J Biol Chem, Vol. 274, Issue 31, 21735-21740, July 30, 1999

The Diadenosine Hexaphosphate Hydrolases from Schizosaccharomyces pombe and Saccharomyces cerevisiae Are Homologues of the Human Diphosphoinositol Polyphosphate Phosphohydrolase
OVERLAPPING SUBSTRATE SPECIFICITIES IN A MutT-TYPE PROTEIN^*

Stephen T. Safrany, Stephen W. Ingram^§, Jared L. Cartwright^¶, J. R. Falck, Alexander G. McLennan^¶, Larry D. Barnes^§, and Stephen B. Shears^**

From the  Inositide Signaling Group, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, the ^§ Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760, the ^¶ School of Biological Sciences, Life Sciences Building, University of Liverpool, Liverpool L69 7ZB, United Kingdom, and the  Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9038

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Aps1 from Schizosaccharomyces pombe (Ingram, S. W., Stratemann, S. A., and Barnes, L. D. (1999) Biochemistry 38, 3649-3655) and YOR163w from Saccharomyces 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Following the discovery of dinucleoside polyphosphates in biological systems over 30 years ago (1), 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,¹ Ap₄A, Ap₅A, and Ap₆A (2-4). 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 (5). Moreover, it was recently proposed (6) that Ap₃A has an antiproliferative role when complexed with the putative tumor suppressor Fhit protein, an Ap₃A hydrolase (7, 8). Thus, the Ap₃A/Ap₄A ratio may be an important factor in determining the alternative cellular fates of proliferation, differentiation, and apoptosis (3, 9). In higher eukaryotes, Ap_nA appear also to be intracellular mediators of certain extracellular stimuli; they respond to glucose in pancreatic -cells (10). Ap_nA may also regulate ATP-sensitive K⁺ channels in -cells and cardiac muscle (11-13) and intracellular ryanodine-binding Ca²⁺-release channels in cardiac and skeletal muscle, and in the brain (14, 15). Finally, extracellular Ap₅A and Ap₆A have also been identified as neurotransmitters (2) and vasomodulators (16, 17).

In addition to these physiological functions, Ap_nA respond to heat shock and oxidative stress with an increase in concentration (18). If allowed to accumulate, they could prove toxic through their ability to inhibit nucleotide kinases (19, 20), protein kinases (21, 22), and other enzymes (23). 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 (24). 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 (24). There is a family of so-called "Ap₄A" hydrolases, which contain the MutT motif; these enzymes hydrolyze Ap₄A in preference to other dinucleotides (25-28). Distinct MutT-type hydrolases that prefer Ap₅A and Ap₆A as substrates have recently been identified in Schizosaccharomyces pombe (Aps1; see Ref. 29) and Saccharomyces cerevisiae (YOR163w; see Ref. 30). 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 (31) and dGTP² are not physiologically significant substrates (6). Instead, this enzyme has been shown to have a quite different catalytic activity; it was identified (31) as a diphosphoinositol polyphosphate phosphohydrolase (DIPP). DIPP's substrates, PP-InsP₅ and [PP]₂-InsP₄ (31), 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-InsP₅ and [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 (32). Second, [PP]₂-InsP₄ turnover is regulated by a cAMP- and cGMP-dependent process that operates independently of A and G kinases (33).

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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Ap₆A was synthesized as described previously (29). Ap₅A, Ap₄A, and Ap₃A were purchased from Sigma. Non-radiolabeled PP-InsP₅ was synthesized as described previously (34). The sources of PP-[³H]InsP₅, [³H]InsP₆, and PP-[³H]InsP₄ were all as described previously (31). [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 mM dithiothreitol) plus 6.25 µg/ml pepstatin, 25 µg/ml aprotinin, 5 µg/ml leupeptin, 25 µg/ml E-64. A 100,000 × g supernatant 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 1 M KCl): 0-10 min, 0% buffer B; 10-20 min, 15-85% B; 20-30 min, 100% B. The InsP₆ kinase (35) and PP-InsP₅ kinase (36) co-eluted from the column. Aliquots of peak fractions were incubated with [³H]InsP₆ in buffer containing: 0.75 mM EGTA, 1.5 mM EDTA, 9 mM MgSO₄, 7.5 mM ATP, 10 mM NaF, 20 mM phosphocreatine, 1 mM dithiothreitol, 20 mM HEPES (pH 6.8), 4 mM CHAPS, 20 Sigma units/ml creatine phosphokinase. The resultant [PP]₂-[³H]InsP₄ (>80% conversion) was purified by HPLC (33) and desalted (37) with 17% recovery. Pure, recombinant preparations of Aps1 (29), YOR163w (30), Fhit (38), human Ap₄A hydrolase (25), IalA protein from Bartonella bacilliformis (28), and DIPP (31) were all obtained as described previously.

Enzyme Assays-- For the analysis of Ap_nA (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 (29). Assay solutions were analyzed by HPLC to resolve individual nucleotides as described previously (29). 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 (31), 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Structural Similarities among Aps1, YOR163w, and DIPP-- Fig. 1 compares the sequences of three enzymes that each contain the MutT motif. Two of these proteins, Aps1 from S. pombe (29) and YOR163w from S. cerevisiae (30), have been characterized as Ap₆A/Ap₅A hydrolases. The third protein is from Homo sapiens and has been characterized as a PP-InsP₅/[PP]₂-InsP₄ phosphohydrolase (31). 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").

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Sequence alignment of Aps1, YOR163w, and DIPP. Two Ap6A hydrolases, namely Aps1 from S. pombe (accession no. Q09790) and YOR163w from S. cerevisiae (Z75071), were aligned with human DIPP (AF062529), using the PILEUP algorithm (with the caveat that the only gap permitted within the MutT motif was to accommodate an additional asparagine residue in YOR163w). The MutT motif is shaded gray. Stars denote amino acids in either of the two yeast proteins that were identical to corresponding residues in DIPP; physicochemically similar residues are marked with a cross.

Reactivity of DIPP toward Diadenosine Polyphosphates-- 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 µM Ap₆A at a rate of 1-2 µmol/min/mg (29, 30). The rank order of relative specific activities of DIPP toward the various diadenosine polyphosphates (Ap₆A > Ap₅A > Ap₄A > Ap₃A; Fig. 2) mirrored that for Aps1 (29). 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) (30). 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.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Relative rates of hydrolysis of ApnA (n = 3-6) by DIPP. DIPP was incubated with 100 µM of either Ap6A, Ap5A, Ap4A or Ap3A, and reactions were quenched and analyzed by HPLC, as described under "Experimental Procedures." Data are the average of two experiments that differed by less than 10%.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   HPLC 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 µM Ap6A (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.

We also determined k_cat and K_m values for the hydrolysis of Ap₆A and Ap₅A by DIPP (Table I). The affinities of DIPP for these particular substrates (K_m = 6-8 µM) were slightly higher than the affinity of Aps1 (approximately 20 µM; Ref. 29) and YOR163w (approximately 60 µM; Ref. 30). The k_cat values for DIPP (0.5 s¹, Table I) were intermediate between those for Aps1 (approximately 2 s¹; Ref. 29) and YOR163w (0.06 s¹ for Ap₅A and 0.4 s¹ for Ap₆A; Ref. 30). It is striking that the Ap₅A/Ap₆A hydrolase activity of DIPP is kinetically very similar to that of both yeast enzymes.

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 (29). YOR163w also generates p₄A and ADP as major products from Ap₆A, but p₅A and AMP are also formed as minor products (30). These different modes of Ap₆A hydrolysis have been attributed to Ap₆A having some mobility within the active site (30).

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 P_i) in previous experiments with YOR163w (30). 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 P_i. 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.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Time course of the hydrolysis of Ap6A and Ap5A by DIPP. DIPP (11.7 µg) was incubated with 100 µM of either Ap6A (panel A) or Ap5A (panel B) in 1.3 ml of buffer as described under "Experimental Procedures." Aliquots (120 µl) were withdrawn at the time points indicated in the figure and were frozen in dry ice. Substrates and products were then resolved by HPLC as described under "Experimental Procedures." , Ap6A; , Ap5A; , p5A; , p4A; , ATP; , ADP; , AMP. Data are from a representative experiment, typical of two.

With regards to Ap₅A, both Aps1 and YOR163w degrade this substrate to two sets of products, namely ADP plus ATP, and p₄A plus AMP (29, 30). However, one difference between these two enzymes is that ADP plus ATP are the major reaction products for Aps1 (29), whereas for YOR163w, p₄A and AMP are the major products (30). 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.

Reactivity of Aps1 and YOR163w toward Diphosphorylated Inositol Polyphosphates-- 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 -phosphate from the diphosphate group of PP-InsP₅, just as is the case for DIPP (31). 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). k_cat values for the three enzymes were also quite similar (Table II).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   HPLC analysis of the hydrolysis of PP-InsP5 and [PP]2-InsP4 by YOR163w. Panel A, YOR163w (0.6 ng) was incubated with 1800 dpm PP-[3H]InsP5 for either 0 () or 20 min (). Panel B, YOR163w (6 ng) was incubated with 1500 dpm [PP]2-[3H]InsP4 for either 0 () or 30 min (). For both experiments, reactions were quenched, neutralized, and analyzed by HPLC as described under "Experimental Procedures." Data are representative of three experiments. Similar results were obtained with Aps1 (data not shown). The arrows mark the elution positions of 3H-labeled standards of InsP5 and PP-InsP4, determined in separate HPLC runs. The inset to panel A shows representative time courses of PP-[3H]InsP5 hydrolysis by YOR163w () and Aps1 (). The inset to panel B shows representative time courses of [PP]2-[3H]InsP4 hydrolysis by YOR163w () and Aps1 ().

[PP]₂-InsP₄ is another diphosphorylated inositol phosphate that is hydrolyzed by DIPP (31). 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 (k¹ = 8.3 ± 1.1 µg¹ min¹, n = 5) than was the case for YOR163w (k¹ = 1.7 ± 0.5 µg¹ min¹, n = 3). In fact, the rate achieved by Aps1 was only 8-fold less than that for DIPP (k¹ = 70 ± 20 µg¹ min¹, n = 4). We were unable to generate more detailed kinetic data for [PP]₂-InsP₄ hydrolysis, because we do not have sufficient mass amounts of this particular substrate.

Reactivity of Other Ap_nA Hydrolases toward PP-InsP₅ and [PP]₂-InsP₄-- The results described above indicate that two MutT motif Ap₆A/Ap₅A hydrolases from yeasts can also hydrolyze PP-InsP₅ or [PP]₂-InsP₄. We therefore examined if this overlapping substrate specificity was also a feature of other MutT-type Ap_nA hydrolases. In Fig. 1, we noted a region of DIPP (from Val³⁴ to Glu⁸⁵) that was 46% identical in sequence to corresponding domains in the yeast Ap₆A/Ap₅A hydrolases. This same region of DIPP was only 33% identical to a human Ap₄A hydrolase (Fig. 6). Although the latter enzyme did hydrolyze PP-InsP₅ and [PP]₂-InsP₄, the reactions proceeded relatively slowly (for PP-InsP₅ hydrolysis, k¹ = 0.005 µg¹min¹ (0.0002% of DIPP activity)²; for [PP]₂-InsP₄ hydrolysis, k¹ = 0.006 µg¹min¹ (0.007% of DIPP activity, see above)).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Sequence comparison of DIPP with IalA from B. bacilliformis and a human Ap4A hydrolase. The region of DIPP (Val34 to Glu85), which is 46% similar to corresponding regions of the yeast Ap6A/Ap5A hydrolases (see Fig. 1), was aligned with the IalA protein (accession no. L25276) and a human MutT motif Ap4A hydrolase (Ap4ase, U30313); the PILEUP algorithm was used. The MutT motif is shaded gray. Stars denote amino acids in either of the ApnA hydrolases that were identical to corresponding residues in DIPP; physicochemically similar residues are marked with a cross.

The IalA invasion protein of B. bacilliformis is another MutT motif protein, and this enzyme expresses broadly similar catalytic activities toward Ap₄A, Ap₅A and Ap₆A (27, 28). The IalA protein, when incubated at up to a 120-fold higher protein concentration than was used for Aps1, showed no detectable hydrolysis of diphosphorylated inositol polyphosphates (data not shown). Indeed, outside the MutT motif, IalA has no significant similarity to DIPP (Fig. 6). Further evidence that the MutT motif does not generally impart the capacity to metabolize PP-InsP₅ and [PP]₂-InsP₄ comes from experiments showing that these compounds are not significant substrates of hMTH1,³ a human homologue of the prototypical MutT protein (which instead hydrolyzes 8-oxo-dGTP; Ref. 39).

Nature has developed more than one solution to the problem of how to regulate cellular levels of diadenosine polyphosphates. As well as Aps1, YOR163w, and DIPP, which belong to the MutT motif family (see above), some Ap_nA hydrolases belong to the HIT protein family and are characterized by the presence of a histidine triad motif, HxHxH (7, 8, 40). We incubated one such enzyme, the human Fhit Ap₃A hydrolase (7) with either PP-InsP₅ or [PP]₂-InsP₄. Neither substrate was metabolized by a concentration of Fhit that was 1000-fold greater than was used for Aps1 (data not shown).

In view of these results, we conclude that the remarkable overlapping substrate specificity seen in Aps1, YOR163w, and DIPP is neither a general property of MutT-type proteins, nor is it a general feature of Ap_nA metabolizing enzymes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The MutT paradigm is based on this motif recurring in a series of nucleoside phosphate hydrolases that act as "guardians" of cellular integrity (24). Members of this enzyme family have been shown to hydrolyze nucleoside phosphates that are directly hazardous, such as the mutagenic 8-oxo-dGTP (39), and they may also protect the cell against the potentially dangerous consequences of inappropriate increases in the levels of intracellular signals, such as dATP (41). To achieve efficiency in this role, it has previously seemed that individual MutT motif proteins have each evolved exclusive catalytic specificities toward restricted groups of nucleoside phosphates (24). Our results with Aps1, YOR163w, and DIPP are therefore exceptional, because the diadenosine polyphosphates, Ap₅A and Ap₆A, and the diphosphorylated inositol phosphates, PP-InsP₅ and [PP]₂-InsP₄, are two unrelated classes of substrates. Furthermore, these two groups of metabolites have independently emerged as participants in various aspects of signal transduction (see the Introduction). Since specificity of action is paramount for an intracellular signal, it is remarkable that one enzyme can hydrolyze these different molecules, thereby potentially regulating their levels and affecting their signaling strength. Thus, our observations not only draw an unexpected link between two disparate areas of signal transduction, but they are also pertinent to the evolution and functions of the MutT motif.

Evidence has accumulated that Ap₆A and Ap₅A are cellular signals that may act both outside (2, 16, 17, 42) and inside (12-15) the cell. There is therefore considerable interest in characterizing the enzymes that are responsible for metabolizing these two diadenosine polyphosphates. DIPP represents the first hydrolase in the animal kingdom shown to prefer Ap₆A and Ap₅A over Ap₄A. In fact, the kinetic parameters associated with DIPP-dependent Ap₆A/Ap₅A hydrolase activity are not only similar to those of Aps1 and YOR163w (Tables I and II), they are also in line with the characteristics of other MutT motif Ap_nA hydrolases (25-28, 30). In that sense, DIPP is a typical Ap₆A/Ap₅A hydrolase. This conclusion raises the important question as to whether this is a function of DIPP in vivo. Our in vitro data suggest that PP-InsP₅ is preferred as a substrate over either Ap₆A or Ap₅A (Tables I and II). However, we do not know the subcellular compartments in which DIPP is present, nor do we have reliable estimates of the concentrations, in these same compartments, of any of DIPP's substrates. Nevertheless, if DIPP were closely associated with the secretory vesicles in which Ap₆A and Ap₅A appear to be concentrated, at least in platelets, chromaffin cells and synapses (2), this enzyme could regulate the signaling strength inside the vesicles. If some DIPP were present on the cell surface, it could contribute to terminating the interactions of Ap₆A and Ap₅A with purinergic receptors (2). Finally, DIPP that is free in the cytosol may be a site for metabolic competition between Ap₆A, Ap₅A, PP-InsP₅, and [PP]₂-InsP₄.

The fact that, to date, yeasts have not been reported to contain either Ap₅A or Ap₆A has raised questions concerning the possible biological functions of Aps1 and YOR163w (29, 30). While the search for Ap₅A and Ap₆A in yeasts continues, it is worth noting that both S. pombe (43) and S. cerevisiae (44) are known to synthesize PP-InsP₅ and [PP]₂-InsP₄. Thus, another important feature of our studies is that PP-InsP₅ and [PP]₂-InsP₄ represent a useful, new focus for further studies into the functions of YOR163w and Aps1. The identification of a PP-InsP₅/[PP]₂-InsP₄ phosphohydrolase in a genetically tractable organism such as yeast also provides us with new opportunities to rapidly increase our insight into the roles of these particular compounds. This is a particularly timely opportunity in the light of a recent observation from studies with S. pombe; the size of this yeast's InsP₆ metabolic reservoir, which in turn dictates the amount of PP-InsP₅ and [PP]₂-InsP₄ inside cells (37), was recently shown to be acutely sensitive to hyperosmotic stress (43).

Another important feature of our study relates to the value of comparing related enzymes from different species, which can provide fresh insight into relationships between protein structure and function. The sequence alignment of DIPP with yeast Ap₆A/Ap₅A hydrolases (Fig. 1) identifies a number of conserved amino acid residues both inside the MutT motif, and in the immediately adjacent flanking regions. A systematic study into the contribution made by these residues, by site-directed mutagenesis for example, will be of great value in future studies into the catalytic process. Thus, our studies represent an important step toward the goal of understanding how the tertiary structures of this class of MutT-type proteins can accommodate two different substrate structures so successfully. Further information on structure/function relationships will also come from the comparison of the sequence of DIPP with the sequences of other MutT-type enzymes that we have now shown not to attack PP-InsP₅ and [PP]₂-InsP₄ (see Fig. 6 and "Results").

A popular viewpoint concerning the evolution of catalytic motifs envisages relatively nonspecific progenitors becoming duplicated and then adapted to perform specific tasks (45). The MutT motif has been proposed to exemplify this evolutionary process (24). It has been proposed that, from a primordial ancestor, a family of proteins with MutT motifs each have evolved subtly different phosphohydrolase specificities toward particular nucleoside phosphates (24). A contrary view states that a protein motif that provides a particular advantage can arise independently, in otherwise unrelated proteins, by convergent evolution (46). It is arguable that the latter idea is supported by the considerable divergence in the protein sequences that lie outside the eponymous motif of the MutT-related proteins (see Figs. 1 and 6 and Ref. 24). However, sequence is less tightly conserved during divergent evolution than is structure (47). In view of these conflicting ideas, Aps1, YOR163w, and DIPP represent models of particular interest for further studies into the evolution of functions and tertiary structures of the MutT family.

It has been suggested that "Nudix" is a more accurate name for the "MutT" motif, in part because of the earlier data indicating that this motif imparted catalytic specificity toward compounds containing a nucleoside diphosphate attached to another moiety, "x" (24). Thus, the original discovery of a MutT motif in DIPP created an important precedent; DIPP became the first member of this class of enzymes shown to actively metabolize substrates that do not conform to the above structure. Our new data with Aps1 and YOR163w extend this exception. The unexpected catalytic promiscuity of this group of enzymes is an important new factor that will have to be taken into account in our ongoing efforts to understand how cells regulate the cellular levels and the biological functions of both diadenosine polyphosphates and diphosphorylated inositol phosphates.

	ACKNOWLEDGEMENTS

We thank Drs. J. Caffrey and X. Yang for their helpful comments during the writing of the manuscript.

	FOOTNOTES

^* This work was supported in part by National Science Foundation Grant MCB-9604124 (to L. D. B.), by a grant from the Leverhulme Trust (to A. G. M.), and by National Institutes of Health Grant GM31278 (to J. R. F.).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.

^** To whom correspondence should be addressed: Inositide Signaling Group, NIEHS, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-0793; Fax: 919-541-0559; E-mail: shears@niehs.nih.gov.

² S. T. Safrany, unpublished data.

³ H. Hayakawa, S. T. Safrany, S. B. Shears, and M. Sekiguchi, unpublished data.

	ABBREVIATIONS

The abbreviations used are: Ap_nA, diadenosine 5',5'''-P¹,Pⁿ-oligophosphate (n = 3-6); Aps1, Ap_sixA hydrolase; p₄A, adenosine 5'-tetraphosphate; p₅A, adenosine 5'-pentaphosphate; PP-InsP₅, diphosphoinositol pentakisphosphate; [PP]₂-InsP₄, bisdiphosphoinositol tetrakisphosphate; InsP₆, inositol hexakisphosphate; DIPP, diphosphoinositol polyphosphate phosphohydrolase; HIT, histidine triad; Fhit, fragile histidine triad; CHAPS, 3-[(cholamidopropyl)dimethylammonio]-1-propane-sulfonate; E-64, trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane; hMTH1, human MutT homologue, type 1; k¹ is the first-order rate constant in the rate equation, [S] = [S]₀e^kt; HPLC, high performance liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES