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J. Biol. Chem., Vol. 277, Issue 49, 47313-47317, December 6, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/49/47313    most recent M209795200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fisher, D. I. Articles by Cartwright, J. L. Search for Related Content PubMed PubMed Citation Articles by Fisher, D. I. Articles by Cartwright, J. L. Social Bookmarking                      What's this?

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^*

David I. Fisher^§, Stephen T. Safrany^¶, Peter Strike^**, Alexander G. McLennan^**, and Jared L. Cartwright^**§§

From the  Cell Regulation and Signalling Group, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom and the ^¶ Division of Cell Signalling, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, United Kingdom

Received for publication, September 24, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 P_i. Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily of Nudix hydrolases, which also degrade the non-nucleotide diphosphoinositol polyphosphates. K_m and k_cat values for PRPP hydrolysis for the Deinococcus 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 s¹, 0.13 mM and 0.057 s¹, and 0.38 mM and 1.0 s¹, respectively. Active site mutants of the Caenorhabditis 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 the D. 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 (1, 2). Thus, nucleoside triphosphates (Npp-p), dinucleoside polyphosphates (Npp-p_nN), 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 (1).

Recently, a subfamily of Nudix hydrolases has been described that hydrolyze the long chain dinucleoside and nucleoside polyphosphates, including diadenosine 5',5-P¹,P⁶-hexaphosphate (Ap₆A),¹ diadenosine 5',5-P¹,P⁵-pentaphosphate (Ap₅A), and adenosine 5'-pentaphosphate (p₅A) but which have relatively low activity with diadenosine 5',5-P¹,P⁴-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 (3, 4), although the latter enzymes do not appear to utilize PP-InsP₅ or [PP]₂-InsP₄ as substrates (5). 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) (6), DIPP-2 and -2 (NUDT4) (7, 8), DIPP-3 (NUDT10, hAps2), and -3 (NUDT11, hAps1) (9, 10), whereas the yeasts Saccharomyces cerevisiae (DDP1) (5, 11) and Schizosaccharomyces pombe (Aps1) (5, 12) 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 (13). Several of these enzymes have high affinities and high k_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 (14), apoptosis (15), DNA repair (16), and in vacuole biogenesis and environmental stress responses in yeast (17); 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 (18-21); in bacteria and lower eukaryotes it is also a precursor for histidine and tryptophan biosynthesis (22-25). 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 (26-28). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials-- Recombinant human Aps1 (DIPP-3) and Aps2 (DIPP-3) (10), S. cerevisiae Ddp1p Ap₆A hydrolase (YOR163w protein) (11), and Npy1p NADH pyrophosphatase (YGL067w protein) (29), Caenorhabditis elegans Ap₄A hydrolase (3), African Swine Fever virus g5R protein (13), human NUDT5 ADP-sugar hydrolase (30), NUDT9 ADP-ribose hydrolase (31), and mouse Nudt7 coenzyme A pyrophosphatase (32) were prepared as previously described. Active site mutants of the C. elegans Ap₄A hydrolase (E52Q and E56Q) were a gift from H. Abdelghany.² The YgdP Ap₄A hydrolase from Salmonella typhimurium was a gift from T. Ismail.³ Recombinant human DIPP-1, DIPP-2, and -2 were prepared as GST-fusion proteins as described for hAps1 and hAps2⁴ (10). Recombinant human Ap₄A hydrolase, Deinococcus radiodurans coenzyme A pyrophosphatase (DR1184 gene product), and D. radiodurans Ap_nA hydrolase (DR2356 gene product) were prepared by procedures similar to those used for the C. elegans Ap₄A hydrolase.⁵ PRPP and all nucleotides were from Sigma. The EnzChek® phosphate assay kit was from Molecular Probes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Assay of PRPP Pyrophosphatase Activity-- Initial screening of enzyme preparations for their ability to release P_i from PRPP was carried out using a phosphomolybdate colorimetric assay (33). 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 (34). 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. radiodurans Ap_nA hydrolase). In each case, controls lacking substrate or enzyme were performed.

HPLC Analysis of PRPP Hydrolysis Products-- The products of PRPP hydrolysis by the D. radiodurans Ap_nA 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 M NH₄HCO₃, 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 (33, 35).

Other Methods-- Ap₄A hydrolase activity was determined luminometrically as previously described (3). Protein concentrations were estimated by the Coomassie Blue binding method (36). Positive ion electrospray mass spectrometry was performed as previously described (11).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hydrolysis of PRPP by Nudix Hydrolases-- 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 P_i produced min¹·micromoles protein¹ 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 (37, 38), 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. elegans Ap₄A 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 s¹, the C. elegans E52Q and E56Q mutants have k_cat values for Ap₄A of 0.0052 and 0.00024 s¹, 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.

K_m and k_cat values for PRPP were determined for one prokaryotic Ap_nA hydrolase, one eukaryotic Ap₄A hydrolase, and one eukaryotic Ap₆A hydrolase/DIPP, using a continuous spectrophotometric assay. D. radiodurans DR2356 Ap_nA hydrolase is an enzyme that hydrolyzes a variety of nucleoside and dinucleoside polyphosphates, including p₄A, p₅A, Ap₄A, Ap₅A, and Ap₆A.⁵ It has no activity toward diphosphoinositol polyphosphates⁴; however, it readily hydrolyzes PRPP with a K_m of 1 mM and a k_cat of 1.5 s¹ (Table II). For comparison, K_m and k_cat values for Ap₄A were determined to be 30 µM and 0.035 s¹, 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 s¹ and a K_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 hydrolysis in vitro with a k_cat of 1.0 s¹ 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 (5). The physiological significance of these data is discussed below.

Identification of Rib-1,5-P₂ as the Product of PRPP Hydrolysis-- Because all Nudix hydrolases cleave pyrophosphate linkages, it was anticipated that they would remove the -phosphate from the pyrophosphate moiety attached to the ribose C1. Using the D. radiodurans DR2356 Ap_nA 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 P_i released determined colorimetrically. Two products, A and B, were observed that co-chromatographed with P_i and PP_i, 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 P_i. Product B could be Rib-1,5-P₂, ribosyl 1-pyrophosphate, 5-phosphoribosyl 1,2-(cyclic) phosphate (37, 38), or, less likely, PP_i 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 (38). 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 Ap_nA hydrolase is a PRPP pyrophosphatase producing Rib-1,5-P₂ and P_i 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 P_i are the products in all cases.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Anion-exchange HPLC analysis of the products of PRPP hydrolysis by the D. radiodurans DR2356 ApnA hydrolase. Samples of PRPP before () and after () incubation with 7 µg of D. radiodurans DR2356 ApnA hydrolase were subjected to anion-exchange chromatography on a Resource-Q column and fractions collected as described under "Experimental Procedures." The positions of Rib-1-P, Rib-5-P, PPi, and Pi standards are indicated by arrows.

View larger version (91K): [in this window] [in a new window]   Fig. 2.   TLC 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. 38.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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/Ap_nA 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 (5). 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 (39) shows that it can fit readily into the substrate-binding cleft with its 5-phosphate located in the exact position occupied by the P¹ phosphate of Ap₄A and the -phosphate of the pyrophosphate moiety located where the attacked P⁴ phosphate of Ap₄A lies (Fig. 3). The same water (or hydroxyl) responsible for nucleophilic attack at P⁴ could attack this -phosphate. This model is consistent with the requirement for the catalytic residues Glu⁵² and Glu⁵⁶ in the C. elegans Ap₄A hydrolase. The -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 the C. 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.

View larger version (58K): [in this window] [in a new window]   Fig. 3.   A model of PRPP in the substrate-binding site of the C. elegans Ap4A hydrolase. PRPP was modeled into the substrate-binding site of the C. elegans Ap4A hydrolase (PDB entry 1KT9) using Turbo-Frodo software and the PRPP coordinates taken from the ternary complex of hypoxanthine guanine phosphoribosyltransferase-PRPP-9-deazaguanuine (PDB entry 1FSG). The positions of Tyr76 and Tyr121, which bind one adenine ring between them in the previously described binary complex, and of Glu52, one of the catalytic Glu residues in the loop-helix-loop active site motif, are indicated. Phosphoryl groups of PRPP are shown as 5-P, P, and P.

A major determinant of the stability of the C. elegans Ap₄A 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 P¹ phosphate of Ap₄A (39). Stacking of the adenosine moiety attached to P¹ 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 lower k_cat (1.1 s¹) 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 P¹) 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 concentrations in vivo. Literature values for the intracellular concentration of PRPP vary considerably and have been reported in different units. However, taking various measurements for prokaryotes (25, 40, 41) and eukaryotes (20, 26, 42-44) and applying the unit conversion factors of Traut (45) 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 (43, 45). The measured K_m values 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 (46), whereas PP-InsP₅ may be in the low micromolar range (47). 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 (48). Indeed, it is likely to be even lower than the sole measured value for Ap₅A of 4 nM in Schizosaccharomyces pombe (52). 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 Ap_nA 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_m for 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 (49) and -independent (50) 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 (37, 38). 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 (51), a cyclic intermediate is unlikely; generation of Rib-1,5-P₂ most probably occurs directly by nucleophilic attack of water on the C1 -phosphate rather than via a complex route involving the initial internal attack of a ribose hydroxyl. Recently, it has been clearly demonstrated (26) 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 (26-28). 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.

	ACKNOWLEDGEMENT

We thank Dr. J. B. Rafferty for assistance with the molecular modeling.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of a studentship from the Biotechnology and Biological Sciences Research Council (BBSRC).

Supported by the Royal Society and Tenovus (Scotland).

^** Supported by the BBSRC (Grant 26/PRS11831) and the Wellcome Trust.

To whom correspondence should be addressed: School of Biological Sciences, University of Liverpool, Life Sciences Bldg., Liverpool L69 7ZB, UK. Tel.: 0151-794-4369; Fax: 0151-794-4349; E-mail: agmclen@liv.ac.uk.

^§§ Present address: Dept. of Biology, University of York, P.O. Box 373, York YO10 5YW, UK.

Published, JBC Papers in Press, October 4, 2002, DOI 10.1074/jbc.M209795200

² H. Abdelghany and A. G. McLennan, unpublished data.

³ T. Ismail and A. G. McLennan, unpublished data.

⁴ S. T. Safrany, unpublished observations.

⁵ D. I. Fisher, J. L. Cartwright, and A. G. McLennan, unpublished data.

	ABBREVIATIONS

The abbreviations used are: Ap₆A, diadenosine 5',5-P¹,P⁶-hexaphosphate; Ap₄A, diadenosine 5',5-P¹,P⁴-tetraphosphate; Ap₅A, diadenosine 5',5-P¹,P⁵-pentaphosphate; Ap_nA, diadenosine 5',5-P¹,Pⁿ-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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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