FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2064433587> ?p ?o ?g. }

• W2064433587 endingPage "21740" @default.
• W2064433587 startingPage "21735" @default.
• W2064433587 abstract "Aps1 from Schizosaccharomyces pombe(Ingram, S. W., Stratemann, S. A., and Barnes, L. D. (1999) Biochemistry 38, 3649–3655) and YOR163w fromSaccharomyces cerevisiae (Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604–8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa- and pentaphosphates (Ap6A and Ap5A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap6A and Ap5A, in preference to other diadenosine polyphosphates. The emergence of Ap6A and Ap5A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process. Aps1 from Schizosaccharomyces pombe(Ingram, S. W., Stratemann, S. A., and Barnes, L. D. (1999) Biochemistry 38, 3649–3655) and YOR163w fromSaccharomyces cerevisiae (Cartwright, J. L., and McLennan, A. G. (1999) J. Biol. Chem. 274, 8604–8610) have both previously been characterized as MutT family hydrolases with high specificity for diadenosine hexa- and pentaphosphates (Ap6A and Ap5A). Using purified recombinant preparations of these enzymes, we have now discovered that they have an important additional function, namely, the efficient hydrolysis of diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition, this finding provides important new information pertinent to the structure, function, and evolution of the MutT motif. Moreover, we report that the human protein previously characterized as a diphosphorylated inositol phosphate phosphohydrolase represents the first example, in any animal, of an enzyme that degrades Ap6A and Ap5A, in preference to other diadenosine polyphosphates. The emergence of Ap6A and Ap5A as extracellular effectors and intracellular ion-channel ligands points not only to diphosphorylated inositol phosphate phosphohydrolase as a candidate for regulating signaling by diadenosine polyphosphates, but also suggests that diphosphorylated inositol phosphates may competitively inhibit this process. Following the discovery of dinucleoside polyphosphates in biological systems over 30 years ago (1Randerath K. Janeway C.M. Stephenson M.L. Zamecnik P.C. Biochem. Biophys. Res. Commun. 1966; 24: 98-105Crossref PubMed Scopus (92) Google Scholar), these compounds have been studied extensively in prokaryotic and eukaryotic organisms. Several important intracellular and extracellular signaling functions have now been ascribed to the diadenosine compounds, Ap3A, 1The abbreviations used are: ApnA, diadenosine 5′,5′′′-P1,P n-oligophosphate (n = 3–6); Aps1, ApsixA hydrolase; p4A, adenosine 5′-tetraphosphate; p5A, adenosine 5′-pentaphosphate; PP-InsP5, diphosphoinositol pentakisphosphate; [PP]2-InsP4, bisdiphosphoinositol tetrakisphosphate; InsP6, inositol hexakisphosphate; DIPP, diphosphoinositolpolyphosphate phosphohydrolase; HIT, histidine triad; Fhit, fragilehistidine triad; CHAPS, 3-[(cholamidopropyl)dimethylammonio]-1-propane-sulfonate; E-64, trans-epoxysuccinyl-l-leucylamido(4-guanidino)-butane; hMTH1, human MutThomologue, type 1; k −1is the first-order rate constant in the rate equation, [S] = [S]0e−kt; HPLC, high performance liquid chromatography.1The abbreviations used are: ApnA, diadenosine 5′,5′′′-P1,P n-oligophosphate (n = 3–6); Aps1, ApsixA hydrolase; p4A, adenosine 5′-tetraphosphate; p5A, adenosine 5′-pentaphosphate; PP-InsP5, diphosphoinositol pentakisphosphate; [PP]2-InsP4, bisdiphosphoinositol tetrakisphosphate; InsP6, inositol hexakisphosphate; DIPP, diphosphoinositolpolyphosphate phosphohydrolase; HIT, histidine triad; Fhit, fragilehistidine triad; CHAPS, 3-[(cholamidopropyl)dimethylammonio]-1-propane-sulfonate; E-64, trans-epoxysuccinyl-l-leucylamido(4-guanidino)-butane; hMTH1, human MutThomologue, type 1; k −1is the first-order rate constant in the rate equation, [S] = [S]0e−kt; HPLC, high performance liquid chromatography. Ap4A, Ap5A, and Ap6A (2Miras-Portugal M.T. Gualix J. Pintor J. FEBS Lett. 1998; 430: 78-82Crossref PubMed Scopus (67) Google Scholar, 3Kisselev L.L. Justesen J. Wolfson A.D. Frolova L.Y. FEBS Lett. 1998; 427: 157-163Crossref PubMed Scopus (184) Google Scholar, 4McLennan A.G. McLennan A.G. Ap4A and Other Dinucleoside Polyphosphates. CRC Press, Boca Raton, FL1992Google Scholar). Indeed, the ultimate fate of cell lineages and the very survival of an organism may depend upon the tight control of cellular diadenosine polyphosphate metabolism. For example, the intracellular level of Ap4A has long been known to be associated with cell proliferation (5Rapaport E. Zamecnik P.C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3984-3988Crossref PubMed Scopus (220) Google Scholar). Moreover, it was recently proposed (6Pace H.C. Garrison P.N. Robinson A.K. Barnes L.D. Draganescu A. Rösler A. Blackburn G.M. Siprashvili Z. Croce C.M. Huebner K. Brenner C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5484-5489Crossref PubMed Scopus (133) Google Scholar) that Ap3A has an antiproliferative role when complexed with the putative tumor suppressor Fhit protein, an Ap3A hydrolase (7Barnes L.D. Garrison P.N. Siprashvili Z. Guranowski A. Robinson A.K. Ingram S.W. Croce C.M. Ohta M. Huebner K. Biochemistry. 1996; 35: 11529-11535Crossref PubMed Scopus (368) Google Scholar, 8Huebner K. Garrison P.N. Barnes L.D. Croce C.M. Annu. Rev. Genet. 1998; 32: 7-31Crossref PubMed Scopus (179) Google Scholar). Thus, the Ap3A/Ap4A ratio may be an important factor in determining the alternative cellular fates of proliferation, differentiation, and apoptosis (3Kisselev L.L. Justesen J. Wolfson A.D. Frolova L.Y. FEBS Lett. 1998; 427: 157-163Crossref PubMed Scopus (184) Google Scholar, 9Vartanian A. Prudovsky I. Suzuki H. DalPra I. Kisselev L. FEBS Lett. 1997; 415: 160-162Crossref PubMed Scopus (75) Google Scholar). In higher eukaryotes, ApnA appear also to be intracellular mediators of certain extracellular stimuli; they respond to glucose in pancreatic β-cells (10Martin F. Pintor J. Rovira J.M. Ripoll C. Miras-Portugal M.T. Soria B. FASEB J. 1998; 14: 1499-1506Crossref Scopus (36) Google Scholar). ApnA may also regulate ATP-sensitive K+ channels in β-cells and cardiac muscle (11Ripoll C. Martin F. Rovira J.M. Pintor J. Miras-Portugal M.T. Soria B. Diabetes. 1996; 45: 1431-1434Crossref PubMed Scopus (65) Google Scholar, 12Jovanovic A. Jovanovic S. Mays D.C. Lipsky J.J. Terzic A. FEBS Lett. 1998; 423: 314-318Crossref PubMed Scopus (39) Google Scholar, 13Jovanovic A. Alekseev A.E. Terzic A. Biochem. Pharmacol. 1997; 54: 219-225Crossref PubMed Scopus (51) Google Scholar) and intracellular ryanodine-binding Ca2+-release channels in cardiac and skeletal muscle, and in the brain (14Morii H. Makinose M. Eur. J. Biochem. 1992; 205: 979-984Crossref PubMed Scopus (18) Google Scholar, 15Holden C.P. Padua R.A. Geiger J.D. J. Neurochem. 1996; 67: 574-580Crossref PubMed Scopus (21) Google Scholar). Finally, extracellular Ap5A and Ap6A have also been identified as neurotransmitters (2Miras-Portugal M.T. Gualix J. Pintor J. FEBS Lett. 1998; 430: 78-82Crossref PubMed Scopus (67) Google Scholar) and vasomodulators (16Ogilvie A. Blasius R. Schulze-Lohoff E. Sterzel R.B. J. Autonom. Pharmacol. 1996; 16: 157-163Crossref Scopus (48) Google Scholar, 17Schlüter H. Offers E. Bruggemann G. van der Giet M. Tepel M. Nordhoff E. Karas M. Spieker C. Witzel H. Zidek W. Nature. 1994; 367: 186-188Crossref PubMed Scopus (204) Google Scholar). In addition to these physiological functions, ApnA respond to heat shock and oxidative stress with an increase in concentration (18Baker J.C. Jacobson M.K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2350-2352Crossref PubMed Scopus (78) Google Scholar). If allowed to accumulate, they could prove toxic through their ability to inhibit nucleotide kinases (19Lienhard G.E. Secemski I.I. J. Biol. Chem. 1973; 248: 1121-1123Abstract Full Text PDF PubMed Google Scholar, 20Bone R. Cheng Y.-C. Wolfenden R. J. Biol. Chem. 1986; 261: 16410-16413Abstract Full Text PDF PubMed Google Scholar), protein kinases (21Shoyab M. Arch. Biochem. Biophys. 1985; 236: 441-444Crossref PubMed Scopus (9) Google Scholar, 22Pype S. Slegers H. Enzyme Protein. 1993; 47: 14-21Crossref PubMed Scopus (8) Google Scholar), and other enzymes (23Sillero M.A.G. Cameselle J.C. McLennan A.G. Ap4A and Other Dinucleotide Polyphosphates. CRC Press, Boca Raton, FL1992: 205-228Google Scholar). Thus, there is considerable interest in the enzymes that control the synthesis and catabolism of diadenosine polyphosphates. The MutT/Nudix motif represents one general solution to the challenge of regulating the levels of metabolic intermediates that either act as cellular signals, or can be deleterious to cell function (24Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). This motif, which appears in a number of proteins from across the phylogenetic spectrum, is characterized by the following (or closely related) sequence: GX5EX7REUXEEXGU, where U is usually either I, L, or V (24Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). There is a family of so-called “Ap4A” hydrolases, which contain the MutT motif; these enzymes hydrolyze Ap4A in preference to other dinucleotides (25Thorne N.M.H. Hankin S. Wilkinson M.C. Nuñez C. Barraclough R. McLennan A.G. Biochem. J. 1995; 311: 717-721Crossref PubMed Scopus (59) Google Scholar, 26Maksel D. Guranowski A. Ilgoutz S.C. Moir A. Blackburn G.M. Gayler K.R. Biochem. J. 1998; 329: 313-319Crossref PubMed Scopus (37) Google Scholar, 27Conyers G.B. Bessman M.J. J. Biol. Chem. 1999; 274: 1203-1206Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 28Cartwright J.L. Britton P. Minnick M.F. McLennan A.G. Biochem. Biophys. Res. Commun. 1999; 256: 474-479Crossref PubMed Scopus (70) Google Scholar). Distinct MutT-type hydrolases that prefer Ap5A and Ap6A as substrates have recently been identified in Schizosaccharomyces pombe (Aps1; see Ref. 29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar) and Saccharomyces cerevisiae (YOR163w; see Ref. 30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, no Ap5A/Ap6A hydrolases have previously been found in higher eukaryotes. The search for mammalian homologues of the yeast Ap5A and Ap6A hydrolases has now drawn us to the observation that there is a human MutT-type protein with some limited sequence similarity to both Aps1 and YOR163w (Fig. 1). However, this particular human enzyme has not previously been shown to have any significant activity toward nucleoside phosphates: for example, dATP (31Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (135) Google Scholar) and dGTP 2S. T. Safrany, unpublished data.2S. T. Safrany, unpublished data. are not physiologically significant substrates (6Pace H.C. Garrison P.N. Robinson A.K. Barnes L.D. Draganescu A. Rösler A. Blackburn G.M. Siprashvili Z. Croce C.M. Huebner K. Brenner C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5484-5489Crossref PubMed Scopus (133) Google Scholar). Instead, this enzyme has been shown to have a quite different catalytic activity; it was identified (31Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (135) Google Scholar) as adiphosphoinositol polyphosphatephosphohydrolase (DIPP). DIPP's substrates, PP-InsP5 and [PP]2-InsP4 (31Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (135) Google Scholar), which are the most highly phosphorylated members of the inositol-based cell-signaling family, are metabolically unrelated to the diadenosine polyphosphates. However, it is of interest that PP-InsP5and [PP]2-InsP4 have themselves been strongly implicated as playing important roles in signal transduction. For example, cellular levels of PP-InsP5 act as a sensor to a specific mode of Ca2+ pool depletion (32Glennon M.C. Shears S.B. Biochem. J. 1993; 293: 583-590Crossref PubMed Scopus (96) Google Scholar). Second, [PP]2-InsP4 turnover is regulated by a cAMP- and cGMP-dependent process that operates independently of A and G kinases (33Safrany S.T. Shears S.B. EMBO J. 1998; 17: 1710-1716Crossref PubMed Scopus (55) Google Scholar). We now describe our discovery that Aps1, YOR163w, and DIPP have overlapping substrate specificities; these enzymes catalyze the hydrolysis of diadenosine polyphosphates and diphosphorylated inositol polyphosphates. This overlapping specificity of an enzyme for two completely different classes of substrate is not only of enzymological significance, but in addition this finding provides important new information pertinent to the structure, function and evolution of the MutT motif. Ap6A was synthesized as described previously (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar). Ap5A, Ap4A, and Ap3A were purchased from Sigma. Non-radiolabeled PP-InsP5 was synthesized as described previously (34Reddy K.M. Reddy K.K. Falck J.R. Tetrahedron Lett. 1997; 38: 4951-4952Crossref Scopus (29) Google Scholar). The sources of PP-[3H]InsP5, [3H]InsP6, and PP-[3H]InsP4 were all as described previously (31Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (135) Google Scholar). [PP]2-[3H]InsP4 was prepared as follows; three rat brains were homogenized in 2 volumes of buffer A (20 mm HEPES, pH 6.8, 2 mm CHAPS, 1 mm EDTA, 1 mm EGTA, 1 mmdithiothreitol) plus 6.25 μg/ml pepstatin, 25 μg/ml aprotinin, 5 μg/ml leupeptin, 25 μg/ml E-64. A 100,000 × gsupernatant was loaded onto a heparin-agarose type I (Sigma) column (5 mm × 5 cm) in buffer A. Proteins were eluted with a gradient generated by mixing buffer A with buffer B (buffer A plus 1m KCl): 0–10 min, 0% buffer B; 10–20 min, 15–85% B; 20–30 min, 100% B. The InsP6 kinase (35Voglmaier S.M. Bembenek M.E. Kaplin A.I. Dormán G. Olszewski J.D. Prestwich G.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4305-4310Crossref PubMed Scopus (129) Google Scholar) and PP-InsP5 kinase (36Huang C.-F. Voglmaier S.M. Bembenek M.E. Saiardi A. Snyder S.H. Biochemistry. 1998; 37: 14998-15004Crossref PubMed Scopus (43) Google Scholar) co-eluted from the column. Aliquots of peak fractions were incubated with [3H]InsP6in buffer containing: 0.75 mm EGTA, 1.5 mmEDTA, 9 mm MgSO4, 7.5 mm ATP, 10 mm NaF, 20 mm phosphocreatine, 1 mmdithiothreitol, 20 mm HEPES (pH 6.8), 4 mmCHAPS, 20 Sigma units/ml creatine phosphokinase. The resultant [PP]2-[3H]InsP4 (>80% conversion) was purified by HPLC (33Safrany S.T. Shears S.B. EMBO J. 1998; 17: 1710-1716Crossref PubMed Scopus (55) Google Scholar) and desalted (37Menniti F.S. Miller R.N. Putney Jr., J.W. Shears S.B. J. Biol. Chem. 1993; 268: 3850-3856Abstract Full Text PDF PubMed Google Scholar) with 17% recovery. Pure, recombinant preparations of Aps1 (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar), YOR163w (30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), Fhit (38Brenner C. Pace H.C. Garrison P.N. Rösler A. Liu X.-H. Blackburn G.M. Huebner K. Barnes L.D. Protein Eng. 1997; 10: 1461-1463Crossref PubMed Scopus (40) Google Scholar), human Ap4A hydrolase (25Thorne N.M.H. Hankin S. Wilkinson M.C. Nuñez C. Barraclough R. McLennan A.G. Biochem. J. 1995; 311: 717-721Crossref PubMed Scopus (59) Google Scholar), IalA protein fromBartonella bacilliformis (28Cartwright J.L. Britton P. Minnick M.F. McLennan A.G. Biochem. Biophys. Res. Commun. 1999; 256: 474-479Crossref PubMed Scopus (70) Google Scholar), and DIPP (31Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (135) Google Scholar) were all obtained as described previously. For the analysis of ApnA (n = 3–6) hydrolysis, the substrate was incubated with enzyme in buffer containing 50 mm HEPES (pH 7.6), 1 mm MnCl2, and 100 μg/ml bovine serum albumin at 37 °C. The mass of enzyme, incubation time, and substrate concentrations were varied as described previously to determine substrate specificity, time courses, and substrate saturation curves (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar). Assay solutions were analyzed by HPLC to resolve individual nucleotides as described previously (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar). For the analysis of PP-InsP5 and [PP]2-InsP4 hydrolysis, the substrate was incubated with enzyme in buffer containing 50 mm KCl, 50 mm HEPES (pH 7.2), 4 mm CHAPS, 50 μg/ml bovine serum albumin, 1 mm Na2EDTA, 2 mm MgSO4. At the appropriate time, aliquots were quenched, neutralized and subsequently analyzed by HPLC as described previously (31Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (135) Google Scholar), except that the gradient was generated by mixing buffer A (1 mm Na2EDTA) and buffer B (buffer A plus 1.3 m(NH4)2HPO4, pH 3.85 with H3PO4) as follows: 0–5 min, 0% B; 5–10 min, 0–50% B; 10–60 min, 50–100% B; 60–70 min, 100% B. Fractions were collected at 1-min intervals, beginning 15 min into the gradient. Fig. 1 compares the sequences of three enzymes that each contain the MutT motif. Two of these proteins, Aps1 fromS. pombe (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar) and YOR163w from S. cerevisiae(30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), have been characterized as Ap6A/Ap5A hydrolases. The third protein is from Homo sapiens and has been characterized as a PP-InsP5/[PP]2-InsP4phosphohydrolase (31Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (135) Google Scholar). Much of the DIPP sequence (around 70%) showed no significant similarity to either of the two yeast proteins (Fig. 1). However, the region from Val34 to Glu85 in DIPP, which includes the MutT motif and short flanking regions, is 46% identical to the corresponding regions of YOR163w (Val47 to Glu99) and Aps1 (Val57 to Lys108). This comparison was of particular interest because DIPP has not been shown to metabolize nucleoside phosphates, and neither Aps1 nor YOR163w has been shown to metabolize diphosphoinositol polyphosphates. We therefore determined if DIPP and the Ap5A/Ap6A hydrolases could metabolize the other enzyme's substrates. For these experiments, we used pure, recombinant preparations of each enzyme (see “Experimental Procedures”). Ap6A was found to be actively metabolized by DIPP (Fig. 2), with multiple products being formed (Fig. 3A). In fact, when incubated with 100 μm Ap6A, DIPP was virtually as efficient as an Ap6A hydrolase (1.1 μmol/min/mg) as is either Aps1 or YOR163w, both of which hydrolyze 100 μmAp6A at a rate of 1–2 μmol/min/mg (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar, 30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The rank order of relative specific activities of DIPP toward the various diadenosine polyphosphates (Ap6A > Ap5A > Ap4A > Ap3A; Fig. 2) mirrored that for Aps1 (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar). In terms of relative substrate affinities, YOR163w is slightly different from both Aps1 and DIPP, since YOR163w does not hydrolyze Ap4A, possibly due to the insertion of an extra asparagine residue within the putative substrate-binding MutT motif (Fig. 1) (30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). It is significant that DIPP provides the first example of a hydrolase in the animal kingdom that expresses a preference for Ap5A and Ap6A over other diadenosine polyphosphates.Figure 3HPLC Analysis of the hydrolysis of Ap6A and Ap5A by DIPP . Either no (dashed line) or 1.08 μg (solid line) of DIPP was incubated with 100 μmAp6A (panel A) or Ap5A (panel B) in a 100 μl reaction for 15 min at 37 °C. The reaction was stopped by freezing on dry ice, and an aliquot was analyzed by HPLC as described under “Experimental Procedures.” The time of elution is shown on the abscissa of panel B, and the elution positions of nucleotide standards are shown above panel A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also determined kcat and Km values for the hydrolysis of Ap6A and Ap5A by DIPP (Table I). The affinities of DIPP for these particular substrates (Km = 6–8 μm) were slightly higher than the affinity of Aps1 (approximately 20 μm; Ref. 29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar) and YOR163w (approximately 60 μm; Ref. 30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Thekcat values for DIPP (0.5 s−1, Table I) were intermediate between those for Aps1 (approximately 2 s−1; Ref. 29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar) and YOR163w (0.06 s−1 for Ap5A and 0.4 s−1 for Ap6A; Ref.30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). It is striking that the Ap5A/Ap6A hydrolase activity of DIPP is kinetically very similar to that of both yeast enzymes.Table IKinetic parameters for the metabolism of Ap5A and Ap6A by DIPPSubstrateKmkcatkcat/Kmμms −1m −1s −1 × 10 5Ap6A5.9 ± 3.0 (5)0.50 ± 0.15 (5)0.85Ap5A7.7 ± 2.2 (3)0.42 ± 0.13 (3)0.55Data were determined as described under “Experimental Procedures”; values are presented as means and standard errors, with the number of experiments given in parentheses. Open table in a new tab Data were determined as described under “Experimental Procedures”; values are presented as means and standard errors, with the number of experiments given in parentheses. The reaction mechanisms of Aps1, YOR163w, and DIPP promise to be interesting to unravel and compare. For the yeast enzymes, it has been noted that Ap6A can be hydrolyzed to three different sets of products. Aps1 primarily hydrolyzes Ap6A asymmetrically to yield p4A and ADP, but there is also some symmetrical hydrolysis to yield 2 ATP as a minor product (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar). YOR163w also generates p4A and ADP as major products from Ap6A, but p5A and AMP are also formed as minor products (30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). These different modes of Ap6A hydrolysis have been attributed to Ap6A having some mobility within the active site (30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The time course of Ap6A hydrolysis by DIPP (Fig. 4A) provides insight into this enzyme's reaction mechanisms. In these assays, the rate of consumption of Ap6A was matched by the rate of accumulation of AMP plus ADP, at a ratio of about 4:1 (Fig. 4A). These results suggest that the predominant route of Ap6A hydrolysis is to AMP plus p5A, with the formation of ADP plus p4A being a more minor reaction. Although we estimate that approximately 80% of the Ap6A was degraded to p5A plus AMP, the p5A did not accumulate, other than for a small amount after 2 min (Fig. 4A). Presumably, p5A is dephosphorylated almost as rapidly as it is formed; p5A that was generated from Ap6A was also rapidly hydrolyzed (to p4A plus Pi) in previous experiments with YOR163w (30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). DIPP also hydrolyzed the p4A that was formed during Ap6A hydrolysis (Fig. 4A). Between the 15- and 35-min time points, the decrease in p4A levels was matched by a corresponding increase in ATP levels. Thus, we conclude DIPP hydrolyzed p4A to ATP and Pi. Furthermore, once all the original Ap6A had been consumed (after approximately 15 min), there were only relatively minor increases in the levels of ADP and AMP. Thus, we also conclude that there is relatively little hydrolysis of either p4A or ATP to either ADP or AMP. Finally, by the end of the time course, the total amount of [AMP + ADP] that was formed was approximately equivalent to the amount of Ap6A added (Fig. 4A). Thus, all the ATP that accumulated can be accounted for by further dephosphorylation of p5A and p4A, rather than direct, symmetrical cleavage of Ap6A. With regards to Ap5A, both Aps1 and YOR163w degrade this substrate to two sets of products, namely ADP plus ATP, and p4A plus AMP (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar, 30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, one difference between these two enzymes is that ADP plus ATP are the major reaction products for Aps1 (29Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Crossref PubMed Scopus (31) Google Scholar), whereas for YOR163w, p4A and AMP are the major products (30Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). When DIPP was incubated with Ap5A, this substrate was primarily hydrolyzed to AMP plus p4A (Figs. 3B and 4B). ADP accounted for no more than 4% of total nucleoside products (Fig. 4B), indicating that there was relatively little cleavage of Ap5A to ADP plus ATP. Thus, the ATP that did accumulate in these reactions (Fig. 4B) must have arisen from the further hydrolysis of p4A. We next discovered that Aps1 and YOR163w expressed phosphohydrolase activity toward PP-InsP5 (Fig. 5A). Reaction products were identified by HPLC. InsP6 was formed (Fig. 5A), which indicates that both yeast enzymes cleaved the β-phosphate from the diphosphate group of PP-InsP5, just as is the case for DIPP (31Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (135) Google Scholar). No InsP5 was produced (Fig. 5A), indicating that the pyrophosphate group was not removed from PP-InsP5; the absence of InsP5 in these reactions also demonstrates that InsP6 was not a substrate. The yeast enzymes also did not hydrolyze any of the monoester phosphates of PP-InsP5, since no PP-InsP4 was formed (Fig. 5A). Both Aps1 and YOR163w had only an 8-fold lower substrate affinity for PP-InsP5, compared with DIPP (Table II).kcat values for the three enzymes were also quite similar (Table II).Table IIKinetic parameters for the metabolism of PP-InsP5 by Aps1, YOR163w, and DIPPEnzymeKmkcatkcat/Kmnms −1m −1s −1 × 10 5Aps131 ± 3 (4)0.17 ± 0.02 (4)53YOR163w31 ± 6 (3)0.06 ± 0.01 (3)20DIPP4.2 ± 0.4 (5)0.2 ± 0.01 (8)474Data were determined as described under “Experimental Procedures”; values are presented as means and standard errors, with the number of experiments given in parentheses. Open table in a new tab Data were determined as described under “Experimental Procedures”; values are presented as means and standard errors, with the number of experiments given in parentheses. [PP]2-InsP4 is another diphosphorylated inositol phosphate that is hydrolyzed by DIPP (31Safrany S.T. Caffrey J.J. Yang X. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Crossref PubMed Scopus (135) Google Scholar). We have also found [PP]2-InsP4 to be metabolized by Aps1 and YOR163w (Fig. 5B). Under first-order conditions, Aps1 hydrolyzed [PP]2-InsP4 5-fold more rapidly (k −1 = 8.3 ±" @default.
• W2064433587 created "2016-06-24" @default.
• W2064433587 creator A5001561535 @default.
• W2064433587 creator A5005563728 @default.
• W2064433587 creator A5013107971 @default.
• W2064433587 creator A5017729387 @default.
• W2064433587 creator A5050099661 @default.
• W2064433587 creator A5077198825 @default.
• W2064433587 creator A5084431379 @default.
• W2064433587 date "1999-07-01" @default.
• W2064433587 modified "2023-10-03" @default.
• W2064433587 title "The Diadenosine Hexaphosphate Hydrolases fromSchizosaccharomyces pombe and Saccharomyces cerevisiae Are Homologues of the Human Diphosphoinositol Polyphosphate Phosphohydrolase" @default.
• W2064433587 cites W1481454511 @default.
• W2064433587 cites W1485488844 @default.
• W2064433587 cites W1497060658 @default.
• W2064433587 cites W1558506222 @default.
• W2064433587 cites W1579399439 @default.
• W2064433587 cites W178228284 @default.
• W2064433587 cites W1789455264 @default.
• W2064433587 cites W1819159746 @default.
• W2064433587 cites W1922824291 @default.
• W2064433587 cites W1966063738 @default.
• W2064433587 cites W1972436355 @default.
• W2064433587 cites W1974020806 @default.
• W2064433587 cites W1974517908 @default.
• W2064433587 cites W1977405908 @default.
• W2064433587 cites W1978992872 @default.
• W2064433587 cites W1982832802 @default.
• W2064433587 cites W1983503188 @default.
• W2064433587 cites W1985744441 @default.
• W2064433587 cites W1989951007 @default.
• W2064433587 cites W2004730354 @default.
• W2064433587 cites W2032882710 @default.
• W2064433587 cites W2034502645 @default.
• W2064433587 cites W2034588677 @default.
• W2064433587 cites W2038194517 @default.
• W2064433587 cites W2038351234 @default.
• W2064433587 cites W2039105952 @default.
• W2064433587 cites W2042894072 @default.
• W2064433587 cites W2057757274 @default.
• W2064433587 cites W2063178731 @default.
• W2064433587 cites W2077928157 @default.
• W2064433587 cites W2087521687 @default.
• W2064433587 cites W2089196635 @default.
• W2064433587 cites W2095074799 @default.
• W2064433587 cites W2101524123 @default.
• W2064433587 cites W2112773862 @default.
• W2064433587 cites W2126076915 @default.
• W2064433587 cites W2126904200 @default.
• W2064433587 cites W2137259838 @default.
• W2064433587 cites W2149967205 @default.
• W2064433587 cites W2152362181 @default.
• W2064433587 cites W2165914529 @default.
• W2064433587 cites W2229995226 @default.
• W2064433587 cites W2276140391 @default.
• W2064433587 cites W2420651668 @default.
• W2064433587 cites W99285315 @default.
• W2064433587 doi "https://doi.org/10.1074/jbc.274.31.21735" @default.
• W2064433587 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10419486" @default.
• W2064433587 hasPublicationYear "1999" @default.
• W2064433587 type Work @default.
• W2064433587 sameAs 2064433587 @default.
• W2064433587 citedByCount "127" @default.
• W2064433587 countsByYear W20644335872012 @default.
• W2064433587 countsByYear W20644335872013 @default.
• W2064433587 countsByYear W20644335872014 @default.
• W2064433587 countsByYear W20644335872015 @default.
• W2064433587 countsByYear W20644335872016 @default.
• W2064433587 countsByYear W20644335872017 @default.
• W2064433587 countsByYear W20644335872018 @default.
• W2064433587 countsByYear W20644335872019 @default.
• W2064433587 countsByYear W20644335872020 @default.
• W2064433587 countsByYear W20644335872021 @default.
• W2064433587 countsByYear W20644335872022 @default.
• W2064433587 countsByYear W20644335872023 @default.
• W2064433587 crossrefType "journal-article" @default.
• W2064433587 hasAuthorship W2064433587A5001561535 @default.
• W2064433587 hasAuthorship W2064433587A5005563728 @default.
• W2064433587 hasAuthorship W2064433587A5013107971 @default.
• W2064433587 hasAuthorship W2064433587A5017729387 @default.
• W2064433587 hasAuthorship W2064433587A5050099661 @default.
• W2064433587 hasAuthorship W2064433587A5077198825 @default.
• W2064433587 hasAuthorship W2064433587A5084431379 @default.
• W2064433587 hasBestOaLocation W20644335871 @default.
• W2064433587 hasConcept C181199279 @default.
• W2064433587 hasConcept C185592680 @default.
• W2064433587 hasConcept C2777132085 @default.
• W2064433587 hasConcept C2777576037 @default.
• W2064433587 hasConcept C2778038992 @default.
• W2064433587 hasConcept C2779222958 @default.
• W2064433587 hasConcept C55493867 @default.
• W2064433587 hasConcept C86803240 @default.
• W2064433587 hasConceptScore W2064433587C181199279 @default.
• W2064433587 hasConceptScore W2064433587C185592680 @default.
• W2064433587 hasConceptScore W2064433587C2777132085 @default.
• W2064433587 hasConceptScore W2064433587C2777576037 @default.
• W2064433587 hasConceptScore W2064433587C2778038992 @default.
• W2064433587 hasConceptScore W2064433587C2779222958 @default.

• next