Arginine 343 and 350 are two active residues involved in substrate binding by human Type I D-myo-inositol 1,4,5,-trisphosphate 5-phosphatase.

The crucial role of two reactive arginyl residues within the substrate binding domain of human Type I D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P) 5-phosphatase has been investigated by chemical modification and site-directed mutagenesis. Chemical modification of the enzyme by phenylglyoxal is accompanied by irreversible inhibition of enzymic activity. Our studies demonstrate that phenylglyoxal forms an enzyme-inhibitor complex and that the modification reaction is prevented in the presence of either Ins(1,4,5)P, D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1, 3,4,5)P) or 2,3-bisphosphoglycerate (2,3-BPG). Direct [3H]Ins(1,4,5)P binding to the covalently modified enzyme is dramatically reduced. The stoichiometry of labeling with 14C-labeled phenylglyoxal is shown to be 2.1 mol of phenylglyoxal incorporated per mol of enzyme. A single [14C]phenylglyoxal-modified peptide is isolated following α-chymotrypsin proteolysis of the radiolabeled Ins(1,4,5)P 5-phosphatase and reverse-phase high performance liquid chromatography (HPLC). The peptide sequence (i.e. M-N-T-R-C-P-A-W-C-D-R-I-L) corresponds to amino acids 340-352 of Ins(1,4,5)P 5-phosphatase. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivatives shows the modified amino acids to be Arg-343 and Arg-350. Furthermore, two mutant enzymes were obtained by site-directed mutagenesis of the two arginyl residues to alanine, and both mutant enzymes have identical UV circular dichroism (CD) spectra. The two mutants (i.e. R343A and R350A) show increased K values for Ins(1,4,5)P (10- and 15-fold, respectively) resulting in a dramatic loss in enzymic activity. In conclusion, we have directly identified two reactive arginyl residues as part of the active site of Ins(1,4,5)P 5-phosphatase. These results point out the crucial role for substrate recognition of a 10 amino acids-long sequence segment which is conserved among the primary structure of inositol and phosphatidylinositol polyphosphate 5-phosphatases.

Arginyl residues are known to act as anionic binding sites in proteins and may thus assist in the binding of substrates or enzyme catalysis. Covalent and irreversible modification with amino acid specific reagents has been used successfully to identify lysyl or arginyl residues in the substrate binding domain in many enzymes, such as tyrocidine synthetase 1 (21), Ca 2ϩ /ATPase (22), 6-phosphofructo-2-kinase (23), and Ins(1,4,5)P 3 3-kinase (24). In addition, two arginyl residues were shown using site-directed mutagenesis to be critical to bind the C-2 phospho group of fructose 2,6-bisphosphate in rat liver fructose 2,6-bisphosphatase (25). We therefore investigated the possibility that active site arginines may play such a role in substrate binding for Type I Ins(1,4,5)P 3 5-phosphatase.
In this study, modification and inactivation of human Type I Ins(1,4,5)P 3 5-phosphatase by phenylglyoxal, an arginine-specific chemical modification reagent (see Ref. 26), was shown to be prevented by Ins(1,4,5)P 3 . We identified two essential arginines, i.e. Arg-343 and Arg-350, taking part of the sequence segment R-X-P-A-W-C-D-R-I-L. This segment appears to be crucial for enzymic activity, especially for substrate binding. The data have been confirmed by site-directed mutagenesis of both arginyl residues. This represents the first direct identification of an inositol polyphosphate 5-phosphatase active site peptide.

Methods
Enzyme Preparation-Native bovine brain enzyme was purified as previously reported (27). The complete 412-amino acid-long coding region of human Type I Ins(1,4,5)P 3 5-phosphatase (plasmid ECH10 in Ref. 19) was subcloned in pTrcHis A expression vector. It generated plasmid ECH11, which was used to transform competent Top 10 host bacteria. An overnight 5-ml culture was used to inoculate 1.2-liter of Luria-Bertani medium containing 50 g/ml ampicillin. After overnight culture at 37°C, it was diluted to an OD of 0.6 at 600 nm. Isopropyl-␤-galactopyranoside was added at 0.5 mM for an 8-h culture at 30°C. The cells were harvested by centrifugation at 5,000 ϫ g for 15 min, resuspended in 50 ml of buffer R (50 mM Tris/HCl (pH 8.0), 12 mM ␤-mercaptoethanol, 50 g/ml Pefabloc, 5 M leupeptin) per liter of culture and lysed by sonication (three times 15 s at 4°C). The cell debris were removed by centrifugation at 10,000 ϫ g for 20 min. The crude lysate was applied to 50 ml of Ni-NTA-agarose resin equilibrated in buffer A (50 mM Tris/HCl (pH 8.0), 300 mM NaCl, 12 mM ␤-mercaptoethanol, 50 g/ml Pefabloc, 5 M leupeptin). The column was washed with buffer A containing 50 mM imidazole. Ins(1,4,5)P 3 5-phosphatase was eluted in buffer A containing 100 mM imidazole to obtain 8 mg of pure enzyme per liter of initial bacteria culture. The protein concentration was determined by the method of Petterson (28). Immunodetection of the enzyme with anti-(human brain Type I Ins(1,4,5)P 3 5-phosphatase) antibodies was performed using alkaline phosphatase-conjugated anti-rabbit IgG and corresponding colorimetric methods (29). When the pure recombinant enzyme was separated by SDS-polyacrylamide gel electrophoresis, a single 43-kDa band was stained by Coomassie Blue and recognized by Western blotting (data not shown). K m for Ins(1,4,5)P 3 was 21 M, which is comparable to the purified native enzyme (see Table I).
Site-directed Mutagenesis-Site-directed mutagenesis was performed by using sequential polymerase chain reactions. Forward and reverse primers were 5Ј-GGCAAAGAGATCTACTCGG-3Ј (BglII restriction site is underlined) and 5Ј-CTAGGGTACCACGTCACTGCACGA-CAC-3Ј (KpnI restriction site is underlined), respectively. In brief, original wild-type DNA (plasmid ECH11) was used as template for a first PCR step (25 cycles). To generate mutant plasmid ECH11R343A, two PCR products were obtained separately using forward primer and mutated primer 1 (5Ј-TGGGCACGCGGTGTTCATGTACTG-3Ј, with the mutations in bold), and reverse primer and mutated primer 2 (5Ј-GAACACCGCGTGCCCAGCCTGG-3Ј, with the mutations in bold), respectively. Both purified PCR fragments encompassing the mutation were annealed with each other and extended by mutually primed synthesis using forward and reverse primers in a second unique PCR step (25 cycles). To generate mutant plasmid ECH11R350A, the procedure was the same, exept that the two mutated primers 1 and 2 were 5Ј-TGAGGATGGCGTCACACCAGGCTG-3Ј and 5Ј-GTGTGACGC-CATCCTCATGTC-3Ј (the mutations are indicated in bold), respectively. Both final mutated PCR fragments were subcloned in wild-type plasmid ECH11 after digestion with BglII and KpnI restriction enzymes. The presence of the mutations and the absence of any undesired mutation was confirmed by nucleotide sequence analysis. The two mutated plasmids were expressed in HB 101 host bacteria. R343A and R350A mutant enzymes were purified on Ni-NTA-agarose resin by the procedure described above.

5-Phosphatase with [ 14 C]Phenylglyoxal and ␣-Chymotryptic Digestion-Expressed
Ins-(1,4,5)P 3 5-phosphatase was concentrated to 1.7 mg/ml using an Amicon Centricon 10 centrifugal concentrator. Enzyme (150 g) was incubated at 23°C for 30 min in a final volume of 200 l in the presence or absence of 70 M Ins(1,4,5)P 3 in buffer M with 10 mM [ 14 C]phenylglyoxal. The modified arginyl residues were then reduced by adding NaBH 4 to a final concentration of 5 mM and allowing the reaction mixture to stand for 2 h at 30°C (32). The reaction was stopped at 4°C with 1 ml of 10% (mass/volume) trichloroacetic acid and left on ice for 45 min. Each sample was centrifuged for 15 min at 13,000 ϫ g (Eppendorf centrifuge), and the protein pellet was washed with 1 ml of acetone. After drying the pellet (SpeedVac concentrator), the protein was dissolved in 50 l of 8 M urea, 0.4 M NH 4 HCO 3 (pH 8.0), diluted to 200 l with water, and digested for 12 h at 30°C with 3.5 g of ␣-chymotrypsin.
Isolation of [ 14 C]Phenylglyoxal-labeled Peptide by Reverse-phase HPLC-Chymotryptic fragments of [ 14 C]phenylglyoxal-labeled Ins-(1,4,5)P 3 5-phosphatase were separated by reverse-phase HPLC on an Alltech Macrosphere 300 A C 18 5U column (2.1 mm ϫ 250 mm) by eluting with a gradient of solvent A (5% acetonitrile, 0.1% heptafluoro-butyric acid) and solvent B (95% acetonitrile, 0.1% heptafluorobutyric acid) at a flow rate of 0.2 ml/min as follows: 100% solvent A for 10 min, followed by a linear acetonitrile gradient (0 -72% solvent B over 85 min) and finally 100% solvent B for 5 min. The elution was followed by measuring the absorbance at 214 nm with an Applied Biosystems 1000S Diode Array detector, and each peak was collected separately. A 3-l aliquot of each peak fraction was counted with 10 ml of scintillation mixture to estimate the radioactivity associated with each peak. The radioactive 115-l peak fraction (corresponding to the [ 14 C]phenylglyoxal-labeled peptide of which radioactive modification was totaly protected by Ins(1,4,5)P 3 ) was concentrated by SpeedVac to 20 l and diluted in 0.5 ml of 5% acetonitrile, 0.1% trifluoroacetic acid. The peptide was further purified onto the same Alltech C 18 column and eluted with the same gradient described above except that 0.1% heptafluorobutyric acid was replaced by 0.1% trifluoroacetic acid. A 3-l aliquot of each peak fraction was counted for radioactivity.
Peptide Microsequencing and Identification of the Modified Arginines-The amino acid sequence of the labeled peptide was determined by Edman degradation using an Applied Biosystems model 477A peptide sequenator, with on-line quantification of the phenylthiohydantoin derivatives by HPLC. Thirty percent of the amino acid phenylthiohydantoin derivatives was collected in the internal fraction collector and counted for radioactivity to identify the labeled amino acid residues.
Determination of Circular Dichroism Spectra-All spectra were collected in a Jasco 710 spectropolarimeter in a 0.01-cm cell at 20°C. Scans were collected with a bandwith of 1 nm, a sensitivity of 10 millidegrees, and a time response of 0.125 s. For each enzyme preparation, the final spectra is the result of 12 accumulated scans. Samples were concentrated using an Amicon Centricon 10 centrifugal concentrator and diluted to 1 mg/ml in 20 mM Tris/HCl (pH 8.0), 100 mM NaCl, 1 mM dithiothreitol, and 10% glycerol. Samples were centrifuged at 13,000 ϫ g for 5 min to remove any precipitated protein.
The A 280 was measured to scale the CD data to the same protein concentration. Secondary structures were estimated by the method outlined in the Jasco manual using seven reference spectra.

Inactivation of Human Type I Ins(1,4,5)P 3 5-Phosphatase by
Phenylglyoxal-The arginine-specific modifying reagent phenylglyoxal inactivated native and recombinant Ins(1,4,5)P 3 5-phosphatase in a time-and dose-dependent manner (Fig. 1). The time course of inactivation was very similar for both native (Fig. 1A) and recombinant enzyme (Fig. 1B). The linear plots of the logarithm of residual enzymic activity versus the reaction time indicate that the time-dependent decrease in activity displayed pseudo first-order kinetics (Fig. 1). This behavior could be indicative of a two-step mechanism of inactivation described in Reaction 1, where a rapid reversible binding of phenylglyoxal (I) to the enzyme (E) precedes the covalent modification to an inactive enzyme-inhibitor complex (EI*).
The first order reaction may be described by Equation 1 where (V/V 0 ) is the residual enzymic activity, k is the observed first order rate constant of inactivation, and t is the time of reaction with phenylglyoxal (33).
A steady state treatment of the reaction described in Reaction 1 yields Equation 2 (34) where The linearity of a secondary plot of (1/k) versus (1/[phenylglyoxal]) using the data from the primary plot of Fig. 1 indicated that phenylglyoxal binding takes place through the twostep mechanism of interaction described by Reaction 1 (Fig. 1C). Substrate Protection against Chemical Modification by Phenylglyoxal-To further investigate the interaction of phenylglyoxal with Ins(1,4,5)P 3 5-phosphatase, protection from labeling was examined by incubating the enzyme with 20 mM phenylglyoxal in the presence of Ins(1,4,5)P 3 (0 -100 M) (Fig.  2). Ins(1,4,5)P 3 almost completely protected against phenylglyoxal-induced inactivation of the enzyme. The rate of inactivation decreased with increasing substrate Ins(1,4,5)P 3 concentration, reaching a limit value at approximately 70 M Ins(1,4,5)P 3 . Protection was also provided by the other substrate Ins(1,3,4,5)P 4 , and the competitive inhibitor 2,3-BPG (data not shown). These results suggest that at least one reactive arginyl residue is involved in substrate binding.
Stoichiometry of Phenylglyoxal Binding to Ins(1,4,5)P 3 5-Phosphatase-Since it appeared that phenylglyoxal interacts with the active site of the enzyme, we established the stoichiometry of this covalent modification using 14 C-radiolabeled phenylglyoxal. Fig. 4A shows a time course of phenylglyoxal incorporation in the presence or absence of 70 M Ins(1,4,5)P 3 . The curve showed an exponential approach to a limiting value of 2.1 arginyl residues modified in the absence of substrate. Ins(1,4,5)P 3 almost completely abolished phenylglyoxal incorporation (Fig. 4A). Ins(1,3,4,5)P 4 and 2,3-BPG also protected against phenylglyoxal incorporation (data not shown). The amount of incorporated phenylglyoxal in the absence of substrate at each time point was measured as a function of the residual Ins(1,4,5)P 3 5-phosphatase activity (Fig. 4B). This linear plot showed a direct correlation between the loss of enzyme activity and the incorporation of phenylglyoxal. Extrapolation of the data to 100% loss of enzyme activity indicated that activity was completely abolished when 2.1 mol of modifying  Fig. 5, A and B, shows the separation of the resulting peptides by reverse-phase HPLC after labeling in the absence of Ins(1,4,5)P 3 and the amount of radioactivity contained within each peak. The appearance of the HPLC profile suggested that complete digestion by the protease was obtained (Fig. 5A). After labeling in the absence of Ins(1,4,5)P 3 , a single major radioactive peak was observed with a retention time of 48.2 min (Fig. 5B). Since two arginyl residues were covalently modified, we supposed that this single peptide contained both modified residues. In the presence of 70 M Ins(1,4,5)P 3 , the HPLC profile was identical to the profile shown in Fig. 5A (data not shown) but the extent of [ 14 C]phenylglyoxal incorporation was greatly reduced (at least 16-fold) (Fig. 5C). Preparation of peptide fraction suitable for sequence analysis required an additional HPLC purification using a different ion pairing agent. The radioactive fraction that was protected by Ins(1,4,5)P 3 gave only one major radioactive peak upon rechromatography (Fig. 6). The sites of modification of the radioactive peptide were elucidated by automated gas phase Edman degradation sequencing. Fig. 7 shows the observed amino acid sequence, the radioactivity present in the first 13 cycles, and the yield quantified for each cycle. The major radioactivity appeared at cycles 4 and 11. Some carryover of radioactivity was observed into the two subsequent Resulting peptides were separated on a C 18 reverse-phase HPLC column using a gradient of acetonitrile in 0.1% heptafluorobutyric acid. An arrow head indicates the position of the major radioactive peak. B, ordinate represents the radioactivity detected in each peak of profile shown in A. C, Ins(1,4,5)P 3 5-phosphatase was incubated as described in A except that labeling was performed in the presence of 70 M Ins(1,4,5)P 3 . Ordinate represents the radioactivity detected in each peak of the HPLC profile, which was identical to the profile shown in A as mentioned under "Results." fractions, which was probably due to incomplete cleavage of the modified residues in cycles 4 and 11. Comparison of the obtained microsequence (M-N-T-R-/-P-A-W-/-D-R-I-L) with the predicted protein sequence of human brain Type I Ins(1,4,5)P 3 5-phosphatase (12) showed that this sequence corresponded to amino acids 340 -352 and that [ 14 C]phenylglyoxal was covalently attached to Arg-343 and Arg-350. The last residue of the peptide is a leucine residue in spite of the fact this peptide is a chymotryptic digest; the most likely explanation is that low specificity protease activity cleaved at leucine under extensive digestion. Moreover, the yield of phenylthiohydantoin-arginines in the amino acid analyzer was much lower than for the other amino acids in the sequence, consistent with their chemical modification by phenylglyoxal (Fig. 7).
Purification and Circular Dichroism Analysis of R343A and R350A Mutant Enzymes-Wild-type and mutant enzymes were induced at 30°C in a pTrcHis A expression system as described under "Experimental Procedures." After purification on Ni-NTA agarose resin, mutant and wild-type enzymes appeared homogeneous on Coomassie Blue-stained SDS gels (data not shown). Secondary structure analysis by circular dichroism revealed no change in the overall structure of the mutants when compared with the wild-type enzyme (Fig. 8). The spectra were also identical when comparing unmodified enzyme and inactive enzyme after modification by phenylglyoxal (data not shown).
Effect of Mutation of Arg-343 and Arg-350 to Alanine on the K m for Ins(1,4,5)P 3 and Maximal Velocity for Type I Ins(1,4,5)P 3 5-Phosphatase-As shown in Table I, the K m for Ins(1,4,5)P 3 for the wild-type enzyme was 21 M. The K m for Ins(1,4,5)P 3 for the R343A and R350A mutants were 205 and 320 M, respectively, i.e. 10-and 15-fold increases as compared with the wild-type value. Wild-type enzyme had a maximal velocity of 191 mol/min/mg (Table I). The R343A and R350A mutants had maximal velocities of 31 and 237 mol/min/mg, which correspond to a 6-fold decrease and a 20% increase, respectively, as compared with the wild-type value.

DISCUSSION
In this study, we aimed to identify active site residues interacting with the substrate for human Type I InsP 3 5-phosphatase. This was investigated in a first step without any assumption concerning the localization of reactive arginyl residues by covalent chemical modification with phenylglyoxal, and in a second step by site-directed mutagenesis of the previously identified arginyl residues.
Inactivation kinetics of the enzyme by phenylglyoxal-induced chemical modification are identical for native and expressed protein. Our results indicate that the amount of phenylglyoxal labeling paralleled the loss in enzyme activity and that the labeling involves two residues. The peptide mapping of the protein, which had been labeled with radioactive phenylglyoxal, enabled us to find a peptide which was preferentially labeled in the absence of substrate. We have identified two reactive arginyl residues within the active site of Ins(1,4,5)P 3 5-phosphatase, i.e. Arg-343 and Arg-350 (Fig. 9). The identification of both reactive amino acids is supported by two lines of evidence. First, the phenylthiohydantoin residues corresponding to the cycles where the modified arginines should elute contained radioactivity. Second, the yield of unmodified arginine in these cycles was particularly low. The covalent modification results in both an inactivation of the enzyme and a drastic decrease in Ins(1,4,5)P 3 binding. This strongly suggests that the Ins(1,4,5)P 3 binding domain of human Type I Ins(1,4,5)P 3  tated to alanine. The similarity in the induction yields and circular dichroism spectra between the mutant and the wildtype enzymes indicate that mutation of these arginyl residues did not affect the gross secondary structure of the enzymes. However, the R343A and R350A mutants display a higher K m for Ins(1,4,5)P 3 as compared to the wild-type enzyme (10-and 15-fold, respectively). The dramatic decrease in affinity for Ins(1,4,5)P 3 for both the R343A and R350A mutant enzymes indicates that both Arg-343 and Arg-350 are involved in binding Ins(1,4,5)P 3 . Although both mutants have decreased affinities for substrate, they exhibit distinct effects on the V max for Ins(1,4,5)P 3 5-phosphatase. The more important change in V max for R343A mutant enzyme suggest that Arg-343 may also be involved in Ins(1,4,5)P 3 5-phosphatase catalysis. Taking together, our data clearly indicate for the first time that Arg-343 and Arg-350 are two reactive residues involved in Ins(1,4,5)P 3 binding by human Type I Ins(1,4,5)P 3 5-phosphatase.
A lysine-rich Ins(1,3,4,5)P 4 -binding motif (R/K-R/K-T-K-X-R/ K-R/K-K-T) has been identified in synaptotagmin II (35). Moreover, a polybasic motif has also been proposed to be necessary for PtdIns(4,5)P 2 binding (R/K-X-X-X-X-K-X-R/K-R/K) and allosteric Ins(1,4,5)P 3 binding to actin-binding proteins and phospholipase C-␦ 1 , respectively (36,37). The pleckstrin homology domain is a noncatalytic protein module of approximately 120 amino acids which present the ability to bind PtdIns(4,5)P 2 and Ins(1,4,5)P 3 (38). The specific function of the pleckstrin homology domain has not yet been elucidated. However, none of those motives are found in the amino acid sequence of Type I Ins(1,4,5)P 3 5-phosphatase, nor in other Ins(1,4,5)P 3 binding proteins such as Ins(1,4,5)P 3 3-kinases and Ins(1,4,5)P 3 receptor/calcium channels. The primary function of the Ins(1,4,5)P 3 binding domain is supposed to anchor the inositol cycle and the three phosphates in position 1, 4, and 5. This probably occurs through locking the inositol cycle in a hydrophobic pocket and binding the phosphates with positively charged residues. Arg-343 and Arg-350 of Type I Ins(1,4,5)P 3 5-phosphatase could play an active role in this last interaction. Indeed, the use of arginine to stabilize the binding of the phosphate moieties of the substrate is quite a common occurrence in proteins for which the three-dimensional structure is known, such as 6-phosphofructo-1-kinase (39), glycogen phosphorylase b (40), and fructose 1,6-bisphosphatase. An arginyl residue of fructose 1,6-bisphosphatase belongs to the active site where the 6-phosphate group is bound: this basic residue is conserved in inositol monophosphatase sequence, which shares a very similar secondary structure topology with fructose 1,6-bisphosphatase (41). Since Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 have a net charge of Ϫ3 and Ϫ4, respectively, both molecules can interact with a cluster of basic amino acids, at least with Arg-343 and Arg-350. Results of the present study indicate that Arg-343 and Arg-350 in Type I Ins(1,4,5)P 3 5-phosphatase take part of an active peptide which may form a core where electrostatic and weak group (inositol ring)-specific interactions take place.
Molecular cloning and structural studies revealed that Ins(1,4,5)P 3 3-kinase and PtdIns 3-kinase (for review, see Ref. 42) present strong substrate specificities and do not share any sequence homology. Although primary structure corresponding to inositol and phosphatidylinositol polyphosphate 5-phosphatases present little amino acid identity, it is intriguing to note that both reactive arginyl residues Arg-343 and Arg-350 (in human Type I Ins(1,4,5)P 3 5-phosphatase sequence) identified in this study take part of a COOH-terminal sequence segment, i.e. 343 R-C-P-A-W-C-D-R-I-L 352 (active site arginines are indicated in bold), which is well conserved between sequences corresponding to inositol and phosphatidylinositol polyphosphate 5-phosphatases (Fig. 9). It would be of interest to investigate the effect of phenylglyoxal on the enzymic activity of other phosphatases involved in the dephosphorylation of inositol and phosphatidylinositol polyphosphate molecules since these enzymes may present structural similarities. These 5-phosphatases bind and hydrolyze the C-5 phospho group of   9. Aligment of human inositol and phosphatidylinositol polyphosphate 5-phosphatase amino acid sequences and location of [ 14 C]phenylglyoxal-labeled arginyl residues in human Type I Ins(1,4,5)P 3 5-phosphatase. 43 kDa 5-phosphatase refers to the amino acid sequence of human Type I Ins(1,4,5)P 3 5-phosphatase (19). 75 kDa 5-phosphatase refers to the amino acid sequence of human inositol polyphosphate 5-phosphatase (11). OCRL protein refers to the amino acid sequence of human Lowe's syndrome PtdIns(4,5)P 2 5-phosphatase gene open reading frame (15). The 10-amino acid-long sequence segment is well conserved between inositol and phosphatidylinositol polyphosphate 5-phosphatase sequences. It includes R-343 and R-350 (double underlined) which were covalently labeled with [ 14 C]phenylglyoxal. Amino acids that are conserved between the three sequences are indicated in bold. Amino acids are represented in the one-letter code.