Sulfatases, Trapping of the Sulfated Enzyme Intermediate by Substituting the Active Site Formylglycine *

Michael Recksiek, Thorsten Selmer, Thomas Dierks, Bernhard Schmidt, and Kurt von FiguraDagger

From the Institut für Biochemie und Molekulare Zellbiologie, Abt. Biochemie II, Universität Göttingen, Gosslerstr. 12d, 37073 Göttingen, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Sulfatases contain an active site formylglycine residue that is generated by post-translational modification. Crystal structures of two lysosomal sulfatases revealed significant similarity to the catalytic site of alkaline phosphatase containing a serine at the position of formylglycine. To elucidate the catalytic mechanism of sulfate ester hydrolysis, the formylglycine of arylsulfatases A and B was substituted by serine. These mutants upon incubation with substrate were covalently sulfated at the introduced serine. This sulfated enzyme intermediate was stable at pH 5. At alkaline pH it was slowly hydrolyzed. These characteristics are analogous to that of alkaline phosphatase which forms a phosphoserine intermediate that is stable at pH 5, but is hydrolyzed at alkaline pH. In wild-type sulfatases the hydroxyl needed for formation of the sulfated enzyme intermediate is provided by the aldehyde hydrate of the formylglycine. The second, non-esterified hydroxyl of the aldehyde hydrate is essential for rapid desulfation of the enzyme at acidic pH, which most likely occurs by elimination. The lack of this second hydroxyl in the serine mutants explains the trapping of the sulfated enzyme intermediate. Thus, in acting as a geminal diol the formylglycine residue allows for efficient ester hydrolysis in an acidic milieu.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Sulfatases are members of a highly conserved gene family, sharing extensive sequence homology (1, 2), a unique post-translational modification that is essential for sulfate ester cleavage (3, 4) and a high degree of structural similarity (5, 6). The post-translational modification involves the oxidation of a conserved cysteine residue at Cbeta , yielding L-Calpha -formylglycine (FGly),1 in which an aldehyde group replaces the thiomethyl group of the side chain (3). This modification occurs in the endoplasmic reticulum and is directed by a short linear sequence surrounding the cysteine to be modified (7). In multiple sulfatase deficiency, a rare human disorder, FGly formation is defective (3) leading to the synthesis of inactive sulfatases.

The crystal structure of arylsulfatase B (ASB, Ref. 5) and arylsulfatase A (ASA, Ref. 6) has been reported to 2.5 and 2.1 Å resolution, respectively. The alpha /beta fold of the two sulfatases shows a striking similarity and significant homology to that of alkaline phosphatase. The FGly residue is located in a positively charged substrate binding pocket, in which a metal ion is coordinated. The electron density at the FGly side chain in crystals of ASA was interpreted as an aldehyde hydrate (6) and in crystals of ASB as a sulfate adduct of the aldehyde (5).

The proposals made for the catalytic mechanisms of the two sulfatases differ. For ASB the reaction sequence is thought to be initiated by formation of a sulfate ester by addition of the substrate to the oxogroup of the FGly (Fig. 1). The substrate alcohol then is supposed to be released with the help of a nucleophile such as water, leaving the sulfate bound to the enzyme. This sulfate ester was found in the crystal suggesting that it is the resting form of ASB (5). For ASA the reaction cycle is proposed to start with the formation of an aldehyde hydrate, which was observed in the crystal. One of the geminal hydroxyl groups cleaves the sulfate ester by nucleophilic substitution of the substrate alcohol (transesterification) leading to the same covalent intermediate as proposed for the resting form of ASB. The second, non-esterified hydroxyl group of the covalent intermediate is proposed to induce an elimination of sulfate resulting in regeneration of the aldehyde (see Fig. 1 and Ref. 6).


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Fig. 1.   Reaction scheme for sulfate or phosphate ester cleavage. The scheme depicts the reaction sequences proposed for ASB (5), ASA (6), ASA-C69S mutant (this article), and alkaline phosphatase (AP) (8-10).

In alkaline phosphatase the active site residue is a serine. The first half-reaction of the catalytic cycle comprises the transesterification of the phosphate from the substrate onto the active site serine. In the second half-reaction the phosphoserine is hydrolyzed (8-10). Thus, the catalytic mechanism of sulfatases and alkaline phosphatase share the formation of a covalent ester intermediate.

To discriminate between the two mechanisms proposed for sulfate ester cleavage by ASA and ASB we constructed mutant proteins of ASA (ASA-C69S) and ASB (ASB-C91S), in which the active site FGly (originating from a cysteine) is replaced by a serine. This substitution leads to a sulfatase protein that can no longer be modified to the FGly containing form (7). If the catalytic cycle is initiated by the addition of the sulfate ester to the 3-oxogroup of the active site FGly as proposed for ASB, only the wild-type sulfatases but not the serine mutants should be able to cleave the sulfate ester and to form a sulfated intermediate. If on the other hand the sulfate ester reacts with an active site aldehyde hydrate as proposed for ASA, the serine mutants could be able to form a covalently sulfated intermediate (see Fig. 1) in analogy to the formation of the phosphoserine intermediate of alkaline phosphatase. Furthermore, if release of the sulfate group from the sulfated intermediate occurs by elimination requiring the second hydroxyl of the aldehyde hydrate, release of the sulfate from the intermediate of the serine mutant should be abolished. Using [35S]p-nitrocatechol sulfate (pNCS) as a substrate we could demonstrate the formation of stable sulfoserine intermediates of ASA-C69S and ASB-C91S. The sulfated intermediate could not be trapped for the wild-type sulfatases. These observations provide functional evidence for a novel reaction mechanism of an enzyme-mediated hydrolysis that resembles the mechanism of alkaline phosphatase, but depends on an aldehyde hydrate in the active site, as proposed for ASA (6).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Synthesis of [35S]pNCS-- Following the procedure of Prosser and Roy (11), 0.2 mmol of [35S]chlorosulfonic acid (Amersham) were dissolved in 250 µl of CS2 and 250 µl of N,N-dimethylaniline. 14 mg of 4-nitrocatechol (Sigma) were added and incubated with stirring for 20 h at room temperature. The mixture was chilled on ice and 250 µl of 5 M KOH was added. By centrifugation (10 min, Eppendorf centrifuge) the organic phase was separated from the aqueous phase and discarded. The reaction product was precipitated from the aqueous phase by adding 10 volumes of ethanol. After centrifugation (as above) the pellet was dissolved in 200 µl of water. The ethanol precipitation was repeated twice and the last pellet was dissolved in 900 µl of water. After addition of 100 µl of 1 M HCl the reaction product was purified by isocratic RP-HPLC on a DELTA PAK C18-300 Å column (19 × 300 mm, Waters) using 50 mM HCl, 10% methanol as a chromatography buffer (flow rate 17 ml/min). [35S]pNCS was detected by absorbance at 280 nm and liquid scintillation counting. The relevant fractions were immediately neutralized with KOH, lyophilized, and dissolved in 300 mM ammonium acetate (pH 5.0). The reaction product was characterized and quantitated spectrophotometrically (epsilon 396 = 18,900 cm2/mmol). The stock solution was 8.5 mM with a specific radioactivity of 294 mCi/mmol. The purity of [35S]pNCS was controlled by thin-layer chromatography (see below; Ref. 21) using unlabeled pNCS as a standard. [35S]pNCS was quantitatively hydrolyzed upon incubation with wild-type ASA (not shown).

Site-directed Mutagenesis, Protein Expression, and Purification-- The cDNAs coding for human ASA (12) or ASB (13) were cloned as KpnI/EcoRI-(ASA) or BamHI/EcoRI fragments (ASB) into the pMPSVHE vector (14) downstream of the myeloproliferative sarcoma virus promoter. The cysteine codons 69 (ASA) or 91 (ASB), respectively, were mutated into serine codons by polymerase chain reaction methods using non-coding mutagenesis primers (ACCGGGAGCCGGCCGGTCAGGAGGGCGGCCCTAGAGGGTGTGCTCAGAGACACAG (ASA) or CTGGTAGCGGCCAGTGAGCAGCTG GCTCCGCGACGGCGTGCTCAGCGG-CTGC (ASB)), comprising an EagI (ASA) or XcmI site (ASB). The polymerase chain reaction products were subcloned as KpnI/EagI (ASA) or BamHI/XcmI fragments (ASB) replacing the corresponding fragments of the template DNAs, which in the case of XcmI had to be digested only partially.

The resulting plasmids carrying wild-type or mutated cDNAs and pGK-hygro as selection marker were used for stable transfection of mouse embryonic fibroblasts deficient in both mannose 6-phosphate receptors, as described (15). The expressed proteins were purified from the secretions of the cells by affinity chromatography (16, 17).

Incorporation of [35S]Sulfate into Sulfatase Proteins-- Unless otherwise indicated, 1.5-5 µg of wild-type ASA or ASA-C69S in 2 µl of TBS (10 mM Tris, 150 mM NaCl, pH 7.4) were incubated with 2 µl of the [35S]pNCS stock solution in 0.3 M ammonium acetate (pH 5.0) (see above) for 3 min at 37 °C. In the case of ASB, 3 µg of protein in 3 µl of TBS and 3 µl of [35S]pNCS were used. The final pH was 5.0. The incubation was stopped by adding 26 µl of electrophoresis sample buffer (18) preheated to 95 °C and further incubation for 5 min at 95 °C. After SDS-PAGE on a high-Tris gel containing 17.5% acrylamide, 0.23% bis-acrylamide (19) the gel was stained with Coomassie Blue, dried, and exposed on a PhosphorImaging plate. 35S-Labeled sulfation of proteins was quantitated on a PhosphorImager (Fujix BAS1000) using the MacBAS software. When investigating the kinetics of 35S-labeled sulfation, the parameters of time, temperature, pH, and substrate concentration were varied, as described in the figure legends. To vary the pH, aliquots of the [35S]pNCS stock solution were lyophilized thoroughly to remove ammonium acetate, and redissolved in a 100 mM ammonium acetate, 50 mM Tris buffer that was appropriate to adjust the pH of the incubation mixture to those values given in Fig. 4.

Peptide Analysis-- 50 µg of ASA-C69S (or 30 µg of ASB-C91S) in 7 µl (24 µl) of TBS were incubated with 7 µl (24 µl) of the [35S]pNCS stock solution for 3 min at 37 °C. Incubation was stopped by adding trichloroacetic acid (final concentration 20%). The precipitated protein was washed with 5% trichloroacetic acid and lyophilized thoroughly. The protein was dissolved in 4 M guanidine hydrochloride, 400 mM Tris, 10 mM EDTA (pH 8.6) and subjected to reductive carboxymethylation and tryptic digestion as described (3). Separation of tryptic peptides by RP-HPLC, digestion with endoproteinase Asp-N, mass spectrometry, and sequencing/radiosequencing of peptides on a 477A sequencer (Applied Biosystems) also was described earlier (3). Mass spectrometry of sulfated peptides was facilitated when using the negative polarity mode.

Desulfation of 35S-Labeled Sulfated Sulfatases-- After incubation with [35S]pNCS (see above), the sulfatase (40 µg) was separated from its substrate by gel filtration on a Fast Desalting column (PC 3.2/10, Pharmacia) equilibrated with 50 mM ammonium acetate (pH 5.0). Aliquots of 2 µg of [35S]sulfatase then were incubated for 16 h at 37 °C in the absence or presence of various acceptor compounds (see Table II) dissolved in gel filtration buffer. The incubation was stopped by trichloroacetic acid precipitation (see above). One aliquot was precipitated directly after gel filtration. The protein pellet was dissolved in electrophoresis sample buffer. After SDS-PAGE the gel was dried and exposed overnight on a PhosphorImaging plate (see above). When investigating the kinetics of galactose-induced desulfation, the parameters of time, temperature, pH, and galactose concentration were varied, as described in the legend to the figures. To adjust the pH of the incubation mixture the sulfated protein during gel filtration was transferred into a 50 mM ammonium acetate, 50 mM Tris buffer of appropriate pH, which also was used to dissolve the galactose.

Analysis of Enzyme-generated Galactose [35S]Sulfate-- After incubation of 25 µg of ASA-C69S with [35S]pNCS and gel filtration (see above) a 4.5-µg aliquot of the protein was precipitated by trichloroacetic acid without further incubation and was analyzed by SDS-PAGE. 16 µg of the protein were incubated for 2 h at 37 °C with 50 mM galactose, another 4.5-µg aliquot with buffer alone. The incubation mixtures and, in addition, standards for [35S]pNCS, [35S]sulfate, galactose, and galactose 6-sulfate were analyzed by high-voltage paper electrophoresis on Whatman 3MM paper at 65 V/cm for 50 min using a buffer containing 80 mM pyridine-HAc (pH 5.5). After electrophoresis the dried paper was analyzed for radioactivity by radiodensitometry. Galactose and galactose 6-sulfate were detected by reaction with aniline phthalate (20).

A good separation of [35S]pNCS and galactose [35S]sulfate was achieved by HPLC. The corresponding 35S-labeled spots were excised from the electrophoresis paper and extracted with water. The extracts were lyophilized, redissolved in 20 µl of water, and analyzed by RP-HPLC on a C18 column (ODS DABS, 4.6 × 250 mm, Beckman) running a 0-84% acetonitrile gradient in 0.1% trifluoroacetic acid.

Enzyme-generated monosaccharide [35S]sulfates, furthermore, were analyzed by thin-layer chromatography. After gel filtration, 14 µg of sulfated ASA-C69S protein (see above) was incubated with either 50 mM galactose or 50 mM N-acetylgalactosamine for 2 h at 37 °C. The samples then were subjected to a second gel filtration to remove the ASA-C69S protein. The salt fraction was lyophilized thoroughly and dissolved in 10 µl of 50 mM ammonium acetate (pH 5). The galactose [35S]sulfate and the N-acetylgalactosamine [35S]sulfate containing samples then were treated with 50 ng of wild-type ASA or wild-type ASB, respectively. After incubation for 3 h at 37 °C the samples were loaded on a thin-layer plate (Silica Gel 60, Merck) together with two standards (1700 dpm of [35S]sulfate and 8 µg of galactose 6-sulfate). Methanol, 1-propanol, ammonia, water (55/50/5/10) served as the mobile phase (21). The plate was dried and exposed on a PhosphorImaging plate overnight. Afterward the galactose 6-sulfate standard was visualized by reaction with aniline phthalate (20). As a control, [35S]pNCS, which like the galactose [35S]sulfates, was subjected to gel filtration and lyophilization, was incubated with ASA or ASB, leading to quantitative formation of [35S]sulfate (not shown).

Continuous Trans-sulfation-- Two aliquots of ASA-C69S protein (28 µg each) were incubated at 37 °C with 5 mM pNCS in 330 µl of 50 mM Tris-HCl (pH 9). One of the samples in addition contained 500 mM galactose. After 3, 6.5, and 9 h, a 100-µl sample was withdrawn and stopped by adding 100 µl of 1 M NaOH. pNC formation was assayed spectrophotometrically at 515 nm.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Formation of a Sulfated Form of ASA-C69S-- To detect the formation of a covalently sulfated intermediate of sulfatases, [35S]pNCS with a specific activity of 294 Ci/mol was prepared and incubated with either wild-type ASA or the ASA-C69S mutant in which FGly 69 was replaced by serine (see Introduction). Substitution of FGly 69 by serine is associated with a loss of catalytic activity. While wild-type ASA cleaves pNCS with a Vmax of 60-80 units/mg, the Vmax of ASA-C69S is reduced to 0.1 units/mg (data not shown). This residual activity is suspected to result from minute amounts of contaminating wild-type ASA (less than 0.2% of total protein) rather than from an intrinsic residual activity. The contamination is attributed to the fact that the same immunoaffinity column had to be used for purification of the mutant and the wild-type form of ASA. In accordance with this interpretation is the observation that the Km for pNCS was 4-7 mM for both the wild-type and mutant enzyme.

After incubating ASA and ASA-C69S for 3 min at 37 °C with [35S]pNCS, the samples were boiled in the presence of SDS, subjected to SDS-PAGE, and analyzed by PhosphorImaging and Coomassie staining (Fig. 2). Incorporation of radioactivity was observed for the ASA-C69S mutant, but not for wild-type ASA. Incubation with sodium [35S]sulfate did not result in sulfation of ASA-C69S (not shown). To localize the radioactivity in [35S]ASA-C69S, the labeled mutant was subjected to tryptic digestion and the peptides were separated by RP-HPLC. A single peptide was found to be labeled (Fig. 3A). The mass of this peptide was 1796 Da (Fig. 3B), which corresponds to the sulfated form of tryptic peptide 2 comprising residues 59-73 of ASA-C69S. To determine the location of the [35S]sulfate group, the sulfated peptide 2 was subjected to radiosequencing. The radioactivity was released in the sequencing cycle 11 (Fig. 3C), which corresponds to serine 69. These data demonstrate that incubation of ASA-C69S with pNCS leads to the formation of a protein that is covalently sulfated at residue 69. 


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Fig. 2.   Sulfation of ASA-C69S in the presence of [35S]pNCS. 3 µg of ASA and ASA-C69S mutant were incubated for 3 min at 37 °C in the presence of [35S]pNCS. The samples then were boiled in the presence of SDS and subjected to SDS-PAGE. The upper part shows the Coomassie staining and the lower part the PhosphorImaging of the gel.


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Fig. 3.   Identification of the sulfated amino acid residue in ASA-C69S. After incubation with [35S]pNCS, ASA-C69S was precipitated with trichloroacetic acid and subjected to reductive carboxymethylation and tryptic digestion. The tryptic peptides were separated by RP-HPLC using a gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid. A, elution profile of the tryptic peptides and of the radioactivity (histogram). B, mass spectrogram of fraction 31 (retention time 35.6 min) containing the bulk of the radioactivity. The mass of 1796 Da corresponds to that predicted for a sulfate ester of the tryptic peptide 2, comprising residues 59-73 of ASA-C69S. C, radiosequencing of the 1796-Da peptide eluting in fraction 31. The radioactivity released in sequencing cycle 11 corresponds to serine 69 of ASA-C69S. The sequence of peptide 2, which was verified by amino acid sequencing of the first 6 residues, is given.

Kinetics of [35S]ASA-C69S Formation-- When ASA-C69S was incubated with [35S]pNCS at 37 °C for 3-30 min, formation of [35S]ASA-C69S had almost reached its maximum level after 3 min. Based on the specific radioactivity of the substrate, about 25% of ASA-C69S was sulfated. This was confirmed by analysis of the tryptic peptides. The non-sulfated and sulfated forms of peptide 2 were separated by RP-HPLC. Amino acid sequencing of the separated peptides revealed a 1:3 ratio for the sulfated and non-sulfated form of peptide 2 (not shown).

When the formation of [35S]ASA-C69S was followed at temperatures ranging from 0 to 37 °C, a linear relation between the ln of [35S]ASA-C69S and 1/T was observed (Fig. 4A). From the slope of the Arrhenius plot an activation energy of 47 kJ/mol was calculated that is compatible with an enzymatically catalyzed formation of [35S]ASA-C69S. At 0 °C half-maximal formation of [35S]ASA-C69S was observed after 20 min (Fig. 4B). The Km for pNCS for ASA-C69S sulfation was 6 mM (Fig. 4C), which is similar to the Km of 4 mM observed for cleavage of pNCS by wild-type ASA. The pH dependence showed a broad activity profile with an optimum at pH 5-6 (Fig. 4D). Also the wild-type showed maximal hydrolytic activity at similar pH values (pH 4.5-5). In contrast to cleavage of pNCS by wild-type ASA, however, a considerable incorporation of sulfate into the ASA-C69S was observed even in the neutral and alkaline range up to pH 9 (Fig. 4D).


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Fig. 4.   Dependence of [35S]ASA-C69S formation on temperature, time, substrate concentration and pH. ASA-C69S was incubated in the presence of [35S]pNCS under variation of one parameter of the standard assay conditions (0 °C, 3 min incubation, 1.6 mM pNCS, pH 5.0), as indicated. After incubation the samples were boiled in the presence of SDS and analyzed by SDS-PAGE and PhosphorImaging, as is shown for A and B. By two-dimensional quantification of the PhosphorImages arbitrary units for [35S]ASA-C69S were obtained. All values represent the mean of duplicates. The variation from the mean is indicated. A, the temperature dependence of [35S]ASA-C69S formation is shown in an Arrhenius plot from which an activation energy of 47 kJ/mol was calculated. B, time dependence of [35S]ASA-C69S formation. C, the dependence of [35S]ASA-C69S formation on [35S]pNCS concentration is shown in a Lineweaver-Burk plot allowing the extrapolation of a Km of 6 mM. D, pH dependence of [35S]ASA-C69S formation (circles). For comparison, the pH dependence of pNCS hydrolysis by wild-type ASA is shown (squares).

Cleavage of pNCS by ASA is inhibited by sulfite, sulfate, and phosphate, but not by nitrate (22-24). Formation of [35S]ASA-C69S showed the same sensitivity toward these anions and was inhibited by 60-80% in the presence of 10 mM sulfite, sulfate, or phosphate (all sodium salts), but not by 10 mM potassium nitrate (Table I).

                              
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Table I
Inhibition of [35S]ASA-C69S formation
ASA-C69S was incubated under standard conditions (see Fig. 4) with [35S]pNCS in the presence of the indicated inhibitor. After separation by SDS-PAGE, [35S]ASA-C69S was quantitated, as described in Fig. 4. The values are given as percentage of [35S]ASA-C69S formed in the absence of inhibitor.

Desulfation of [35S]ASA-C69S-- [35S]ASA-C69S was separated from [35S]pNCS by gel filtration and then incubated at pH 5 and 37 °C for up to 5 days. The radioactivity remained stably associated with the sulfatase polypeptide (shown for a 16-h incubation period in Figs. 5 and 6). Thus, under these conditions the ASA-C69S is completely inactive and therefore cannot account for the residual hydrolytic activity ascribed to contaminating wild-type ASA (see above). At higher pH values a slow desulfation of [35S]ASA-C69S was noted (Fig. 5), which led to desulfation of 30-40% of [35S]ASA-C69S during an incubation for 16 h at 37 °C and pH 9. 


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Fig. 5.   Hydrolysis of [35S]ASA-C69S at alkaline pH. After incubation with [35S]pNCS, [35S]ASA-C69S was separated from the low molecular weight labeled material by gel filtration using 0.05 M ammonium acetate (pH 5) as a chromatography buffer. The pH then was adjusted by addition of 5 volumes of 0.5 M ammonium acetate (pH 5), 0.5 M sodium phosphate (pH 7), or 0.5 M ammonium acetate (pH 9, 9.5, or 10). After incubating the samples for 16 h at 37 °C the protein was precipitated with trichloroacetic acid and analyzed by SDS-PAGE, Coomassie staining, and PhosphorImaging. [35S]ASA-C69S was quantified as described in the legend to Fig. 4. The values obtained were corrected for losses of protein, which to some extent occurred during precipitation, and which were quantitated by scanning of the Coomassie-stained gel.

Desulfation was greatly accelerated by supplementing the incubation medium with galactose. In the presence of 30 mM galactose, complete desulfation of ASA-C69S was observed after an incubation for 16 h at 37 °C and pH 5.0 (Fig. 6). The time course of the desulfation revealed that 50% desulfation was accomplished within 30 min (Fig. 7A). The galactose-induced desulfation reaction exhibited saturation kinetics, as is shown in a Lineweaver-Burk plot (Fig. 7B). The Km for galactose was 38 mM. The temperature dependence revealed an activation energy of 36 kJ/mol as was calculated from an Arrhenius plot (Fig. 7C). Desulfation in the presence of galactose was fastest between pH 8 and 9 (Fig. 7D). The reaction could not be inhibited by sulfate, sulfite, or phosphate (not shown).


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Fig. 6.   Desulfation of [35S]ASA-C69S. After gel filtration (see Fig. 5) [35S]ASA-C69S was incubated for 16 h at 37 °C in the absence or presence of 30 mM galactose. The protein was precipitated with trichloroacetic acid and analyzed by SDS-PAGE, Coomassie staining, and PhosphorImaging, as indicated. As a control, [35S]ASA-C69S was precipitated directly after gel filtration (no incubation).


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Fig. 7.   Dependence of [35S]ASA-C69S desulfation on time, galactose concentration, temperature, and pH. After gel filtration (see Fig. 5) [35S]ASA-C69S was incubated in the presence of galactose under variation of one parameter of the standard assay conditions (25 min, 30 mM galactose, 37 °C, pH 5.0), as indicated. [35S]ASA-C69S was quantitated as in Fig. 5. For A and B, the phosphoimage analysis of [35S]ASA-C69S is shown. The values of desulfated ASA-C69S given in B-D represent the percentage of [35S]ASA-C69S that was desulfated during the assay. All values represent the mean of duplicate samples. The variation from the mean is indicated. A, time dependence of [35S]ASA-C69S desulfation. B, the dependence of [35S]ASA-C69S desulfation on galactose concentration is shown in a Lineweaver-Burk plot allowing the extrapolation of a Km of 38 mM. C, the temperature dependence of [35S]ASA-C69S desulfation is shown in an Arrhenius plot from which an activation energy of 36 kJ/mol was calculated. D, pH dependence of [35S]ASA-C69S desulfation during an incubation of 4 min in the presence of 10 mM galactose.

Desulfation was induced also by other carbohydrates and non-carbohydrate nucleophiles (Table II). At 3 mM concentration and pH 5.0 galactose and mannose were the most effective compounds, followed by fructose and glucose. Intermediate desulfation efficiencies were observed in the presence of ethanolamine, ascorbate, p-nitrocatechol, fucose, o-nitrophenol, and N-acetylgalactosamine, which at 30 mM concentrations led to desulfation of 30-90% of [35S]ASA-C69S. Saccharose, inositol, p-nitrophenol, 4-methylumbelliferone, ethanol, ascorbate 2-sulfate, galactose 1- or 6-phosphate, and galactose 6-sulfate at 30 or 50 mM concentrations did not induce desulfation (Table II).

                              
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Table II
Desulfation of [35S]ASA-C69S in the presence of various nucleophilic compounds
After gel filtration (see Fig. 5) [35S]ASA-C69S was incubated for 16 h at 37 °C and pH 5 in the presence of 3, 30, or 50 mM concentrations of the indicated compounds. After separation by SDS-PAGE, [35S]ASA-C69S was quantitated, as described in Fig. 5. The values are given as percentage of [35S]ASA-C69S incubated with buffer alone.

We next examined whether desulfation of [35S]ASA-C69S in the presence of galactose is linked to the formation of a sulfated galactose. About 40% of the radioactivity present in the [35S]ASA-C69S preparation obtained after gel filtration was free [35S]SO42-, as became evident after high-voltage electrophoresis of [35S]ASA-C69S (Fig. 8A, left lane). The presence of [35S]sulfate is ascribed to the cleavage of [35S]pNCS by traces of wild-type ASA contaminating our ASA-C69S preparation (see above) and the incomplete removal of the released [35S]sulfate during gel filtration. When the [35S]ASA-C69S preparation was incubated in the presence of galactose and subjected to high-voltage electrophoresis, all radioactivity initially associated with the protein migrated with a mobility comparable to that of pNCS and galactose 6-sulfate standards (Fig. 8A, right lane). This material was eluted from the paper and subjected to RP-HPLC under conditions that resolve pNCS and galactose 6-sulfate. All radioactivity coeluted with the galactose 6-sulfate standard (Fig. 8B).


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Fig. 8.   Formation of [35S]galactose sulfate. [35S]ASA-C69S was incubated in the presence or absence of 50 mM galactose for 2 h at 37 °C and pH 5. A, the incubation mixtures were subjected to high-voltage paper electrophoresis followed by radiodensitometry. The migration of standards of [35S]sulfate, [35S]pNCS, galactose 6-sulfate (Gal6SO4), and [35S]ASA-C69S is indicated. Galactose 6-sulfate was detected by reaction with aniline phthalate. B, the labeled material comigrating in high-voltage electrophoresis with pNCS and galactose 6-sulfate (A, right lane) was extracted from the paper and subjected to RP-HPLC (see "Experimental Procedures") using an acetonitrile gradient of 0-84% (dashed line). The fractions were analyzed for radioactivity (histogram). The elution of galactose 6-sulfate and pNCS standards is indicated.

In a separate experiment the low molecular weight 35S-labeled components were subjected to thin layer chromatography under conditions that separate galactose 6-sulfate from sulfate and pNCS (see "Experimental Procedures"). Again about 60% of the radioactive material comigrated with the galactose 6-sulfate standard (not shown). Treatment of the low molecular weight 35S-labeled material with wild-type ASA prior to thin layer chromatography indicated that the galactose [35S]sulfate is resistant to hydrolysis by ASA (not shown). In similar experiments with N-acetylgalactosamine as acceptor, we could demonstrate formation of N-acetylgalactosamine [35S]sulfate concomitant to desulfation of [35S]ASA-C69S. The N-acetylgalactosamine [35S]sulfate was resistant to hydrolysis by wild-type ASB (not shown). These results clearly indicate that ASA-C69S has a sulfotransferase activity transferring sulfate from pNCS via a sulfated enzyme intermediate onto acceptor compounds such as galactose or N-acetylgalactosamine. The corresponding sulfate derivatives of these compounds generated in the trans-sulfation reaction are no substrates for ASA or ASB.

Cleavage of pNCS by ASA-C69S in the Presence of Galactose (Continuous Trans-sulfation)-- The experiments shown so far demonstrate that ASA-C69S reacts with pNCS to sulfated ASA-C69S and p-nitrocatechol. In a reaction that occurs much slower than the formation of sulfated ASA-C69S, the sulfate group can be transferred from the protein to galactose. Addition of galactose to a mixture of ASA-C69S and pNCS should therefore result in the continuous formation of p-nitrocatechol and galactose sulfate (according to the scheme shown in Fig. 9). In an experiment testing this assumption, the pH was raised to 9.0, where the rate-limiting desulfation reaction has its optimum. When ASA-C69S was incubated for up to 9 h at pH 9 and 37 °C in the presence of 5 mM pNCS but without galactose, 1.38 mol of p-nitrocatechol/h were formed per mol of enzyme. The presence of 500 mM galactose stimulated the production of p-nitrocatechol formation 4-fold (Fig. 9). The basal formation of p-nitrocatechol (in the absence of galactose) can be accounted for by the contaminating wild-type ASA, which is strongly inhibited at pH 9 (0.2% residual activity when compared with pH 5). The galactose-induced formation of p-nitrocatechol indicates, that under the conditions tested, each ASA-C69S monomer catalyzes the cleavage of at least 4 molecules of pNCS/h. The cleavage rate is probably four times higher, since only one-fourth of the ASA-C69S preparation was active (see above).


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Fig. 9.   Cleavage of pNCS by ASA-C69S in the presence of galactose. ASA-C69S was incubated with 5 mM pNCS for 0-9 h at 37 °C and pH 9 in the absence (open circles) or presence of 0.5 M galactose (filled circles). Formation of p-nitrocatechol (pNC) was followed spectrophotometrically and is given as mole of pNC/mol of ASA-C69S.

Sulfation of ASB and ASB-C91S-- Similar experiments as with wild-type ASA and the ASA-C69S mutant were performed with wild-type ASB and a ASB-C91S mutant, in which FGly residue 91, the homologue of FGly 69 in ASA, was substituted by serine. This substitution is known to destroy the ASB activity (25). When wild-type ASB or ASB-C91S were incubated with [35S]pNCS, only ASB-C91S incorporated radioactivity (Fig. 10). [35S]ASB-C91S was subjected to tryptic digestion and RP-HPLC of its tryptic peptides. Mass spectrometry of the radioactive fractions revealed a mass of 2951 Da, which is predicted for the sulfated form of tryptic peptide 3 of ASB. Peptide 3 contains residues 69-95 of ASB and upon digestion with endoproteinase Asp-N was converted into the 13-mer peptide 3C (residues 83-95, see Ref. 3). After purification of this peptide by RP-HPLC all radioactivity was associated with a 1622-Da peptide, which is the mass predicted for the sulfated form of peptide 3C. Radiosequencing of this peptide released the radioactivity in cycle 9 which corresponds to residue 91 of ASB (data not shown). This clearly demonstrates that incubation with pNCS results in sulfation of serine 91 in ASB-C91S similar to the sulfation of the active site serine in ASA-C69S.


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Fig. 10.   Sulfation of ASB-C91S in the presence of [35S]pNCS. 3 µg of wild-type ASB, ASB-C91S, and ASA-C69S were incubated in the presence of 4.25 mM [35S]pNCS for 3 min at 37 °C and pH 5. The samples then were boiled in the presence of SDS and subjected to SDS-PAGE. The gel was analyzed by staining with Coomassie and PhosphorImaging, as indicated.

Incorporation of sulfate into ASB-C91S was almost quantitative, i.e. 3-4 times more efficient than observed for ASA-C69S (Fig. 10). This was also confirmed by amino acid sequencing of sulfated and non-sulfated peptide 3C obtained after digestion of [35S]ASB-C91S with trypsin and endoproteinase Asp-N. The molar ratio of sulfated and non-sulfated peptide 3C was 9:1 (not shown). In contrast to [35S]ASA-C69S, the sulfated form of ASB-C91S was fully stable during an incubation for 16 h at 37 °C and pH 5 or 9 and also in the presence of 30 mM galactose or N-acetylgalactosamine (not shown).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The active sites of sulfatases and alkaline phosphatase share striking structural homology (5, 6). The active site of ASA carries a Mg2+ ion and the aldehyde function of FGly 69, where in alkaline phosphatase a Zn2+ and the hydroxyl of serine 102 are found, which are involved in catalysis. The key feature of the proposed mechanism for sulfate ester cleavage by ASA is the participation of FGly 69 as an aldehyde hydrate. The electron density around the FGly side chain in ASA is in agreement with an aldehyde hydrate with one of the two geminal hydroxyls coordinating the Mg2+ ion (6). The geminal hydroxyl groups of the aldehyde hydrate are supposed to serve functions which in alkaline phosphatase are carried out by the hydroxyl of serine 102 and an activated water molecule. Thus, one aldehyde hydrate hydroxyl would attack the sulfur of the substrate to form a covalently sulfated enzyme intermediate in a transesterification step analogous to that observed in alkaline phosphatase, where serine 102 becomes phosphorylated (Fig. 1). While hydrolysis of the phosphorylated alkaline phosphatase intermediate occurs with the help of a water molecule activated by a Zn2+ cation (8-10), the sulfate is released from ASA by elimination. This elimination requires the second hydroxyl of the aldehyde hydrate (for details, see Ref. 6).

If the proposed similarity between the mechanisms of sulfate and phosphate ester cleavage holds true, we reasoned that substitution in sulfatases of the active site FGly residue by serine would generate sulfatases that are still capable of catalyzing the initial transesterification step which yields the covalently sulfated enzyme intermediate. Due to the missing second hydroxyl group the catalytic cycle would be arrested, unless a water molecule could be activated to hydrolyze the sulfated enzyme intermediate.

Sulfate Ester Cleavage Is Initiated by Transesterification of the Sulfate from the Substrate onto an Active Site Hydroxyl-- When ASA-C69S and ASB-C91S were incubated in the presence of pNCS a sulfated derivative was formed, in which the sulfate was covalently attached to the serine residue in the active site. The reaction was characterized in some detail for ASA-C69S and found to require an activation energy typical of enzymatically catalyzed reactions. Furthermore, the pH optimum, the dependence on substrate concentration, and the sensitivity to inhibitors was similar to that known for cleavage of pNCS by wild-type ASA. In contrast to ASB-C91S, where essentially all sulfatase polypeptides became sulfated, only about 25% of the ASA-C69S polypeptides were sulfated. This may be due to a higher affinity of ASB-C91S for pNCS as compared with ASA-C69S, since the pNCS concentration used in these experiments was below the Km of ASA-C69S. In addition, a partial denaturation of the enzyme during purification, which includes desorption at pH 2.8 from the affinity matrix, cannot be avoided. Purified wild-type ASA rapidly inactivates at pH 2.4.2

The observation that the active site serine mutants of ASA and ASB make one catalytic half-cycle, thereby cleaving pNCS under formation of sulfoserine containing derivatives in analogy to the phosphoserine containing intermediate of alkaline phosphatase, strongly suggests that also wild-type sulfatases initiate sulfate ester cleavage by a transesterification step (Fig. 1). This transesterification requires a nucleophilic hydroxyl at the catalytic residue, which in sulfatases can only be generated by hydratation of the FGly residue. If formation of the sulfated intermediate would be initiated by addition of the sulfate ester to the oxogroup as proposed for ASB (Ref. 5, see Fig. 1), substitution of the FGly by serine should abolish the reaction of sulfatases with pNCS. Therefore, the reaction of pNCS with ASA-C69S and ASB-C91S strongly supports the reaction scheme outlined in Fig. 1 for ASA. Sulfated intermediates could not be detected for wild-type ASA and wild-type ASB. We ascribe this to the fast release of the sulfate group from the intermediate.

Release of Sulfate from the Enzyme Intermediate by Elimination or Hydrolysis-- At pH 5, where cleavage of pNCS by wild-type ASA or ASB as well as the transesterification of pNCS by ASA-C69S and ASB-C91S is optimal, the sulfated forms of ASA-C69S and ASB-C91S are stable. The failure of the serine mutants to release the sulfate and thereby completing the catalytic cycle is ascribed to the absence of a second, non-esterified hydroxyl group at the Cbeta atom of the sulfated residue. In the sulfated intermediate of wild-type ASA and ASB this hydroxyl is present due to hydratation of the FGly residue. This hydroxyl polarizes the Cbeta -O bond of the sulfate ester intermediate which leads to the elimination of sulfate by cleavage of this bond. The elimination regenerates concomitantly the aldehyde group (shown in Fig. 1 for ASA).

As observed for the sulfated intermediates of ASA-C69S and ASB-C91S, also the phosphoserine intermediate of alkaline phosphatase is stable at pH 5 (9). At elevated pH this phosphoserine ester is hydrolyzed by the nucleophilic attack of a water molecule, which is coordinated and activated by a Zn2+ cation and which cleaves a P-O bond (8-10). A slow hydrolytic release of the sulfate group from sulfated ASA-C69S could also be observed at alkaline pH. At pH 9, 30-40% of [35S]ASA-C69S lost its sulfate group during an incubation for 16 h at 37 °C.

In the presence of a variety of monosaccharides including galactose or of other nucleophiles such as ethanolamine or p-nitrocatechol, [35S]ASA-C69S was desulfated also at pH 5. The desulfation was characterized in more detail for galactose, which among the compounds tested was the most potent inducer of desulfation. Desulfation in the presence of galactose was shown to occur via transesterification. Thus, the ASA-C69S has a sulfotransferase activity involving two transesterification steps that lead to sulfation and desulfation of the protein. The pH optimum for the second transesterification leading to desulfation was in the alkaline range similar to the pH dependence of the transphosphorylation catalyzed by alkaline phosphatase (26, 27). The affinity of ASA-C69S for galactose is low (Km 38 mM). The position which is sulfated could not be demonstrated directly. The C-3 hydroxyl of galactose and the C-4 hydroxyl of N-acetylgalactosamine can be excluded, as the sulfated products were resistant to ASA, which cleaves galactose 3-sulfate (28), and to ASB, which cleaves N-acetylgalactosamine 4-sulfate (29). Since N-acetylgalactosamine has no C-2 hydroxyl, and fucose, which also induced desulfation (Table II), lacks the C-6 hydroxyl, the C-1 hydroxyl is the most likely candidate for accepting the sulfate group. This hydroxyl has the highest nucleophily among the carbohydrate hydroxyls.

Although the present data demonstrate that the sulfate can be released by hydrolysis and that certain acceptors can accelerate this desulfation, it should be noted that the rate is too low to allow adequate catalysis of sulfate ester cleavage. One ASA-C69S monomer in the presence of saturating amounts of galactose would cleave less than 20 pNCS molecules per h.

Conclusions-- Phosphate ester cleavage by alkaline phosphatase, which carries a catalytic serine residue, and sulfate ester cleavage by sulfatases, in which the active site FGly residue was substituted by serine, share the initial transesterification step resulting in a protein-sulfate (ASA and ASB) or protein-phosphate (alkaline phosphatase) ester intermediate. The hydroxyl group needed for transesterification in wild-type sulfatases is provided by the FGly hydrate. The major difference between sulfatases and alkaline phosphatase resides in the cleavage of the enzyme-ester intermediate. In alkaline phosphatase this occurs by hydrolysis (nucleophilic attack by an activated water molecule). The sulfated intermediates of sulfatases apparently are unable to activate a water molecule. Efficient release of sulfate occurs by an elimination reaction that is induced by the presence of a hydroxyl in the sulfated enzyme intermediate. Also this hydroxyl originates from the water molecule that is added to the aldehyde function of FGly. Thus, the post-translational generation of this residue that is unique to the active site of sulfatases allows the enzyme to engage a water molecule by aldehyde hydrate formation before undergoing covalent interactions with the substrate. The hydratation of the aldehyde function generates the hydroxyl needed for the initial transesterification step and, in addition, the hydroxyl allowing the elimination of sulfate from the enzyme in the second half-reaction.

    ACKNOWLEDGEMENTS

We thank C. Peters and M. Pauly-Evers (Freiburg) for providing the affinity column for purification of ASB.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.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.

Dagger To whom correspondence should be addressed. Tel.: 49-551-395948; Fax: 49-551-395979; E-mail: kfigura@ukb2-00.uni-bc.gwdg.de.

1 The abbreviations used are: FGly, Calpha -formylglycine; ASA, arylsulfatase A (cerebroside-3-sulfate 3-sulfohydrolase, EC 3.1.6.8); ASB, arylsulfatase B (N-acetylgalactosamine-4-sulfate 4-sulfohydrolase, EC 3.1.6.9); pNCS, p-nitrocatechol sulfate; RP-HPLC, reversed-phase high-performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

2 T. Selmer, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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