From the Institut für Biochemie und Molekulare Zellbiologie,
Abt. Biochemie II, Universität Göttingen, Gosslerstr. 12d,
37073 Göttingen, Germany
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.
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INTRODUCTION |
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
C
, yielding L-C
-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
/
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).
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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).
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EXPERIMENTAL PROCEDURES |
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
(
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.
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RESULTS |
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.
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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).
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DISCUSSION |
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 C
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
C
-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.
We thank C. Peters and M. Pauly-Evers
(Freiburg) for providing the affinity column for purification of
ASB.