Comparative Mechanistic and Substrate Specificity Study of Inositol Polyphosphate 5-Phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2*

Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative ino-sitol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg 2 (cid:1) , transition metals such as Mn 2 (cid:1) , Co 2 (cid:1) , and Ni 2 (cid:1) are also effective activators of SPsynaptojanin. In contrast, Ca 2 (cid:1) and Cu 2 (cid:1) are inhibitory. We provide evidence that Mg 2 (cid:1) binds the same site occu-pied by Ca 2 (cid:1) observed in the crystal structure of SPsynaptojanin Experiments were carried out at 30 °C in a 1.6-ml reaction mixture containing 100 m M NaCl, 50 m M HEPES, 1 m M Mg 2 (cid:2) , 0.25 m M EDTA, pH 7.4, 0.1 mg/ml purine nucleoside phosphorylase (Sigma), and 200 (cid:1) M 7-methyl-6-thioguanosine. The spectrophotometric measurements were conducted using a PerkinElmer Life Sciences Lambda 14 spectropho- tometer equipped with a magnetic stirrer in the cuvette holder. NMR Measurements— 0.6 ml of 1.5 m M Ins(1,4,5)P 3 was dissolved in a buffer containing 100 m M NaCl, 50 m M deuterated Tris, 1 m M MgCl 2 , 0.25 m M EDTA in D 2 O at pH 7.1. The spectra for Ins(1,4,5)P 3 both before and after the addition of SPsynaptojanin were acquired. NMR experiments were performed at 26 °C on a Bruker DRX 300 spectrom- eter. Phosphorus chemical shifts were relative to an external standard (85% phosphoric acid) set at 0.0 ppm. Two-dimensional 1 H- 1 H total correlation spectroscopy (TOCSY) spectra were collected using an 80-ms MLEV-17 spin lock and WATERGATE water suppression with 4000 and 256 points in F2 and F1, respectively, with 16 scans per t 1 point and a recycle delay of 1.5 s. The experiments used a proton sweep width of 14 ppm with the carrier set to 4.7 ppm. Spectra were processed with a cosine bell window function and zero-filled to yield data sets with 4000 and 512 points in F2 and F1, respectively. 31 P-Decoupled two-dimensional 1 H- 1 H TOCSY spectra were collected as above and used GARP 31 P-decoupling during the acquisition period. Proton chemical shifts were referenced to 3-(trimethylsilyl)propionate. Proton coupled and decoupled 31 P NMR spectra were collected at 121.5 MHz, with an acquisition time of 1.347 s, and 512 transition scans.

Eukaryotic cells contain both water-soluble inositol phosphates and the corresponding phosphoinositide lipids. These molecules reside in different cellular compartments and regulate the localization and activity of proteins through their interaction with specific binding domains. Inositol and phosphatidylinositol phosphates are key modulators of cellular processes including signal transduction, cell proliferation, and apoptosis, vesicle trafficking, cell motility and cytoskeletal organization, and transcription (1)(2)(3)(4)(5). The best known inositolbased signaling pathway is the cell surface receptor-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-(4,5)P 2 ) 1 by phospholipase C to yield the second messengers inositol 1,4,5-triphosphate (Ins(1,4,5)P 3 ), a regulator of Ca 2ϩ release, and diacylglycerol, an activator of protein kinase C. The activity of the inositol-based molecules depends on which site(s) on the inositol ring is phosphorylated, and the level of phosphorylation is maintained by the combined action of a host of inositol kinases and phosphatases. Although considerable effort has been directed toward the analysis of the kinases that produce the phosphorylated inositol molecules, the study of phosphatases that oppose the activity of these kinases remains limited.
There are several distinct families of inositol phosphatases that are capable of removing the phosphoryl group from the inositol ring (3,6). The inositol phosphatases are classified according to the position of the phosphate that they hydrolyze. For instance, dephosphorylation of the D1 position is carried out by the inositol polyphosphate 1-phosphatase, which is a member of an Mg 2ϩ -dependent, Li ϩ -inhibited gene family (7). The hydrolysis of the D3 phosphate is catalyzed by the inositol polyphosphate-3-phosphatases that include the tumor suppressor PTEN and the myotubularin family of phosphatases (6). PTEN and myotubularins have the same active site sequence CX 5 R that characterize the protein-tyrosine phosphatase superfamily (8). Interestingly, the newly identified inositol polyphosphate 4-phosphatases responsible for the hydrolysis of the D4 phosphate also contain the CX 5 R catalytic motif (9). Finally, the removal of the D5 phosphate is accomplished by a large family of inositol polyphosphate-5-phosphatases, which can be further classified into type I enzymes that hydrolyze only water-soluble substrates, type II enzymes that hydrolyze both water-soluble and lipid substrates, type III enzymes that hydrolyze only substrates with a 3-position phosphate, and type IV enzymes that are poorly characterized and are active only toward PtdIns(3,4,5)P 3 (3,10). The crystal structure of a type II 5-phosphatase SPsynaptojanin has been determined, revealing a fold similar to metal-dependent endonucleases (11).
Recent studies have documented the critical roles of inositol polyphosphate-5-phosphatases in normal physiology and disease. The synaptojanin-catalyzed dephosphorylation of PtdIns(4,5)P 2 is essential for proper synaptic vesicle recycling (12). The insulin-stimulated production of PtdIns(3,4,5)P 3 is central to glucose homeostasis. Mice deficient of SHIP2, an SH2 domain-containing inositol-5-phosphatase that dephosphorylates PtdIns(3,4,5)P 3 , are hypoglycemic and sensitized to insulin, suggesting that SHIP2 may be a potential therapeutic target for the treatment of type II diabetes (13). Certain human diseases are also associated with genetic mutations affecting the 5-phosphatase activity. The Lowe oculocerebrorenal syndrome is an X-linked disorder characterized by congenital cataracts, mental retardation, and renal Fanconi syndrome. The oculocerebrorenal gene encodes a 5-phosphatase that exhibits diminished enzyme activity in oculocerebrorenal patients (14).
Elucidating the molecular determinants for inositol polyphosphate-5-phosphatase catalysis and substrate specificity is crucial to our understanding of how this family of enzymes regulates cellular signaling. In the following we describe two convenient, non-radioactive methods for assaying inositol phosphatase activity. We report a detailed kinetic analysis of the reaction catalyzed by a putative mammalian synaptojanin homologue from Schizosaccharomyces pombe (SPsynaptojanin) with regard to metal ion and pH dependence. SPsynaptojanin was chosen as a model system since it is the only 5-phosphatase with a known crystal structure. To provide further insight into the function of 5-phosphatases, we also describe an investigation of SPsynaptojanin and SHIP2 substrate specificity with more than 50 water-soluble and lipid inositol phosphates. SHIP2 was chosen as a subject of study because of its importance in insulin signaling and its potential as a therapeutic target. Knowledge of the substrate specificity of inositol-5phosphatases may shed new light on the roles played by these enzymes in cellular physiology and furnish valuable information for the design of potent small molecule inhibitors for biological studies and therapeutic development.
Mutagenesis-The SPsynaptojanin mutants were constructed with the QuikChange site-directed mutagenesis kit from Stratagene using a pET22b plasmid containing the coding sequence for the SPsynaptojanin 5-phosphatase domain (residues 534 -880) (11). Mutations were confirmed by DNA sequencing.
Expression and Purification of SPsynaptojanin and SHIP2-The inositol polyphosphate-5-phosphatase domain (Ipp5c) of SPsynaptojanin was constructed into the pET22b vector with a His 6 tag and a tobacco etch virus protease site at its N terminus. The plasmid was transformed into BL21/DE3 competent cells by standard procedure. A single colony was selected and grown in LB medium (supplemented with 100 g/ml ampicillin) at 37°C overnight. A 10-ml overnight culture was transferred to 1 liter of LB medium (supplemented with 100 g/ml ampicillin) and allowed to grow at 37°C until the absorbance at 600 nm was 0.6 -0.8. Isopropyl-1-thio-␤-D-galactopyranoside (1 mM) was added to induce SPsynaptojanin expression. After further incuba-tion at room temperature with shaking for 16 h, the cells were harvested by centrifugation at 5000 rpm for 5 min. The cell pellet from 1 liter of culture was resuspended in 30 ml of lysate buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole). The cells were lysed by passage through a French press at 12,000 p.s.i. twice. Cellular debris was removed by centrifugation at 16,000 rpm for 30 min. A 2-ml 50% slurry of nickel nitrilotriacetic acid-agarose (Qiagen) was equilibrated with lysate buffer and added to the lysate supernatant. After incubation with gentle agitation at 4°C for 1 h, the matrix was transferred into a column and washed with 20 ml of lysate buffer. The His 6 -tagged protein was eluted by buffers containing concentrations of imidazole of 5, 20, 50, and 110 mM in 20 mM Tris-HCI, pH 7.9, 500 mM NaCl. Fractions containing SPsynaptojanin were pooled together and concentrated with a Centriprep-30 filtration unit (Amicon). Mutants were further purified by fast protein liquid chromatography with a Superdex G-75 column using an elution buffer containing 50 mM Tris, pH 7.9, 300 mM NaCl. Protein purity was checked by 10% SDS-PAGE. Protein concentration was determined by a Bio-Rad protein assay.
The SHIP2 catalytic domain (residues 419 -732) was constructed into the pET22b vector, with a His 6 tag and a tobacco etch virus protease site at its N terminus. The plasmid was transformed into BL21/DE3 competent cell by standard procedure. A 10-ml overnight culture was transferred to 1 liter of LB medium (supplemented with 100 g/ml ampicillin) and allowed to grow at 37°C until the absorbance at 600 nm was 0.6. Without the addition of isopropyl-1-thio-␤-D-galactopyranoside, the culture was incubated at room temperature with shaking for an additional 6 h. The cells were harvested by centrifugation at 5000 rpm for 5 min. The cell pellets from 1 liter of culture was resuspended in 30 ml of lysate buffer (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 5 mM imidazole). The cells were lysed by passage through a French press cell at 12,000 p.s.i. twice. Cellular debris was removed by centrifugation at 16,000 rpm for 30 min. A 2-ml 50% slurry of nickel nitrilotriacetic acid-agarose (Qiagen) was equilibrated with the lysate buffer and added to the lysate supernatant. After incubating with gentle agitation at 4°C for 1 h, the matrix was transferred into a column and washed with 20 ml of lysate buffer. The His 6 -tagged protein was eluted by an imidazole gradient from 5 to 150 mM in 20 mM Tris-HCI, pH 7.9, 500 mM NaCl. Fractions the SHIP2 protein were pooled together and concentrated with a Centriprep-30 filtration unit (Amicon), and the buffer was changed to 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol. The purified protein was made to 20% glycerol and stored at Ϫ80°C.
Ammonium Molybdate Inorganic Phosphate Assay-In a typical assay (22) the inositol phosphate was incubated at 30°C in a buffer containing 50 mM HEPES, 100 mM NaCl, 1 mM MgCl 2 , with or without 0.25 mM EDTA at pH of 7.4 for 10 min. To determine the steady state kinetic parameters K m and k cat , the substrate concentrations were in the range of 3-300 M. For activity comparison, the concentrations of inositol phosphates were fixed at 200 M. To ensure initial rate conditions, the product versus time progress curves were routinely checked for linearity at different substrate and enzyme concentrations. In all cases the enzyme concentration was at least 100-fold lower than that of the substrate, and the quantity of substrate consumed was less than 10% of the initial value, so that the steady state assumption was fulfilled. There was no product inhibition under these conditions. Reactions were started by the addition of an appropriate amount of SPsynaptojanin (4 l) to the reaction mixture containing the substrate (100 l). After an appropriate amount of time, usually 5 min, 25 l of 10% (w/v) trichloroacetic acid was used to quench the reaction. Then, 65 l of solution B was added to the mixture. 2.5 min later solution C was added. The reaction mixture was well mixed every time after the addition of reagents. UV-visible absorbance at 700 nm was measured 5 min after final mix. Here, solution B is a mixture of 3.4 ml of 2% (w/v) ammonium molybdate and 1 g of L-ascorbic acid in 4.9 ml of 50% trichloroacetic acid and made freshly daily. Solution C contains 2% (w/v) sodium arsenite in 2% (v/v) acetic acid. The measurements of activity in the absence of EDTA were conducted in the same buffer, except there was no EDTA.
Continuous Spectrophotometric Enzyme-coupled Assay-Kinetic parameters for the dephosphorylation of inositol phosphate were also determined using a continuous spectrophotometric assay. This assay incorporates a coupled enzyme system, which uses purine nucleoside phosphorylase and its chromogenic substrate 7-methyl-6-thioguanosine for the quantification of inorganic phosphate produced in the phosphatase reaction (23,24). The change in absorbance at 360 nm was due to the conversion of 7-methyl-6-thioguanosine to 7-methyl-6-thioguanine in the presence of inorganic phosphate. Quantitation of the inorganic phosphate produced in the phosphatase reaction was determined using the extinction coefficient of 11,200 M Ϫ1 cm Ϫ1 at 360 nm and pH 7.0. Experiments were carried out at 30°C in a 1.6-ml reaction mixture containing 100 mM NaCl, 50 mM HEPES, 1 mM Mg 2ϩ , 0.25 mM EDTA, pH 7.4, 0.1 mg/ml purine nucleoside phosphorylase (Sigma), and 200 M 7-methyl-6-thioguanosine. The spectrophotometric measurements were conducted using a PerkinElmer Life Sciences Lambda 14 spectrophotometer equipped with a magnetic stirrer in the cuvette holder.
NMR Measurements-0.6 ml of 1.5 mM Ins(1,4,5)P 3 was dissolved in a buffer containing 100 mM NaCl, 50 mM deuterated Tris, 1 mM MgCl 2 , 0.25 mM EDTA in D 2 O at pH 7.1. The spectra for Ins(1,4,5)P 3 both before and after the addition of SPsynaptojanin were acquired. NMR experiments were performed at 26°C on a Bruker DRX 300 spectrometer. Phosphorus chemical shifts were relative to an external standard (85% phosphoric acid) set at 0.0 ppm. Two-dimensional 1 H-1 H total correlation spectroscopy (TOCSY) spectra were collected using an 80-ms MLEV-17 spin lock and WATERGATE water suppression with 4000 and 256 points in F2 and F1, respectively, with 16 scans per t 1 point and a recycle delay of 1.5 s. The experiments used a proton sweep width of 14 ppm with the carrier set to 4.7 ppm. Spectra were processed with a cosine bell window function and zero-filled to yield data sets with 4000 and 512 points in F2 and F1, respectively. 31 P-Decoupled twodimensional 1 H-1 H TOCSY spectra were collected as above and used GARP 31 P-decoupling during the acquisition period. Proton chemical shifts were referenced to 3-(trimethylsilyl)propionate. Proton coupled and decoupled 31 P NMR spectra were collected at 121.5 MHz, with an acquisition time of 1.347 s, and 512 transition scans.

RESULTS AND DISCUSSION
Characterization of SPsynaptojanin as an Inositol Polyphosphate-5-phosphatase-Recently, the crystal structure of a previously uncharacterized putative mammalian synaptojanin homologue from S. pombe (SPsynaptojanin) was reported (11). This was the first three-dimensional structure of a catalytic domain from the inositol-5-phosphatase family. This structure was solved after soaking the crystals of SPsynaptojanin for 20 h in 2 mM Ins(1,4,5)P 3 and 1 mM CaCl 2 . The electron difference density indicated that the putative hydrolysis product Ins(1,4)P 2 , not Ins(1,4,5)P 3 , was bound at the active site. Given the fact that the catalytic domain of SPsynaptojanin shows significant sequence identity to those of mammalian inositol polyphosphate-5-phosphatases, the presence of Ins(1,4)P 2 in the active site is consistent with the expectation that SPsynaptojanin is an inositol-5-phosphatase.
To further characterize the regiospecificity of SPsynaptojanin for the 5 position of Ins(1,4,5)P 3 , NMR measurements were conducted on a reaction containing SPsynaptojanin and Ins(1,4,5)P 3 to identify the position(s) of dephosphorylation. The 1 H-coupled 31 P NMR spectrum of the starting material Ins(1,4,5)P 3 shows that there are three phosphorus peaks, each split by one proton, consistent with the three phosphates attached to the inositol ring (Fig. 1Aa). Fig. 1Ab is the corresponding 1 H-decoupled 31 P spectrum of Ins(1,4,5)P 3 . Upon the addition of SPsynaptojanin, distinct spectral changes (Fig. 1A, c to e) occurred consistent with the disappearance of Ins(1,4,5)P 3 and the coincident appearance of a second set of peaks. Fig. 1Af is the 1 H-coupled 31 P NMR spectrum for the reaction upon prolonged incubation with SPsynaptojanin, which shows two peaks apparently split by a proton and a third peak that is insensitive to proton coupling, consistent with a single inositol bisphosphate and inorganic phosphate as the products of the reaction. To provide evidence that the inositol bisphosphate produced in the SPsynaptojanin-catalyzed Ins(1,4,5)P 3 reaction is Ins(1,4)P 2 , we also collected the 31 P NMR spectra of authentic Ins(1,4)P 2 under identical conditions (Fig. 1B). Spectral comparison (Fig. 1, A, e and f, and B, a and  b) suggests that the inositol bisphosphate produced in the SPsynaptojanin reaction is Ins(1,4)P 2 .
The regiochemistry of the inositol bisphosphate produced in the SPsynaptojanin-catalyzed dephosphorylation of Ins (1,4,5)P 3 was further confirmed by 1 H TOCSY measurements. The overlay of the 31 P coupled (black) and decoupled (red) 1 H TOCSY spectra shows that the inositol phosphate product has two phosphates that are linked at the 1 and 4 positions of inositol (Fig. 2), based upon the previous one-dimensional 1 H NMR spectral assignments of Ins(1,4)P 2 (25). Collectively, the NMR results establish that the inositol phosphate product observed in the SPsynaptojanin-catalyzed Ins(1,4,5)P 3 reaction is Ins(1,4)P 2 , in accord with the classification of SPsynaptojanin as a 5-phosphatase.
Non-radioactive Spectroscopic Assays for Inositol 5-Phosphatases-Traditionally, inositol-5-phosphatases are assayed with radioisotope-labeled substrates followed by chromatographic separation of the products from the substrates. Having established the regiospecificity of SPsynaptojanin, we set out to develop non-radioactive colorimetric procedures that would be more suitable for detailed mechanistic investigation and substrate specificity analysis. Because there are no significant absorbance or fluorescence spectral changes associated with the dephosphorylation of inositol phosphates, our assays have been focused on the determination of inorganic phosphate produced in the reaction. In this regard we adopted two methods for inorganic phosphate determination to measure inositol-5phosphatase activity. The first method relies on the use of an ammonium molybdate reagent that reacts with inorganic phosphate to produce a bright blue color at 700 nm (22). No interference was observed with various inositol phosphates. With this assay, the steady state kinetic parameters k cat and K m for the SPsynaptojanin-catalyzed dephosphorylation of Ins(1,4,5)P 3 were determined to be 11.2 Ϯ 0.4 s Ϫ1 and 57.8 Ϯ 3.2 M, respectively, at pH 7.4 and 30°C (Fig. 3A). The second method for the quantitation of inorganic phosphate is a continuous spectrophotometric enzyme-coupled assay in which the coupling enzyme, purine nucleoside phosphorylase, uses the inorganic phosphate, generated by the action of the 5-phosphatase, to convert 7-methyl-6-thioguanosine to 7-methyl-6thioguanine and ribose-1-phosphate, resulting in an increase in absorbance at 360 nm (23). With the continuous coupled assay, the k cat and K m values for the same reaction under identical conditions were determined to be 11.0 Ϯ 0.2 s Ϫ1 and 55.7 Ϯ 2.9 M, respectively (Fig. 3B). Thus, there is excellent agreement between these two methods, which allows some flexibility in assay choices and eliminates the need for radioactivity.
Metal Ion Dependence-It is known that inositol-5-phosphatases are Mg 2ϩ -dependent phosphomonoesterases (3,10). As expected, SPsynaptojanin displays no measurable activity in the absence of metal ions. The effect of Mg 2ϩ concentration on the SPsynaptojanin reaction at 200 M Ins(1,4,5)P 3 is shown in Fig. 4. As the concentration of Mg 2ϩ is raised, the activity of SPsynaptojanin increases to a maximum at 2 mM metal ion concentration. Further increase in Mg 2ϩ concentration leads to a decline in SPsynaptojanin activity, suggesting the existence of a second metal ion binding site that is inhibitory. Fitting the initial rate versus [Mg 2ϩ ] to Equation 1, which includes the inhibitory term, yielded the k cat , K m , and K i values for Mg 2ϩ . As listed in Table I, the K m for Mg 2ϩ is 530 M, which is 16-fold lower than the K i value, consistent with the fact that the binding of Mg 2ϩ to the second inhibitory metal binding site is much weaker than the activation site. To explore whether other divalent cations are also effective in activating SPsynaptojanin, we examined the effect of Ca 2ϩ , Mn 2ϩ , Co 2ϩ , Ni 2ϩ , and Cu 2ϩ on the enzyme-catalyzed dephosphorylation of Ins(1,4,5)P 3 . We found that neither Ca 2ϩ nor Cu 2ϩ was able to activate SPsynaptojanin. In contrast, transition metal ions Mn 2ϩ , Co 2ϩ , and Ni 2ϩ were all activators of SPsynaptojanin. In addition, Mn 2ϩ , Co 2ϩ , and Ni 2ϩ exhibited similar metal ion dependence on the SPsynaptojanin reaction as observed for Mg 2ϩ , i.e. enzyme activity increased as a function of metal ion concentration to a maximum, after which additional metal ion led to enzyme inhibition (data not shown). The kinetic parameters for these metal ions are summarized in Table I. It appears that Mn 2ϩ , Co 2ϩ , Ni 2ϩ , and Mg 2ϩ can activate SPsynaptojanin to the same extent at saturating metal ion concentrations. However, the K m values of Mn 2ϩ , Co 2ϩ , and Ni 2ϩ for SPsynaptojanin are 30 -80-fold lower than that of Mg 2ϩ . Despite the higher affinities exhibited by Mn 2ϩ , Co 2ϩ , and Ni 2ϩ , these metal ions are unlikely to be physiological relevant because cellular concentrations of free transition metals are significantly lower than those (6 -15 M) required for SPsynaptojanin activation. In contrast, the K m for Mg 2ϩ (530 M) is within the physiological range, and thus, Mg 2ϩ is likely the co-catalyst for SPsynaptojanin inside the cell.
Finally, we noted that although Mg 2ϩ is absolutely required for SPsynaptojanin activity, sub-stoichiometric amounts of EDTA in the reaction could further enhance the 5-phosphatase activity. For example, when activity measurements were made in 50 mM HEPES buffer containing 100 mM NaCl, 1 mM Mg 2ϩ , and 200 M Ins(1,4,5)P 3 , maximal SPsynaptojanin activity was observed at 0.25 mM EDTA (Fig. 5A). This rate-enhancing effect by EDTA may indicate the presence of inhibitory substances in the assay system, most likely trace amounts of heavy metals such as Cu 2ϩ . Indeed, as shown in Fig. 5B, Cu 2ϩ caused a concentration-dependent decrease in the 5-phosphatase activity in the presence of 1 mM Mg 2ϩ , and the presence of 10 M Cu 2ϩ could completely abolish the Mg 2ϩ -dependent 5-phosphatase activity. The addition of 0.25 mM EDTA to the reaction fully restored the SPsynaptojanin activity. Thus, unless stated otherwise, all assay systems contained 0.25 mM EDTA to sequester the inhibitory heavy metals in the buffer.
The Primary Metal Ion Binding Site in SPsynaptojanin-The x-ray crystal structure of SPsynaptojanin in complex with Ca 2ϩ and Ins(1,4)P 2 reveals a Ca 2ϩ binding site in the vicinity of the 4-phosphate group in Ins(1,4)P 2 (11). The bound Ca 2ϩ is directly coordinated by three ligands from SPsynaptojanin and four water molecules (Fig. 6). Both carboxylate oxygens of Glu-597 and the amide oxygen of Asn-568 participate in Ca 2ϩ binding. Of the four water molecules, two form bridging interactions with the 4-phosphate of Ins(1,4)P 2 , one with the side chain oxygens of Asp-566 and Asp-838 and one with the main chain carbonyl of Leu-601. Although the Ca 2ϩ binding site is well defined, its significance to SPsynaptojanin catalysis is unclear because unlike Mg 2ϩ , no activity was detected for SPsynaptojanin in the presence of Ca 2ϩ at concentrations up to 8 mM. In fact, we found that Ca 2ϩ is inhibitory to the Mg 2ϩ -dependent activity, in agreement with an earlier observation with a different inositol-5-phosphatase (26). The Ca 2ϩ inhibition constant and its mode of inhibition were determined by varying the Mg 2ϩ concentration at fixed levels of Ca 2ϩ . As shown in Fig. 7, the effect of Ca 2ϩ on the Mg 2ϩ -dependent SPsynaptojanin-catalyzed dephosphorylation of Ins(1,4,5)P 3 displayed the characteristic intersecting line pattern for competitive inhibition. The K i value for Ca 2ϩ inhibition is 668 Ϯ 78 M.
The fact that Ca 2ϩ inhibits the SPsynaptojanin reaction competitively with respect to the Mg 2ϩ co-factor suggests that they may compete for the same binding site. To test this hypothesis, we assessed the effect of structural perturbations to the Ca 2ϩ binding site on SPsynaptojanin activity. Because both carboxylate oxygens of Glu-597 are involved in coordinating the Ca 2ϩ , we chose to replace Glu-597 with either an Ala or Gln. The catalytic activity of the E597A and E597Q mutants were found to be 4 and 3 orders of magnitude lower than that of the wild-type enzyme at 1 mM Mg 2ϩ , a concentration that fully activates the wild-type enzyme. To determine whether the lower activity for E597Q is a result of lost affinity for Mg 2ϩ , we measured the Mg 2ϩ concentration dependence of the E597Q- catalyzed Ins(1,4,5)P 3 dephosphorylation. The results indicated that the Mg 2ϩ concentration obeys Michaelis-Menten saturation kinetics (data not shown). Kinetic analysis shows that the E597Q-catalyzed reaction displays a k cat of 0.078 Ϯ 0.002 s Ϫ1 and a K m of 1.30 Ϯ 0.14 mM for Mg 2ϩ . Thus, replacement of the carboxylate at residue 597 by a carboxyamide causes a greater than 140-fold decrease in SPsynaptojanin catalytic efficiency, whereas the affinity for Mg 2ϩ is reduced by only 2.4-fold. This result suggests that although Glu-597 is not essential for metal ion binding affinity, the precise interactions between the metal and Glu-597 may be more important for proper positioning and orientation of the metal ion relative to the reactive center for catalysis. The dramatic effects on the Mg 2ϩ -dependent SPsynaptojanin activity observed as a result of a subtle change to the Ca 2ϩ binding site provide further evidence that Mg 2ϩ and Ca 2ϩ bind to the same site.
The drastically different behaviors of Mg 2ϩ and Ca 2ϩ in enzyme catalysis are quite common. Ca 2ϩ and Mg 2ϩ belong to the same elemental group in the periodic table, and the main difference between the two metals is ionic size. Size variation of the metal ion can result in different coordination chemistries and spatial arrangements between the metal ion and the substrate, which could be either activating or inhibitory to enzyme activity. Thus, although Mg 2ϩ and Ca 2ϩ bind to the same site in SPsynaptojanin, their interactions with the enzyme and the substrate may be different. It is of interest to note that the free intracellular Ca 2ϩ concentration in resting cells is very low, ϳ10 -100 nM. In this concentration range, it is unlikely that Ca 2ϩ will exert any influence on SPsynaptojanin activity. However, free Ca 2ϩ concentration within the cell can rise up to the low millimolar range upon Ca 2ϩ release from internal stores, which is triggered by the binding of Ins(1,4,5)P 3 to its receptors (27). At low millimolar concentrations, Ca 2ϩ can effectively block the action of inositol-5-phosphatase, therefore potentially prolonging Ca 2ϩ signaling.
pH-rate Profiles-To determine the ionization constants important for SPsynaptojanin catalysis and substrate binding, we obtained the k cat and k cat /K m values using Ins(1,4,5)P 3 as a substrate at various pH values and 1 mM Mg 2ϩ concentration. The k cat /K m parameter is the apparent second-order rate constant for the reaction of free enzyme and free substrate and includes the binding of substrate through the first irreversible step. The kinetic parameter k cat describes the rate-limiting step under saturating concentrations of the substrate. Plots of k cat /K m versus pH yield critical pK a values for free enzyme and/or substrate, which are essential for binding and/or catalysis, and plots of k cat versus pH provide the essential pK a values of the enzyme-substrate complex, whose decomposition is rate-limiting. The plot of k cat /K m versus pH (Fig. 8A) shows a bell-shaped curve with a maximum at pH 7.4 and with a limiting slope of ϩ1 on the acidic side and Ϫ2 on the basic side. Thus, the k cat /K m versus pH profile for Ins(1,4,5)P 3 indicates that in the free enzyme or substrate, one group with a pK a of 7.59 Ϯ 0.65 must be deprotonated and two groups with a pK a of 6.66 Ϯ 0.12 and 8.41 Ϯ 0.58, respectively, must be protonated for optimal catalysis. The plot of k cat versus pH (Fig. 8B) also shows a bell-shaped curve with pH maximum of 7.2 and a slope of ϩ1 on the acidic limb and Ϫ1 on the basic limb. The data indicate that two groups in the enzyme-substrate complex are involved in catalysis, one group with a pK a value of 6.60 Ϯ 0.10 must be deprotonated and one group with a pK a value of 7.75 Ϯ 0.08 must be protonated. Because several groups in the enzyme and substrate can ionize in the pH range of 6 -8.5, the identities of the critical ionizations for k cat /K m and k cat will require further investigation.
To gain further insights into the molecular determinants for SPsynaptojanin and SHIP2 substrate specificity, we decided to analyze additional inositol phosphates. There are 63 possible water-soluble inositol phosphates, whereas the upper limit for the lipid counterparts, in which only the 3-, 4-, and 5-hydroxyls are involved in generating isomers, is 7 (2). We examined all 4 possible isomers of lipid inositol molecules bearing a 5-phosphate (Table II). More structural variations are possible with water-soluble inositol phosphates. Indeed, more than 20 inositol phosphates have been identified in eukaryotic cells, many of which have not yet been assigned a function (28). In the following, we have determined the activity of SPsynaptojanin and SHIP2 toward 54 inositol phosphates.
For SPsynaptojanin, Ins(4,5)P 2 , Ins(1,4,5)P 3 , Ins(2,4,5)P 3 , Ins(4,5,6)P 3 , Ins(1,2,4,5)P 4 , Ins(1,3,4,5)P 4 , Ins(2,4,5,6)P 4 , and Ins(1,2,4,5,6)P 5 were found to be excellent substrates with k cat /K m values in the range of 10 4 -10 6 M Ϫ1 s Ϫ1 . Several conclusions are apparent upon examination of the results listed in Table III. The common structural feature for SPsynaptojanin substrates is the presence of a 4-phosphate in the inositol ring. Interestingly, the 4-phosphate in Ins(1,4)P 2 , the hydrolysis product from Ins(1,4,5)P 3 , was found in close proximity to the bound Ca 2ϩ (Fig. 6 and Ref. 11). Our kinetic and mutagenesis data suggest that Mg 2ϩ can also occupy the Ca 2ϩ site, although the exact coordination spheres for the two metal ions may be different. We also found that Ins(1,4)P 2 is a competitive inhibitor of SPsynaptojanin with respect to Ins(1,4,5)P 3 (K i ϭ 306 Ϯ 25 M, data not shown). Thus, it is possible that the binding mode observed between the 4-phosphate group in Ins(1,4)P 2 and Ca 2ϩ may be related to that between the 4-phosphate in the substrate and the Mg 2ϩ cofactor. The interactions between the 4-phosphate in the substrate and the Mg 2ϩ binding site in SPsynaptojanin may be essential to achieve precise transition state complementarity in the active site, accounting for the absolute requirement of 4-phosphate for SPsynaptojanin 5-phosphatase activity.
In the structural context of Ins(4,5)P 2 , the presence of 1-(i.e. Ins(1,4,5)P 3 ) or 6-phosphate (i.e. Ins(4,5,6)P 3 ) appears inconsequential. However, introduction of a phosphate at the 3 position strongly hinders the reaction because no measurable activity was observed with Ins(3,4,5)P 3 as a substrate. To FIG. 8. Effect of pH on k cat /K m (A) and k cat (B) for the SPsynaptojanincatalyzed dephosphorylation of Ins(1,4,5)P 3 at 1 mM Mg 2؉ concentration. The catalytically essential ionization constants for the free enzyme and substrate were determined by fitting the data for k cat /K m versus pH to the equation  The substrate specificity for SHIP2 is more restricted than that of SPsynaptojanin. Unlike SPsynaptojanin, no significant activity was detected for SHIP2 with inositol phosphates having less than four phosphate groups. Only Ins(1,3,4,5)P 4 , Ins(1,4,5,6)P 4 , Ins(2,4,5,6)P 4 , and Ins(1,2,3,4,5)P 5 were found to be excellent substrates for SHIP2 (Table III). Similar to SPsynaptojanin, in addition to the 5-phosphate, a common feature for the SHIP2 substrates is also the presence of a 4-phosphate group in the inositol ring. It is possible that the 4-phosphate group engages the catalytic machinery in SHIP2, including the Mg 2ϩ binding site, in a manner similar to that proposed for SPsynaptojanin to promote SHIP2 catalysis. Surprisingly, in contrast to the notion that SHIP2 only hydrolyzes substrates with a 3-position phosphate group (3), both Ins(1,4,5,6)P 4 and Ins(2,4,5,6)P 4 , inositol polyphosphates lacking a 3-phosphate, can also be effectively hydrolyzed by SHIP2. The best substrate identified for SHIP2 is inositol pentakisphosphate Ins(1,2,3,4,5)P 5 , having a k cat /K m value that is 8-fold higher than that of Ins(1,3,4,5)P 4 . Thus, the 2-position axial phosphate may have an activity enhancing effect on the SHIP2 reaction as well.
Conclusions and Biological Implications-We have established the regiospecificity for SPsynaptojanin. Using two nonradioactive spectroscopic assays we have characterized the metal ion and pH dependence of the SPsynaptojanin reaction. In addition to Mg 2ϩ , transition metals such as Mn 2ϩ , Co 2ϩ , and Ni 2ϩ are also effective in promoting SPsynaptojanin activity. In contrast, Ca 2ϩ and Cu 2ϩ are inhibitory. We provide evidence that Mg 2ϩ occupies the same Ca 2ϩ binding site observed in the crystal structure of the complex between SPsynaptojanin and Ins(1,4)P 2 . The k cat /K m versus pH profile indicates that three ionizations of the free enzyme and/or free substrate are involved in the reaction of Ins(1,4,5)P 3 ; one group with a pK a of 7.59 must deprotonated, and two groups with pK a values of 6.66 and 8.41, must be protonated for optimal catalysis. The k cat versus pH profile suggests that two groups in the enzymesubstrate complex are involved in catalysis; one group with a pK a value of 6.60 must be deprotonated, and one group with a pK a value of 7.75 must be protonated.