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• W2024708954 abstract "A total of 17 Nudix hydrolases were tested for their ability to hydrolyze 5-phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates did not degrade PRPP. The products of hydrolysis were ribose 1,5-bisphosphate and Pi. Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily of Nudix hydrolases, which also degrade the non-nucleotide diphosphoinositol polyphosphates. K m andk cat values for PRPP hydrolysis for theDeinococcus radiodurans DR2356 (di)nucleoside polyphosphate hydrolase, the human diadenosine tetraphosphate hydrolase, and human DIPP-1 (diadenosine hexaphosphate and diphosphoinositol polyphosphate hydrolase) were 1 mm and 1.5 s−1, 0.13 mm and 0.057 s−1, and 0.38 mm and 1.0 s−1, respectively. Active site mutants of theCaenorhabditis elegans diadenosine tetraphosphate hydrolase had no activity, confirming that the same active site is responsible for nucleotide and PRPP hydrolysis. Comparison of the specificity constants for nucleotide, diphosphoinositol polyphosphate, and PRPP hydrolysis suggests that PRPP is a significant substrate for theD. radiodurans DR2356 enzyme and for the DIPP subfamily. In the latter case, generation of the glycolytic activator ribose 1,5-bisphosphate may be a new function for these enzymes. A total of 17 Nudix hydrolases were tested for their ability to hydrolyze 5-phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates did not degrade PRPP. The products of hydrolysis were ribose 1,5-bisphosphate and Pi. Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily of Nudix hydrolases, which also degrade the non-nucleotide diphosphoinositol polyphosphates. K m andk cat values for PRPP hydrolysis for theDeinococcus radiodurans DR2356 (di)nucleoside polyphosphate hydrolase, the human diadenosine tetraphosphate hydrolase, and human DIPP-1 (diadenosine hexaphosphate and diphosphoinositol polyphosphate hydrolase) were 1 mm and 1.5 s−1, 0.13 mm and 0.057 s−1, and 0.38 mm and 1.0 s−1, respectively. Active site mutants of theCaenorhabditis elegans diadenosine tetraphosphate hydrolase had no activity, confirming that the same active site is responsible for nucleotide and PRPP hydrolysis. Comparison of the specificity constants for nucleotide, diphosphoinositol polyphosphate, and PRPP hydrolysis suggests that PRPP is a significant substrate for theD. radiodurans DR2356 enzyme and for the DIPP subfamily. In the latter case, generation of the glycolytic activator ribose 1,5-bisphosphate may be a new function for these enzymes. The Nudix hydrolases are members of an enzyme family that was named after their ability to hydrolyze predominantly the pyrophosphate linkage in a variety of compounds having the general structure of a nucleoside diphosphate (Npp) linked to another moiety, X, with varying degrees of specificity (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Google Scholar, 2McLennan A.G. Int. J. Mol. Med. 1999; 4: 79-89Google Scholar). Thus, nucleoside triphosphates (Npp-p), dinucleoside polyphosphates (Npp-pnN), nucleotide sugars (Npp-sugar), NADH, and coenzyme A are examples of Nudix hydrolase substrates that fall into this category. In general terms, the members of this protein family are believed to rid the cell of potentially deleterious endogenous nucleotide metabolites and to modulate the accumulation of metabolic intermediates by diverting them into alternative pathways in response to biochemical need, although specific regulatory functions may also be associated with individual members (1Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Google Scholar). Recently, a subfamily of Nudix hydrolases has been described that hydrolyze the long chain dinucleoside and nucleoside polyphosphates, including diadenosine 5′,5‴-P 1The abbreviations used are: Ap6A, diadenosine 5′,5‴-P 1,P 6-hexaphosphate; Ap4A, diadenosine 5′,5‴-P 1,P 4-tetraphosphate; Ap5A, diadenosine 5′,5‴-P 1,P 5-pentaphosphate; ApnA, diadenosine 5′,5‴-P 1,P n-polyphosphate; DIPP, diphosphoinositol polyphosphate phosphohydrolase; NUDT, Nudix-type gene; PP-InsP5, diphosphoinositol pentakisphosphate; [PP]2-InsP4, bisdiphosphoinositol tetrakisphosphate; PRPP, 5-phosphoribosyl 1-pyrophosphate; Rib-1-P, ribose 1-phosphate; Rib-5-P, ribose 5-phosphate; Rib-1, 5-P2, ribose 1,5-bisphosphate ,P 6-hexaphosphate (Ap6A), 1diadenosine 5′,5‴-P 1,P 5-pentaphosphate (Ap5A), and adenosine 5′-pentaphosphate (p5A) but which have relatively low activity with diadenosine 5′,5‴-P 1,P 4-tetraphosphate (Ap4A). Most interestingly, these enzymes also act as phosphohydrolases toward the non-nucleotide substrates, diphosphoinositol pentakisphosphate (PP-InsP5) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4), with varying degrees of efficiency relative to the nucleotide substrates. Structurally and mechanistically, they are closely related within the Nudix family to the well studied Ap4A hydrolases (3Abdelghany H.M. Gasmi L. Cartwright J.L. Bailey S. Rafferty J.B. McLennan A.G. Biochim. Biophys. Acta. 2001; 1550: 27-36Google Scholar, 4Guranowski A. Pharmacol. Ther. 2000; 87: 117-139Google Scholar), although the latter enzymes do not appear to utilize PP-InsP5 or [PP]2-InsP4 as substrates (5Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Google Scholar). So far, four distinct genes (excluding pseudogenes) and five distinct gene products in this Ap6A hydrolase/diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily have been described in mammalian cells, DIPP-1 (NUDT3) (6Safrany S.T. Caffrey J.J. Yang X.N. Bembenek M.E. Moyer M.B. Burkhart W.A. Shears S.B. EMBO J. 1998; 17: 6599-6607Google Scholar), DIPP-2α and -2β (NUDT4) (7Caffrey J.J. Safrany S.T. Yang X.N. Shears S.B. J. Biol. Chem. 2000; 275: 12730-12736Google Scholar, 8Caffrey J.J. Shears S.B. Gene. 2001; 269: 53-60Google Scholar), DIPP-3α (NUDT10, hAps2), and -3β (NUDT11, hAps1) (9Hidaka K. Caffrey J.J. Hua L. Zhang T. Falck J.R. Nickel G.C. Carrel L. Barnes L.D. Shears S.B. J. Biol. Chem. 2002; 277: 32730-32738Google Scholar, 10Leslie N.R. McLennan A.G. Safrany S.T. BMC Biochem. 2002; 3: 20Google Scholar), whereas the yeastsSaccharomyces cerevisiae (DDP1) (5Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Google Scholar, 11Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Google Scholar) andSchizosaccharomyces pombe (Aps1) (5Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Google Scholar, 12Ingram S.W. Stratemann S.A. Barnes L.D. Biochemistry. 1999; 38: 3649-3655Google Scholar) appear to have one each. PP-InsP5 and [PP]2-InsP4 are also substrates for the g5R Nudix hydrolase encoded by African Swine Fever virus (13Cartwright J.L. Safrany S.T. Dixon L.K. Darzynkiewicz E. Stepinski J. Burke R. McLennan A.G. J. Virol. 2002; 76: 1415-1421Google Scholar). Several of these enzymes have high affinities and highk cat/K m ratios for the diphosphoinositol polyphosphates, suggesting that these compounds may be important substrates in vivo. They may be involved in the regulation of vesicle trafficking (14Ye W.L. Ali N. Bembenek M.E. Shears S.B. Lafer E.M. J. Biol. Chem. 1995; 270: 1564-1568Google Scholar), apoptosis (15Morrison B.H. Bauer J.A. Kalvakolanu D.J. Lindner D.J. J. Biol. Chem. 2001; 276: 24965-24970Google Scholar), DNA repair (16Hanakahi L.A. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Google Scholar), and in vacuole biogenesis and environmental stress responses in yeast (17Dubois E. Scherens B. Vierendeels F. Ho M.M. Messenguy F. Shears S.B. J. Biol. Chem. 2002; 277: 23755-23763Google Scholar); hence, the DIPP Nudix hydrolases have also been implicated in these processes. The ability of this subset of Nudix hydrolases to utilize a sugar pyrophosphate as a substrate prompted us to test another such compound of known biological importance, 5-phosphoribosyl 1-pyrophosphate (PRPP). PRPP is both a substrate and regulator of purine, pyrimidine, and pyridine nucleotide synthesis (18Bagnara A.S. Letter A.A. Henderson J.F. Biochim. Biophys. Acta. 1974; 374: 259-270Google Scholar, 19Holmes E.W. Wyngaarden J.B. Kelley W.N. J. Biol. Chem. 1973; 248: 6035-6040Google Scholar, 20Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Google Scholar, 21Becker M.A. Prog. Nucleic Acid Res. Mol. Biol. 2001; 69: 115-148Google Scholar); in bacteria and lower eukaryotes it is also a precursor for histidine and tryptophan biosynthesis (22Voll M.J. Appella E. Martin R.G. J. Biol. Chem. 1967; 242: 1760-1767Google Scholar, 23Alifano P. Fani R. Lio P. Lazcano A. Bazzicalupo M. Carlomagno M.S. Bruni C.B. Microbiol. Rev. 1996; 60: 44-69Google Scholar, 24Hütter R. Niederberger P. DeMoss J.A. Annu. Rev. Microbiol. 1986; 40: 55-77Google Scholar, 25Hove-Jensen B. J. Bacteriol. 1988; 170: 1148-1152Google Scholar). Furthermore, a potential product of pyrophosphatase activity acting upon PRPP is ribose 1,5-bisphosphate (Rib-1,5-P2), which has recently been shown to be a physiological regulator of glycolysis and the fructose 6-phosphate/fructose 1,6-bisphosphate cycle (26Kawaguchi T. Veech R.L. Uyeda K. J. Biol. Chem. 2001; 276: 28554-28561Google Scholar, 27Sawada M. Mitsui Y. Sugiya H. Furuyama S. Int. J. Biochem. Cell Biol. 2000; 32: 447-454Google Scholar, 28Ogushi S. Lawson J.W.R. Dobson G.P. Veech R.L. Uyeda K. J. Biol. Chem. 1990; 265: 10943-10949Google Scholar). Here, we show that Nudix hydrolases of the DIPP subfamily and the related Ap4A hydrolases all exhibit PRPP pyrophosphatase activity, whereas Nudix hydrolases previously shown to be specific for NDP-sugars, pyridine nucleotides, and coenzyme A are unable to hydrolyze PRPP. Recombinant human Aps1 (DIPP-3β) and Aps2 (DIPP-3α) (10Leslie N.R. McLennan A.G. Safrany S.T. BMC Biochem. 2002; 3: 20Google Scholar), S. cerevisiae Ddp1p Ap6A hydrolase (YOR163w protein) (11Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Google Scholar), and Npy1p NADH pyrophosphatase (YGL067w protein) (29AbdelRaheim S.R. Cartwright J.L. Gasmi L. McLennan A.G. Arch. Biochem. Biophys. 2001; 388: 18-24Google Scholar), Caenorhabditis elegansAp4A hydrolase (3Abdelghany H.M. Gasmi L. Cartwright J.L. Bailey S. Rafferty J.B. McLennan A.G. Biochim. Biophys. Acta. 2001; 1550: 27-36Google Scholar), African Swine Fever virus g5R protein (13Cartwright J.L. Safrany S.T. Dixon L.K. Darzynkiewicz E. Stepinski J. Burke R. McLennan A.G. J. Virol. 2002; 76: 1415-1421Google Scholar), human NUDT5 ADP-sugar hydrolase (30Gasmi L. Cartwright J.L. McLennan A.G. Biochem. J. 1999; 344: 331-337Google Scholar), NUDT9 ADP-ribose hydrolase (31Lin S.R. Gasmi L. Xie Y. Ying K. Gu S.H. Wang Z. Jin H. Chao Y.Q. Wu C.Q. Zhou Z.X. Tang R. Mao Y.M. McLennan A.G. Biochim. Biophys. Acta. 2002; 1594: 127-135Google Scholar), and mouse Nudt7 coenzyme A pyrophosphatase (32Gasmi L. McLennan A.G. Biochem. J. 2001; 357: 33-38Google Scholar) were prepared as previously described. Active site mutants of the C. elegans Ap4A hydrolase (E52Q and E56Q) were a gift from H. Abdelghany. 2H. Abdelghany and A. G. McLennan, unpublished data. The YgdP Ap4A hydrolase from Salmonella typhimurium was a gift from T. Ismail. 3T. Ismail and A. G. McLennan, unpublished data.Recombinant human DIPP-1, DIPP-2α, and -2β were prepared as GST-fusion proteins as described for hAps1 and hAps2 4S. T. Safrany, unpublished observations. (10Leslie N.R. McLennan A.G. Safrany S.T. BMC Biochem. 2002; 3: 20Google Scholar). Recombinant human Ap4A hydrolase, Deinococcus radioduranscoenzyme A pyrophosphatase (DR1184 gene product), and D. radiodurans ApnA hydrolase (DR2356 gene product) were prepared by procedures similar to those used for the C. elegans Ap4A hydrolase. 5D. I. Fisher, J. L. Cartwright, and A. G. McLennan, unpublished data. PRPP and all nucleotides were from Sigma. The EnzChek® phosphate assay kit was from Molecular Probes. Initial screening of enzyme preparations for their ability to release Pi from PRPP was carried out using a phosphomolybdate colorimetric assay (33Ames B.N. Methods Enzymol. 1966; 8: 115-118Google Scholar). Purified enzymes (5 μg) were incubated for 15 min at 37 °C in 50 mm Tris-HCl, pH 8.0, 5 mm Mg acetate, 1 mm dithiothreitol, and 0.2 mm PRPP in a total volume of 200 μl and the reactions stopped by the addition of the molybdate detection reagent. Enzymes testing negative in an initial screen were retested with up to 12 μg of protein per assay. Kinetic constants for PRPP hydrolysis by selected enzymes were calculated from initial rates determined using a sensitive continuous spectrophotometric assay based on the phosphate-dependent conversion of 2-amino-6-mercapto-7-methylpurine riboside to 2-amino-6-mercapto-7-methylpurine and ribose-1-phosphate catalyzed by purine nucleoside phosphorylase (34Webb M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4884-4887Google Scholar). For this, the EnzChek® assay kit was used according to the manufacturer's instructions with the following modifications: reactions were preincubated for 5 min without substrate at 37 °C in 50 mm BisTrisPropane buffer, pH 8.5, 5 mm Mg acetate, and 1 mm dithiothreitol and then incubated with substrate for up to 2 min. Final enzyme concentrations were 75 μg/ml (human Ap4A hydrolase), 15.2 μg/ml (DIPP-1), and 7 μg/ml (D. radioduransApnA hydrolase). In each case, controls lacking substrate or enzyme were performed. The products of PRPP hydrolysis by the D. radiodurans ApnA hydrolase were generated by incubation of 0.2 mm PRPP with 7 μg of enzyme in 50 mm Tris-HCl, pH 8.0, 5 mm Mg acetate, 1 mm dithiothreitol for 10 min at 37 °C. Reactions (200 μl) were stopped by freezing and then applied to a 1-ml Resource-Q column (Pharmacia) at 2 ml/min in 35 mm NH4HCO3, pH 9.6. The elution system consisted of a gradient of 5–100% buffer A (0.7 mNH4HCO3, pH 9.6) in water over 10 min. Fractions (0.5 ml) were collected and the presence of products determined by colorimetric determination of phosphate released after incubation with alkaline phosphatase or inorganic pyrophosphatase as required (33Ames B.N. Methods Enzymol. 1966; 8: 115-118Google Scholar, 35Canales J. Pinto R.M. Costas M.J. Hernández M.T. Miró A. Bernet D. Fernández A. Cameselle J.C. Biochim. Biophys. Acta. 1995; 1246: 167-177Google Scholar). Ap4A hydrolase activity was determined luminometrically as previously described (3Abdelghany H.M. Gasmi L. Cartwright J.L. Bailey S. Rafferty J.B. McLennan A.G. Biochim. Biophys. Acta. 2001; 1550: 27-36Google Scholar). Protein concentrations were estimated by the Coomassie Blue binding method (36Peterson G.L. Methods Enzymol. 1983; 91: 95-119Google Scholar). Positive ion electrospray mass spectrometry was performed as previously described (11Cartwright J.L. McLennan A.G. J. Biol. Chem. 1999; 274: 8604-8610Google Scholar). A total of 17 different Nudix hydrolases were tested for their ability to hydrolyze PRPP. The assay employed measures the release of inorganic phosphate. As can be seen from Table I, all 11 enzymes that can utilize dinucleoside polyphosphates with 4 or more phosphate groups as substrates are also able to hydrolyze PRPP, whereas the 6 that cannot and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates do not degrade PRPP. It should be stressed that the rates quoted of micromoles of Pi produced min−1·micromoles protein−1 are not directly convertible to true k cat values because a standard set of conditions, including a fixed PRPP concentration of 200 μm, was employed for all assays and so is not necessarily optimal for each enzyme. Therefore, the rank order of PRPP hydrolase activity should only be taken as a guide. Because PRPP is known to undergo spontaneous, Mg2+-dependent non-enzymic hydrolysis (37Dennis A.L. Puskas M. Stasaitis S. Sandwick R.K. J. Inorg. Biochem. 2000; 81: 73-80Google Scholar, 38Trembacz H. Jezewska M.M. Biochem. J. 1990; 271: 621-625Google Scholar), it was important to establish that the observed activity was enzyme-catalyzed. This is clearly demonstrated by the lack of degradation by two active site mutants of the C. elegansAp4A hydrolase (Table I). In common with other Nudix hydrolases, substitution of Glu residues in the Nudix motif by Gln dramatically reduced activity of this enzyme toward Ap4A. Compared with the wild type value of 23 s−1, the C. elegans E52Q and E56Q mutants have k catvalues for Ap4A of 0.0052 and 0.00024 s−1, respectively.2 They are also completely inactive with PRPP. This establishes that the same active site is responsible for Ap4A and PRPP hydrolysis. The same is assumed to hold true for the other enzymes.Table IUtilization of PRPP as a substrate by several Nudix hydrolasesEnzymeActivity (μmol of Pi produced/min−1 · μmol protein−1)D. radioduransApnA hydrolase8.8Human DIPP-13.9Human Ap4A hydrolase2.1Human DIPP-2α1.8Human Aps1 (DIPP-3β)1.5C. elegans Ap4A hydrolase (wild type)0.64C. elegans Ap4A hydrolase (E52Q)0C. elegans Ap4A hydrolase (E56Q)0S. typhimurium YgdP Ap4A hydrolase0.60Human Aps2 (DIPP-3α)0.56Human DIPP-2β0.44S. cerevisiae Ddp1p Ap6A hydrolase0.29African Swine Fever virus g5R protein+aTested positive in an initial screen but was not quantified because of lack of material.D. radiodurans DR1184 coenzyme A pyrophosphatase0S. cerevisiae Pcd1p coenzyme A pyrophosphatase0Mouse Nudt7 coenzyme A pyrophosphatase0S. cerevisiae Npy1p NADH pyrophosphatase0Human NUDT5 ADP-sugar pyrophosphatase0Human NUDT9 ADP-ribose pyrophosphatase0The sources of the enzymes listed are shown in “Experimental Procedures.”a Tested positive in an initial screen but was not quantified because of lack of material. Open table in a new tab The sources of the enzymes listed are shown in “Experimental Procedures.” K m and k cat values for PRPP were determined for one prokaryotic ApnA hydrolase, one eukaryotic Ap4A hydrolase, and one eukaryotic Ap6A hydrolase/DIPP, using a continuous spectrophotometric assay. D. radiodurans DR2356 ApnA hydrolase is an enzyme that hydrolyzes a variety of nucleoside and dinucleoside polyphosphates, including p4A, p5A, Ap4A, Ap5A, and Ap6A.5It has no activity toward diphosphoinositol polyphosphates4; however, it readily hydrolyzes PRPP with aK m of 1 mm and ak cat of 1.5 s−1 (TableII). For comparison,K m and k cat values for Ap4A were determined to be 30 μm and 0.035 s−1, respectively, resulting in similar specificity constants (k cat /K m) for both substrates (Table II). Human Ap4A hydrolase also hydrolyzed PRPP with a k cat of 0.057 s−1 and aK m of 0.13 mm (Table II). In this case, however, the specificity constant with PRPP was some 20,000-fold lower than that found with Ap4A as substrate. Finally, human DIPP-1 was the most efficient of the three enzymes at PRPP hydrolysisin vitro with a k cat of 1.0 s−1 and a K m of 0.38 mm(Table II). The specificity constant with PRPP of 2,600 was 325- and 18,000-fold lower than those previously measured with Ap6A and PP-InsP5, respectively (5Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Google Scholar). The physiological significance of these data is discussed below.Table IIKinetic constants for substrate hydrolysis by selected Nudix hydrolasesEnzymeSubstrateConcn. in vivo(S).K mk catk cat/K mS(k cat/K m)mms−1m−1s−1s−1D. radiodurans ApnA hydrolasePRPP10−310−31.51.5 × 1031.5Ap4A10−63.0 × 10−50.0351.2 × 1031.2 × 10−3Human Ap4A hydrolasePRPP10−41.3 × 10−40.0574.4 × 1020.044Ap4A10−65.0 × 10−75.010710Human DIPP-1PRPP10−43.8 × 10−41.02.6 × 1030.26Ap6A10−95.9 × 10−60.58.5 × 1048.5 × 10−5PP-InsP510−64.2 × 10−90.24.7 × 10747.4Kinetic constants for the hydrolysis of Ap6A and PP-InsP5 by DIPP-1 are taken from published data (5Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Google Scholar). All other constants are derived from original data. Values quoted for substrate concentrations in vivo are typical literature values but may vary by one order of magnitude in either direction depending on physiological conditions. Open table in a new tab Kinetic constants for the hydrolysis of Ap6A and PP-InsP5 by DIPP-1 are taken from published data (5Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Google Scholar). All other constants are derived from original data. Values quoted for substrate concentrations in vivo are typical literature values but may vary by one order of magnitude in either direction depending on physiological conditions. Because all Nudix hydrolases cleave pyrophosphate linkages, it was anticipated that they would remove the β-phosphate from the pyrophosphate moiety attached to the ribose C1. Using theD. radiodurans DR2356 ApnA hydrolase as an example enzyme, the products of hydrolysis were first separated by anion-exchange HPLC, fractions collected and incubated with alkaline phosphatase, and the Pi released determined colorimetrically. Two products, A and B, were observed that co-chromatographed with Pi and PPi, respectively (Fig. 1). Peak area integration showed the ratio of phosphate released from PRPP, B, and A to be exactly 3:2:1. Because A did not co-chromatograph with ribose-1-phosphate (Rib-1-P) or ribose 5-phosphate (Rib-5-P) it must be Pi. Product B could be Rib-1,5-P2, ribosyl 1-pyrophosphate, 5-phosphoribosyl 1,2-(cyclic) phosphate (37Dennis A.L. Puskas M. Stasaitis S. Sandwick R.K. J. Inorg. Biochem. 2000; 81: 73-80Google Scholar, 38Trembacz H. Jezewska M.M. Biochem. J. 1990; 271: 621-625Google Scholar), or, less likely, PPi itself. Therefore, a sample of product B was subjected to TLC before and after acid treatment. Acid removes phosphate from the anomeric C1 but not from C5, and ribose and derivatives with an unesterified C1 hydroxyl can be detected after TLC by AgNO3 treatment (38Trembacz H. Jezewska M.M. Biochem. J. 1990; 271: 621-625Google Scholar). Fig.2 shows the TLC plate after AgNO3 treatment. It can clearly be seen that product B was not detected by AgNO3 before acid treatment because it has an esterified C1-OH (lane C); however, after acid treatment, product B generated a visible spot that co-chromatographed with Rib-5-P and acid-treated PRPP (lane F). This indicates that product B must be Rib-1,5-P2. This identification was confirmed by positive ion electrospray mass spectrometry. B had a mass of 333 Da, corresponding to the monosodium salt of Rib-1,5-P2 (not shown). A cyclic phosphate product would have had a mass 17 Da less. Therefore it can be concluded that the D. radiodurans DR2356 ApnA hydrolase is a PRPP pyrophosphatase producing Rib-1,5-P2 and Pi from PRPP. The products of PRPP hydrolysis generated by the human Ap4A hydrolase had the same HPLC retention times as those produced by the D. radiodurans DR2356 Ap4A hydrolase. Therefore, given the conserved reaction mechanism employed by all Nudix hydrolases, it seems highly likely that Rib-1,5-P2 and Pi are the products in all cases.Figure 2TLC analysis of the ribose-containing product of PRPP hydrolysis by the D. radiodurans DR2356 ApnA hydrolase. A, cellulose TLC plate (Merck, 0.1 mm) was spotted with five 200-nl aliquots each of 10 mm Rib-1-P (lane A), PRPP (lane B), ribose (lane D), and Rib-5-P (lane E), and also samples of the same compounds previously treated for 1 h at 37 °C with 25 mm HCl: PRPP (lane G), Rib-1-P (lane H), ribose (lane I), and Rib-5-P (lane J). The peak fraction of product B from Fig. 1 was freeze-dried, dissolved in 20 μl of H2O; 10 μl of this was treated with HCl as above. Five 200-nl aliquots of untreated (lane C) and acid-treated (lane F) product were applied to the TLC plate. The plate was developed and spots located as described in Ref. 38Trembacz H. Jezewska M.M. Biochem. J. 1990; 271: 621-625Google Scholar.View Large Image Figure ViewerDownload (PPT) The results reported here are important for two reasons. First, they have implications for the specificity and physiological function(s) of Nudix hydrolases, particularly the DIPP/ApnA hydrolases, and secondly, they impact on the metabolism of PRPP and the generation from it of the regulatory molecule Rib-1,5-P2. Regarding the enzymes themselves, the surprising ability of the active sites of the DIPPs to accommodate two seemingly unrelated sets of substrates, the diadenosine and diphosphoinositol polyphosphates, has already been highlighted (5Safrany S.T. Ingram S.W. Cartwright J.L. Falck J.R. McLennan A.G. Barnes L.D. Shears S.B. J. Biol. Chem. 1999; 274: 21735-21740Google Scholar). The use of PRPP as a substrate by these enzymes makes this easier to understand. Modeling of PRPP onto the crystal structure of the C. elegans Ap4A hydrolase (39Bailey S. Sedelnikova S.E. Blackburn G.M. Abdelghany H.M. Baker P.J. McLennan A.G. Rafferty J.B. Structure. 2002; 10: 589-600Google Scholar) shows that it can fit readily into the substrate-binding cleft with its 5-phosphate located in the exact position occupied by the P 1 phosphate of Ap4A and the α-phosphate of the pyrophosphate moiety located where the attacked P4phosphate of Ap4A lies (Fig.3). The same water (or hydroxyl) responsible for nucleophilic attack at P4 could attack this α-phosphate. This model is consistent with the requirement for the catalytic residues Glu52 and Glu56 in theC. elegans Ap4A hydrolase. The β-phosphate of the pyrophosphate group can readily occupy the position of the ribose moiety of the ‘AMP product’ of Ap4A because this is already known to accommodate the P5 phosphate of Ap5A and to lie outside the protein structure. The ribose ring of PRPP occupies the same position as the P2 and P3 phosphates of Ap4A. The substrate-binding cleft is wide at this point, and the residue side chains in this region are either small (Ala5, Ala25, Thr33, and Gly37) or are highly mobile and lack electron density in the crystal structure of theC. elegans hydrolase (Tyr27); therefore, a variety of structures may be accommodated in this region because it appears to provide few, if any, phosphate-specific contacts. A major determinant of the stability of the C. elegansAp4A hydrolase-PRPP complex is probably the binding of the 5-phosphate by salt bridges/H-bonds to His31, Lys36, Tyr76, and Lys83. These residues are important for binding the P 1 phosphate of Ap4A (39Bailey S. Sedelnikova S.E. Blackburn G.M. Abdelghany H.M. Baker P.J. McLennan A.G. Rafferty J.B. Structure. 2002; 10: 589-600Google Scholar). Stacking of the adenosine moiety attached to P 1 between the aromatic rings of Tyr76 and Tyr121 does not seem to be as critical for Ap4A binding as was predicted from the crystal structure. We have found that mutation of both Tyr residues to Ala still yields active Ap4A hydrolase with a 20-fold lowerk cat (1.1 s−1) and only a 4-fold higher K m (33 μm) for Ap4A.2 Thus, the binding of the PRPP phosphates on C5 and C1 as in Ap4A and the accommodation of the ribose in a relatively nonspecific region of the binding cleft appear to be sufficient to allow PRPP to behave as a substrate for the C. elegans Ap4A hydrolase. Similar arguments presumably apply to the other Ap4A hydrolases and DIPPs and to the ability of the DIPPs to bind diphosphoinositol polyphosphates. This model also explains why the Nudix hydrolases with specificities for NADH, NDP-sugars, and coenzyme A do not accept PRPP as a substrate. These enzymes do not have a second phosphate binding site (like P 1) located the required distance away from the catalytic binding site (like P4). Only enzymes able to hydrolyze nucleotide substrates with four or more phosphates in the polyphosphate chain should bind PRPP. These results emphasize the point that certain Nudix hydrolases can also accept non-nucleotide substrates and should prompt the search for other such substrates that satisfy these minimal binding requirements. Is PRPP a physiologically relevant substrate for any of the enzyme studies here? According to the measured specificity constants, at least for the eukaryotic enzymes, the diadenosine and diphosphoinositol polyphosphates appear to be highly favored over PRPP. However, this does not take into account the relative substrate concentrationsin vivo. Literature values for the intracellular concentration of PRPP vary considerably and have been reported in different units. However, taking various measurements for prokaryotes (25Hove-Jensen B. J. Bacteriol. 1988; 170: 1148-1152Google Scholar, 40Petersen C. J. Biol. Chem. 1999; 274: 5348-5356Google Scholar, 41Stuer-Lauridsen B. Nygaard P. J. Bacteriol. 1998; 180: 457-463Google Scholar) and eukaryotes (20Becker M.A. Kim M. J. Biol. Chem. 1987; 262: 14531-14537Google Scholar, 26Kawaguchi T. Veech R.L. Uyeda K. J. Biol. Chem. 2001; 276: 28554-28561Google Scholar, 42Hisata T. Anal. Biochem. 1975; 68: 448-457Google Scholar, 43May S.R. Krooth R.S. Anal. Biochem. 1976; 75: 389-401Google Scholar, 44Peters G.J. Veerkamp J.H. Int. J. Biochem. 1979; 10: 885-888Google Scholar) and applying the unit conversion factors of Traut (45Traut T.W. Mol. Cell. Biochem. 1994; 140: 1-22Google Scholar) suggests that prokaryotes typically have a PRPP steady-state concentration of around 1 mm,whereas in eukaryotes (excluding erythrocytes) it is 1–2 orders of magnitude lower, although it can be as high as 0.4 mm in mouse fibroblasts deficient in adenine and hypoxanthine phosphoribosyltransferases (43May S.R. Krooth R.S. Anal. Biochem. 1976; 75: 389-401Google Scholar, 45Traut T.W. Mol. Cell. Biochem. 1994; 140: 1-22Google Scholar). The measured K mvalues for PRPP for all three enzymes are, therefore, within acceptable ranges if PRPP were to be considered a physiologically relevant substrate. The intracellular concentration of Ap4A in unstressed cells is typically 0.1–1.0 μm (46Garrison P.N. Barnes L.D. McLennan A.G. Ap4A and Other Dinucleoside Polyphosphates. CRC Press, Boca Raton, Fl1992: 29-61Google Scholar), whereas PP-InsP5 may be in the low micromolar range (47Menniti F.S. Miller R.N. Putney J.W. Shears S.B. J. Biol. Chem. 1993; 268: 3850-3856Google Scholar). There are no measurements of cytoplasmic Ap6A in mammalian cells; an estimate of 32 nm in platelets is an intracellular average because the Ap6A is concentrated in the dense granules (48Jankowski J. Potthoff W. vanderGiet M. Tepel M. Zidek W. Schlüter H. Anal. Biochem. 1999; 269: 72-78Google Scholar). Indeed, it is likely to be even lower than the sole measured value for Ap5A of 4 nm inSchizosaccharomyces pombe (52Ingram S.W. Safrany S.T. Barnes L.D. Biochem. J. 2002; (in press)Google Scholar). Taking the product of the specificity constant and substrate concentration as a better indication of potential substrate utilization in vivo shows that PRPP hydrolysis is likely to be a much more significant reaction in vivo for the D. radiodurans ApnA hydrolase than is Ap4A hydrolysis (Table II). In contrast, the human Ap4A hydrolase is more likely to act upon Ap4A than on PRPP. For DIPP-1, PP-InsP5 still appears to be the favored substrate by virtue of its extremely low K mfor this compound. Nevertheless, the combined activities of members of the DIPP subfamily could still have a significant impact on PRPP hydrolysis in vivo in tissues where more than one is expressed. PRPP pyrophosphatase activity in cell extracts has been detected before. Divalent ion-dependent (49Tax W.J. Veerkamp J.H. Comp. Biochem. Physiol. B. 1978; 59: 219-222Google Scholar) and -independent (50Fox I.H. Marchant P.J. Can. J. Biochem. 1974; 52: 1162-1166Google Scholar) activities were ascribed to acid and alkaline phosphatase, respectively. Like the spontaneous degradation of PRPP, these reactions are believed to proceed via 1,2 and 1,5 cyclic derivatives to Rib-1-P and -5-P and ultimately to ribose with Rib-1,5-P2 as a possible minor intermediate in one pathway (37Dennis A.L. Puskas M. Stasaitis S. Sandwick R.K. J. Inorg. Biochem. 2000; 81: 73-80Google Scholar, 38Trembacz H. Jezewska M.M. Biochem. J. 1990; 271: 621-625Google Scholar). In view of the established mechanisms of Nudix hydrolases and the fact that Ap4A hydrolysis is known to proceed by direct in-line attack of water (51Guranowski A. Brown P. Ashton P.A. Blackburn G.M. Biochemistry. 1994; 33: 235-240Google Scholar), a cyclic intermediate is unlikely; generation of Rib-1,5-P2 most probably occurs directly by nucleophilic attack of water on the C1 α-phosphate rather than via a complex route involving the initial internal attack of a ribose hydroxyl. Recently, it has been clearly demonstrated (26Kawaguchi T. Veech R.L. Uyeda K. J. Biol. Chem. 2001; 276: 28554-28561Google Scholar) that the rapid rise in Rib-1,5-P2 that occurs in macrophages under hypoxic conditions in parallel with the switch to anaerobic glycolysis is due to a rise in PRPP accompanied by the activation of an unidentified PRPP pyrophosphatase. This activity appears to be divalent ion-independent and may be activated by protein kinase C. Its possible relationship to any of the mammalian Nudix PRPP pyrophosphatases described here is unknown. Nevertheless, it is clear that, at least in mammalian cells, several Nudix hydrolases exist that have the ability to generate Rib-1,5-P2 from PRPP. Like fructose 2,6-bisphosphate, this molecule is a potent activator of phosphofructokinase, is also an inhibitor of fructose 1,6-bisphosphatase, and is believed to be an important regulator of glycolysis (26Kawaguchi T. Veech R.L. Uyeda K. J. Biol. Chem. 2001; 276: 28554-28561Google Scholar, 27Sawada M. Mitsui Y. Sugiya H. Furuyama S. Int. J. Biochem. Cell Biol. 2000; 32: 447-454Google Scholar, 28Ogushi S. Lawson J.W.R. Dobson G.P. Veech R.L. Uyeda K. J. Biol. Chem. 1990; 265: 10943-10949Google Scholar). The Nudix PRPP pyrophosphatases must be considered potential generators of Rib-1,5-P2 in vivo and, therefore, regulators of glucose metabolism. Verification of this possibility will require measurements of PRPP and Rib-1,5-P2 in cells in which the relevant Nudix hydrolase activities have been reduced by gene disruption or knockdown. We thank Dr. J. B. Rafferty for assistance with the molecular modeling." @default.
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• W2024708954 title "Nudix Hydrolases That Degrade Dinucleoside and Diphosphoinositol Polyphosphates Also Have 5-Phosphoribosyl 1-Pyrophosphate (PRPP) Pyrophosphatase Activity That Generates the Glycolytic Activator Ribose 1,5-Bisphosphate" @default.
• W2024708954 cites W1480394609 @default.
• W2024708954 cites W1485488844 @default.
• W2024708954 cites W1509297261 @default.
• W2024708954 cites W1514092148 @default.
• W2024708954 cites W1573664132 @default.
• W2024708954 cites W1581281722 @default.
• W2024708954 cites W1600339587 @default.
• W2024708954 cites W1782999734 @default.
• W2024708954 cites W1894695910 @default.
• W2024708954 cites W1912410317 @default.
• W2024708954 cites W1965005351 @default.
• W2024708954 cites W1966812211 @default.
• W2024708954 cites W1971668526 @default.
• W2024708954 cites W1974020806 @default.
• W2024708954 cites W1983888724 @default.
• W2024708954 cites W1987337899 @default.
• W2024708954 cites W1990668649 @default.
• W2024708954 cites W1995816032 @default.
• W2024708954 cites W1995958182 @default.
• W2024708954 cites W2009886610 @default.
• W2024708954 cites W2016794832 @default.
• W2024708954 cites W2018385053 @default.
• W2024708954 cites W2019044175 @default.
• W2024708954 cites W2029584311 @default.
• W2024708954 cites W2031458979 @default.
• W2024708954 cites W2032019882 @default.
• W2024708954 cites W2034588677 @default.
• W2024708954 cites W2035177231 @default.
• W2024708954 cites W2042127371 @default.
• W2024708954 cites W2045074917 @default.
• W2024708954 cites W2061660741 @default.
• W2024708954 cites W2064393764 @default.
• W2024708954 cites W2064433587 @default.
• W2024708954 cites W2067393589 @default.
• W2024708954 cites W2069732328 @default.
• W2024708954 cites W2073515768 @default.
• W2024708954 cites W2075738743 @default.
• W2024708954 cites W2080419891 @default.
• W2024708954 cites W2094944666 @default.
• W2024708954 cites W2099400747 @default.
• W2024708954 cites W2122872686 @default.
• W2024708954 cites W2123882945 @default.
• W2024708954 cites W2126076915 @default.
• W2024708954 cites W2131367352 @default.
• W2024708954 cites W2142420001 @default.
• W2024708954 cites W2165914529 @default.
• W2024708954 cites W4242781393 @default.
• W2024708954 cites W2097158283 @default.
• W2024708954 cites W2336007313 @default.
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