocclusion (1Meijer A. Verheugt F.W. Werter C.J. Lie K.I. van der Pol J.M. van Eenige M.J. Circulation. 1993; 87: 1524-1530Crossref PubMed Scopus (203) Google Scholar, 2Jarvis B. Simpson K. Drugs. 2000; 60: 347-377Crossref PubMed Scopus (178) Google Scholar). Aspirin inhibits cyclooxygenase, thereby decreasing synthesis of thromboxane A2. Clopidogrel, a P2Y12 antagonist, blocks the ability of ADP to inhibit stimulated adenylate cyclase. However, despite prophylaxis with these anti-platelet drugs, reocclusion of coronary arteries occurs in 20-30% of patients after thrombolytic therapy or angioplasty probably because of the inability of these drugs to inhibit thrombin-induced platelet activation (3Gum P.A. Kottke-Marchant K. Welsh P.A. White J. Topol E.J. J. Am. Coll. Cardiol. 2003; 41: 961-965Crossref PubMed Scopus (977) Google Scholar, 4Matetzky S. Shenkman B. Guetta V. Shechter M. Bienart R. Goldenberg I. Novikov I. Pres H. Savion N. Varon D. Hod H. Circulation. 2004; 109: 3171-3175Crossref PubMed Scopus (1283) Google Scholar). At low concentrations of thrombin, platelet aggregation depends in part on ADP and thromboxane A2, which are released by platelets and exert autocrine-mediated enhancement. At high concentrations of thrombin, platelets are aggregated and activated by pathways independent of both ADP and thromboxane A2. In contrast, elevation of intracellular cAMP produces potent inhibition of all pathways of platelet activation including increase in intracellular Ca2+, shape change, aggregation, secretion, and the effects of phospholipases A2 and C, as well as their responses of platelets to thrombin.Cyclic nucleotide PDE3A 4The abbreviations used are: PDE, phosphodiesterase; Sp-cAMPS-BDB, Sp-adenosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl) monophosphorothioate; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid; 8-BDB-TcAMP, 8-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 3′,5′-cyclic monophosphate. 4The abbreviations used are: PDE, phosphodiesterase; Sp-cAMPS-BDB, Sp-adenosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl) monophosphorothioate; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid; 8-BDB-TcAMP, 8-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 3′,5′-cyclic monophosphate. is the most abundant cAMP PDE in platelets. PDE3A hydrolyzes cAMP resulting in lowering the intracellular cAMP levels, which in turn potentiates platelet activation. Drugs that inhibit PDE3A raise cAMP levels in platelets, thereby increasing the phosphorylation of proteins by cAMP- and cGMP-dependent protein kinases (5Movsesian M.A. J. Card. Fail. 2003; 9: 475-480Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Currently two PDE3A competitive inhibitors cilostazol and milrinone have respectively been used for treating patients with intermittent claudication and acute congestive heart failure (6Levy J.H. Bailey J.M. Deeb G.M. Ann. Thorac. Surg. 2002; 73: 325-330Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 7Beebe H.G. Dawson D.L. Cutler B.S. Herd J.A. Strandness Jr., D.E. Bortey E.B. Forbes W.P. Arch. Intern. Med. 1999; 159: 2041-2050Crossref PubMed Scopus (286) Google Scholar). Unfortunately cilostazol is contraindicated in patients with congestive heart failure, and milrinone is associated with undesirable cardiac arrhythmias. Examination of the inhibitory mechanism of PDE3A is important to exploit other ways of inhibiting this enzyme to minimize side effects.The available PDE family crystal structures known to date are those of the catalytic domains cAMP-PDE (PDE4B2B and PDE4D) (8Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (317) Google Scholar, 9Lee M.E. Markowitz J. Lee J.O. Lee H. FEBS Lett. 2002; 530: 53-58Crossref PubMed Scopus (123) Google Scholar), cGMP-PDE (PDE5A and PDE9A) (10Huai Q. Liu Y. Francis S.H. Corbin J.D. Ke H. J. Biol. Chem. 2004; 279: 13095-13101Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 11Huai Q. Wang H. Zhang W. Colman R.W. Robinson H. Ke H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9624-9629Crossref PubMed Scopus (87) Google Scholar), and dual cAMP/cGMP-PDE (PDE1B and PDE3B) (12Zhang K.Y. Card G.L. Suzuki Y. Artis D.R. Fong D. Gillette S. Hsieh D. Neiman J. West B.L. Zhang C. Milburn M.V. Kim S.H. Schlessinger J. Bollag G. Mol. Cell. 2004; 15: 279-286Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 13Scapin G. Patel S.B. Chung C. Varnerin J.P. Edmondson S.D. Mastracchio A. Parmee E.R. Singh S.B. Becker J.W. Van der Ploeg L.H. Tota M.R. Biochemistry. 2004; 43: 6091-6100Crossref PubMed Scopus (97) Google Scholar). The overall crystal structures of the catalytic domains of these PDEs contain a compact structure consisting of 16 α-helices. Each PDE has three subdomains with a deep hydrophobic pocket at the interface and two conserved metal-binding sites within that pocket. The hydrogen bond network of the neighboring residues His948 and Trp1072 in PDE3B (His956 and Trp1085 in PDE3A) serves to orient the absolutely conserved residue Gln988 (Gln1001 in PDE3A) to accept or donate hydrogen bonds to the purine ring, thereby determining the nucleotide recognition specificity of the enzyme (13Scapin G. Patel S.B. Chung C. Varnerin J.P. Edmondson S.D. Mastracchio A. Parmee E.R. Singh S.B. Becker J.W. Van der Ploeg L.H. Tota M.R. Biochemistry. 2004; 43: 6091-6100Crossref PubMed Scopus (97) Google Scholar). In PDE3B, residues Phe991 (Phe1004 in PDE3A) and Ile955 (Ile967 in PDE3A) on each side of the purine ring and Tyr960 and Pro941 (Try973 and Pro954 in PDE3A) form the hydrophobic clamp (13Scapin G. Patel S.B. Chung C. Varnerin J.P. Edmondson S.D. Mastracchio A. Parmee E.R. Singh S.B. Becker J.W. Van der Ploeg L.H. Tota M.R. Biochemistry. 2004; 43: 6091-6100Crossref PubMed Scopus (97) Google Scholar). Residues His741, His821, Asp822, and Asp937 (His756, His836, Asp837, and Asp950 in PDE3A) and one water molecule in PDE3B are involved in the first metal Mg2+ binding. The second Mg2+ forms hydrogen bonds with Asp822 and five water molecules (13Scapin G. Patel S.B. Chung C. Varnerin J.P. Edmondson S.D. Mastracchio A. Parmee E.R. Singh S.B. Becker J.W. Van der Ploeg L.H. Tota M.R. Biochemistry. 2004; 43: 6091-6100Crossref PubMed Scopus (97) Google Scholar). Water molecules coordinated to the metal ions may act as the nucleophile in the hydrolysis reaction to mediate catalysis.The presence of a 44-amino acid insert within the catalytic domain is a unique feature of the PDE3 gene family. In the PDE3B crystal structure, the 44-amino acid insertion (Pro758-Cys801; Fig. 1) is located between helices 6 and 7 (13Scapin G. Patel S.B. Chung C. Varnerin J.P. Edmondson S.D. Mastracchio A. Parmee E.R. Singh S.B. Becker J.W. Van der Ploeg L.H. Tota M.R. Biochemistry. 2004; 43: 6091-6100Crossref PubMed Scopus (97) Google Scholar). This insertion lacks any clear secondary structure organization, and residues 767-781 are not visible in the electron density maps. This insert in human PDE3A is comprised of amino acid residues 773-816 (Fig. 1). Within the insert there is 38.6% identity between PDE3A and PDE3B including conserved triplets at the N terminus, C terminus, and the middle of the insert. Tang et al. (14Tang K.M. Jang E.K. Haslam R.J. Biochem. J. 1997; 323: 217-224Crossref PubMed Scopus (24) Google Scholar) showed that removal of this insert from PDE3A resulted in the complete loss of activity. Double mutants, P773A/G774A and Y814A/G815A, from each of the N and C termini of the PDE3A insert, which are β turns, display markedly reduced activity (14Tang K.M. Jang E.K. Haslam R.J. Biochem. J. 1997; 323: 217-224Crossref PubMed Scopus (24) Google Scholar). However, knowledge of the role of the 44-amino acid insert of PDE3A in the regulation of enzyme activity or interactions with substrate and/or inhibitor is incomplete.Previously, we have synthesized a nonhydrolyzable, reactive substrate analog, Sp-cAMPS-BDB, which irreversibly inactivates PDE3A in a time-dependent fashion with KI = 10.1 ± 1.7 μm and kmax = 0.0116 ± 0.0004 min-1 (15Hung S.H. Madhusoodanan K.S. Beres J.A. Boyd R.L. Baldwin J.L. Zhang W. Colman R.W. Colman R.F. Bioorg. Chem. 2002; 30: 16-31Crossref PubMed Scopus (4) Google Scholar). We have demonstrated that Sp-cAMPS-BDB targets both the cAMP- and cGMP-binding sites but favors the cAMP site. The protection studies indicate effectiveness of protectants in decreasing rate of inactivation by Sp-cAMPS-BDB is: Sp-cAMPS (Kd = 24 μm) > Rp-cGMPS (1360), Sp-cGMPS (1460) > GMP (4250), AMP (10600), Rp-cAMPS (22170 μm). Sp-cAMPS-BDB has proven to be an effective active site-directed affinity label for PDE3A.In this paper, we describe specific incorporation of PDE3A by a reactive substrate analog, Sp-cAMPS-BDB, isolation of a peptide in the unique insert of PDE3A, and construction of mutant enzymes that identify the amino acid targeted by Sp-cAMPS-BDB. In addition, the role of the insert was further explored by kinetic analyses of nine additional insert mutants. The results define a new functional mechanism by which binding of cAMP to the flexible loop of platelet PDE3A may induce a local conformational change that allows interaction with catalytic residues.EXPERIMENTAL PROCEDURESMaterials—Adenosine 3′,5′-cyclic phosphate ammonium salt [2,8-3H]cAMP) was purchased from PerkinElmer Life Sciences. The nonhydrolyzable, reactive cAMP analog, Sp-adenosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphoro-thioate (Sp-cAMPS-BDB) was synthesized as previously described (15Hung S.H. Madhusoodanan K.S. Beres J.A. Boyd R.L. Baldwin J.L. Zhang W. Colman R.W. Colman R.F. Bioorg. Chem. 2002; 30: 16-31Crossref PubMed Scopus (4) Google Scholar). Sf9 insect cell lines, Sf-900 II SFM medium, BaculoDirect transfection and expression system, the ProBound resin, and Anti-HisG antibody were purchased from Invitrogen. Protease inhibitor mixture set III (PIC III) was purchased from EMD Biosciences (San Diego, CA). A Coomassie Plus protein assay reagent kit was purchased from Pierce. Gentamicin sulfate, cAMP, and N-ethylmaleimide were purchased from Sigma. HPLC grade acetonitrile was obtained from Fisher.Measurement of the Incorporation of Sp-cAMPS-BDB into PDE3A—PDE3A was incubated with 100 μm Sp-cAMPS-BDB ina50mm Hepes buffer at pH 7.3 containing 20 mm MES, 10 mm MgCl2, and 0.5 m NaCl. At various times of incubation (0, 20, 30, 40, 60, and 80 min, respectively), the aliquots were removed, and the residual enzyme activity of PDE3A was determined to correlate with the incorporation (see “Enzyme Activity Assay”). At each time interval, 100 mm [3H]NaBH4 (dissolved in 20 mm NaOH) was added consecutively to reach a final concentration of 2 mm at 4 °C for a total of 1.5 h. [3H]NaBH4 reduces the two oxygens of the diketo group from Sp-cAMPS-BDB to two [3H] hydroxyl groups. The excess [3H]NaBH4 and the free Sp-cAMPS-BDB were removed by four consecutive centrifugations using Microcon centrifugal devices (Millipore, Billerica, MA) at 14,000 × g for 20 min. Aliquots were removed from the retentate to measure the protein concentration using the Coomassie Plus protein assay. The amount of Sp-cAMPS-BDB incorporated into PDE3A from reduction of the affinity labeled enzyme by [3H]NaBH4 was calculated by measuring the radioactive (3H) content by using a Beckman Coulter liquid scintillation analyzer (model LS6500; Fullerton, CA). Control samples were tested using a similar procedure with the pretreatment of cold NaBH4 with Sp-cAMPS-BDB prior to the addition of enzyme.Trypsin Digestion of the Sp-cAMPS-BDB-modified Enzyme—PDE3A (0.8 mg) was incubated with 100 μm Sp-cAMPS-BDB at 25°C for 3 h(∼10% residual activity remained). The incubated mixture was treated twice with 100 mm [3H]NaBH4 for a total of 1.5 h (final concentration, 2 mm), followed by a carboxylation of free SH groups with 10 mm N-ethylmaleimide for 10 min. After removal of the excess reagents by centrifugation using Microcon centrifugal devices, the modified enzyme was digested at 37 °C by 2 consecutive additions of 5% (w/w) tosylphenylalanyl chloromethyl ketone-treated bovine pancreatic trypsin for a total of 2 h.Purification and Determination of the Sequence of Modified Peptide—The radioactive tryptic digest was lyophilized, redissolved in 250 μl of 0.1% trifluoroacetic acid, and applied to an HPLC system using a reverse phase Vydac (Hesperia, CA) C18 column (0.46 × 25 cm). Separation was conducted at the elution rate of 1 ml/min using solvent A (0.1% trifluoroacetic acid in water) for the first 10 min, followed by a linear gradient from solvent A to 45% solvent B (0.1% trifluoroacetic acid in acetonitrile) for 220 min, a linear gradient from 45% solvent B to 100% Solvent B for 20 min, and solvent B for 10 min, successively. The eluent was monitored at 220 nm. Fractions of 1 ml were collected, from which 400 μl was counted for radioactivity. The amino acid sequence of isolated radioactive peptides was determined using an automated gas phase peptide sequence analyzer from Applied Biosystems (model 470A; Foster City, CA) equipped with an on-line phenylthiohydantoin analyzer (model 120) and computer (model 900A). The sequencing results were used to identify the location of the modified peptide in the active site of the catalytic region of PDE3A. This process was repeated twice with identical results.Construction and Purification of PDE3A Mutants—A deletion mutant of PDE3A cDNA coding for the amino acid residues 665-1141 (16Cheung P.P. Xu H. McLaughlin M.M. Ghazaleh F.A. Livi G.P. Colman R.W. Blood. 1996; 88: 1321-1329Crossref PubMed Google Scholar) was subcloned into a pENTER-TOPO vector (Invitrogen) to produce two sites for linear recombination. PDE3A insert mutants H782A, H796A, H798A, S804A, K805A, Y807A, Y807C, T810A, D811A, D812A, Y814A, G815A, and C816S were constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All of the mutants were confirmed by nucleotide sequence analysis (Sidney Kimmel Nucleic Acid Facility, Thomas Jefferson University, Philadelphia, PA). Recombinant mutant baculoviruses were produced by linear combination using BaculoDirect Transfection kit (Invitrogen). Expression of the catalytic region (residues 665-1141) of PDE3A wild type and mutant enzymes using a baculovirus/insect cell Sf9 system and protein purification using a ProBond Nickel resin column has been previously described (17Zhang W. Colman R.W. Blood. 2000; 95: 3380-3386Crossref PubMed Google Scholar, 18Zhang W. Ke H. Tretiakova A.P. Jameson B. Colman R.W. Protein Sci. 2001; 10: 1481-1489Crossref PubMed Scopus (31) Google Scholar).Protein Concentration Determination—Protein concentration of the purified enzymes and purified anti-insert antibody were determined using Coomassie Plus protein assay reagent using bovine serum albumin as standard. The absorbance at 595 nm was measured using a Bio-Tek automatic microplate reader equipped with KC4 module for data analysis (Bio-Tek Instruments, Inc., Winooski, VT).Western Blot Analysis—The PDE3A wild type and mutants were separated on 10% Bis-Tris gel electrophoresis purchased from Invitrogen. The proteins were transferred to a polyvinylidene difluoride membrane using the Xcell II module at a constant voltage of 30 volts for 1 h at room temperature for Western blotting. The membranes were processed using the Chromogenic WesternBreeze system and probed with anti-insert PDE3A antibody (see effects of anti-insert antibody) to detect the presence of PDE3A.Enzyme Activity Assay—PDE3A activity was measured by the amount of cAMP hydrolyzed as previously described (19Grant P.G. Colman R.W. Biochemistry. 1984; 23: 1801-1807Crossref PubMed Scopus (108) Google Scholar). Enzyme was added to a buffer containing 50 mm Tris-HCl, pH 7.8, 10 mm MgCl2, and 0.8 μm [3H]cAMP. Reaction mixtures both with and without enzymes were incubated at 30 °C for 15 min. Catalysis was terminated by serial addition of 0.2 m of ZnSO4 and 0.2 m Ba(OH)2, which precipitates AMP but not cAMP. Samples were vortexed and centrifuged at 10,000 × g for 5 min. The BaSO4 pellets containing the [3H]5′-AMP precipitant were discarded. Aliquots of supernatants containing unreacted [3H]cAMP were removed and counted in a Beckman Coulter liquid scintillation analyzer. Enzyme activity was measured by comparing the amount of cAMP hydrolyzed in PDE3A containing samples to no enzyme controls. These data were then used to calculate enzyme specific activity in nmol of cAMP hydrolyzed per mg of protein per min.Kinetic Constants Determination—The rates (nmol/s) of cAMP hydrolysis for the PDE3A wild type and mutant enzymes were determined using various concentrations of substrate cAMP from 0.02 to 14 μm. The values of Km and Vmax for each of the enzymes were determined by Michaelis-Menten equation as calculated by Enzyme Kinetics Module 1.1 software (Systat Software, Point Richmond, CA). The kcat (s-1) was obtained by dividing Vmax (nmol/s) by the molar enzyme concentration (nmol).Reaction of Sp-cAMPS-BDB with Mutant Enzymes—Purified PDE3A mutant enzyme (Y807A, Y807C, D811A, or D812A) was incubated at 25 °C with various concentrations of Sp-cAMPS-BDB in a 50 mm Hepes buffer at pH 7.3 containing 20 mm MES, 10 mm MgCl2, and 0.5 m NaCl. At timed intervals (0, 5, 10, 20, 30 45, and 60 min), aliquots of the reaction mixture were withdrawn, diluted in a buffer containing 47.5 mm Hepes, pH 7.04, 20 mm MgCl2, 4 mm MES, and assayed in triplicate for residual PDE3A activity. Control samples were performed under identical conditions without the presence of affinity label Sp-cAMPS-BDB.Effect of Anti-insert Antibody on Enzyme Activity—A rabbit polyclonal antibody against the synthetic peptide 802VFSKTYNVTDDKYGC816, the C-terminal 15 amino acids of the PDE3A insert (Fig. 1), which also contain the octapeptide, was prepared by Sigma Genosys and designated as an anti-insert antibody. PDE3A, and mutants Y807A and Y807C were incubated respectively with various concentrations of the anti-insert antibody to a enzyme to antibody ratio of 1.3, 2.0, or 4.0 for 1 h at 37 °C. After incubation, enzyme activity was determined according to the “Enzyme Activity Assay” procedure. The activity of PDE3A wild type, Y807A, and Y807C without antibody was set as 100% activity. The preimmune IgG was used as a control to compare the activity of wild type, Y807A, and Y807C. All of the experiments were performed in triplicate.Molecular Modeling—A homology model of PDE3A based on the crystal structure of PDE4B2B has been published (8Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (317) Google Scholar). However, the model did not contain the additional 44-amino acid insert found in PDE3A. We have now refined the PDE3A model using the recently published PDE3B structures (13Scapin G. Patel S.B. Chung C. Varnerin J.P. Edmondson S.D. Mastracchio A. Parmee E.R. Singh S.B. Becker J.W. Van der Ploeg L.H. Tota M.R. Biochemistry. 2004; 43: 6091-6100Crossref PubMed Scopus (97) Google Scholar) that contain the 44-amino acid insert unique to PDE3. Sybyl 6.91 FlexX docking module (Tripos) was then used to dock the affinity label Sp-cAMPS-BDB to PDE3A. Because mutant Y807A affected the Km, Tyr807 was included in the defined cAMP-binding pocket to construct the model. Residues involved in cAMP binding (17Zhang W. Colman R.W. Blood. 2000; 95: 3380-3386Crossref PubMed Google Scholar, 18Zhang W. Ke H. Tretiakova A.P. Jameson B. Colman R.W. Protein Sci. 2001; 10: 1481-1489Crossref PubMed Scopus (31) Google Scholar) were used as a defined cAMP binding pocket (Tyr807, Asn845, Glu866, Glu971, Phe972, and Phe1004). This docking model was utilized to illustrate and further evaluate the kinetic results obtained from the mutants of insert amino acids of PDE3A.RESULTSIncorporation of Sp-cAMPS-BDB into PDE3A Is Time-dependent—To quantify the amount of the affinity label Sp-cAMPS-BDB incorporated into PDE3A, the enzyme (0.38 mg/ml) was incubated with 100 μm Sp-cAMPS-BDB at pH 7.3, as described under “Experimental Procedures.” Fig. 2 (left panel) shows that the incorporation of PDE3A by Sp-cAMPS-BDB is linear as a function of time. The addition of [3H]NaBH4 to an incubation mixture of enzyme and Sp-cAMPS-BDB stops the reaction by reducing the diketo group of Sp-cAMPS-BDB to a[3H]diol group. Fig. 2 (right panel) shows that the residual enzymatic activity is inversely proportional to the incorporation. At 80 min, 0.86 mol of Sp-cAMPS-BDB was incorporated for each mol of enzyme which corresponded 19% of residual enzymatic activity or 81% inactivation. Thus, 1.08 mol of Sp-cAMPS-BDB was required to inactivate each mole of enzyme indicating a stoichiometry close to 1.0 of the affinity label and the enzyme.FIGURE 2Incorporation of Sp-cAMPS-BDB into PDE3A. The left panel shows the time-dependent incorporation of Sp-cAMPS-BDB into PDE3A. The enzyme was incubated in 50 mm Hepes buffer, pH 7.3, at 25 °C. At the indicated times, the incorporation was stopped by two additions of [3H]NaBH4 (2 mm). The excess reagent was removed by four consecutive centrifugations using Microcon centrifugal devices. The aliquots were removed from the retentate to measure the protein concentration and radioactivity. The right panel shows the relationship between inactivation and incorporation of Sp-cAMPS-BDB into PDE3A. The residual activity of the unmodified enzyme was determined at 20, 30, 40, 60, and 80 min. Enzymes treated with the same procedure but without Sp-cAMPS-BDB in the incubation mixture were used as controls for activity. The data were fitted to linear regression equation and plotted. The results are the means of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Isolated Sp-cAMPS-BDB-modified Peptide in PDE3A Is Located in the Unique 44-Amino Acid Insert—PDE3A (11 nmol) was incubated with 100 μm Sp-cAMPS-BDB for 3 h and treated with [3H]NaBH4 as described under “Experimental Procedures.” The modified enzyme was digested by trypsin for 2 h as described under “Experimental Procedures.” Fig. 3 (solid line) shows that on the reverse phase HPLC separation of the tryptic digest, most of the peptides elute between 0 and 160 min (0 and 30% solvent B). Two major radioactive peaks were observed as shown in Fig. 3 (dashed line, labeled I and II).FIGURE 3Isolation of trypsin-digested peptides by HPLC. The affinity labeled PDE3A was digested with trypsin. The trypsin-digested peptides were separated by a reverse phase HPLC C18 column under the conditions described under “Experimental Procedures.” The curve with the solid line shows the A220 nm HPLC profile. The gradient of acetonitrile of the tryptic digested peptides is shown with the solid line. The curve with the dotted line shows the radioactivity pattern of the same chromatograph. The two radioactive peaks are labeled I and II. This experiment was performed twice with the results in close agreement. This figure represents one of the two experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The amino acid sequence of the purified peptides (Fig. 3, peaks I and II) was determined by Edman degradation using an automated gas phase sequencer. Peak I contains small peptides (data not shown). The amino acid sequence of the peptide from peak II exhibits a single octapeptide, assigned as 806TYNVTDDK813 within the unique 44-amino acid insert of PDE3A (Fig. 1). This peptide results from enzyme cleavage after Lys805 and Lys813, consistent with the specificity of the trypsin recognition sites. The yield of each phenylthiohydantoin-derivative was recorded and ranged from 40 to 20 pmol (data not shown). As expected, the yield decreases as the cycle number increases. Peptide 806-813 is located C-terminal of the first metal-binding motif, 752HNRIH756.Residue Tyr807 in PDE3A Is the Amino Acid Modified by the Affinity Label Sp-cAMPS-BDB—Sp-cAMPS-BDB reacts with nucleophilic amino acids. Thus, Tyr807, Asp811, and Asp812 from peptides 806-813 (determined from the tryptic cleavage study) are candidates for interacting with the affinity label. Lys813 was not considered because this is the trypsin cleavage site, and cleavage would not have occurred if that lysine were modified. To identify which amino acid is being modified by Sp-cAMPS-BDB, mutant enzymes Y807A, D811A, and D812A were constructed, expressed, and purified.To evaluate the effect of mutations on the reaction with Sp-cAMPS-BDB, the mutant enzymes were incubated with the affinity label, and their activity was tested as a function of time. Fig. 4 (A-D) shows the results of reaction of wild type and mutant enzymes, Y807A, D811A, and D812A, respectively, with Sp-cAMPS-BDB. Sp-cAMPS-BDB irreversibly inactivates both mutants D811A and D812A exhibiting saturation kinetics (Fig. 4, H-I). The kmax values for D811A and D812A are 0.005 ± 0.0002 and 0.003 ± 0.0001 min-1, and the KI values are 29.9 ± 2.9 and 24.9 ± 2.5 μm, respectively. The KI values of both D811A and D812A is 2.5-3-fold larger than that of wild type (KI = 10.1 ± 1.7 μm; Fig. 4F). The kmax values of D811A and D812A are one-half and one-third, respectively, that of wild type (kmax = 0.0116 ± 0.0004 min-1; Fig. 4F). These relatively minor changes in kinetics indicate that residues Asp811 and Asp812 are not the modified amino acid of the wild type enzyme that reacts with Sp-cAMPS-BDB. In contrast, Y807A is not inactivated by Sp-cAMPS-" @default.
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• W2015996622 title "New Insights from the Structure-Function Analysis of the Catalytic Region of Human Platelet Phosphodiesterase 3A" @default.
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