From the Zentrum für Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, Gosslerstrasse 12d, 37073 Göttingen, Germany
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ABSTRACT |
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Arylsulfatase A belongs to the sulfatase family
whose members carry a C Mammalian sulfatases are involved in the degradation of sulfated
substrates like mucopolysaccharides, cerebroside sulfates, and sulfated
steroids. The key residue for catalytic activity in sulfate ester
cleavage has recently been shown to be a C These data led to a proposal for the catalytic mechanism of sulfate
ester cleavage (6) as follows (Fig. 1).
In the first half-cycle, one of the two geminal oxygens of the aldehyde
hydrate attacks the sulfur of the sulfate ester leading to a
transesterification of the sulfate group onto the aldehyde hydrate.
Simultaneously the substrate alcohol is released. In the second
half-cycle, sulfate is eliminated from the enzyme-sulfate intermediate
by an intramolecular rearrangement induced by the second oxygen. By
this "intramolecular hydrolysis" the aldehyde group is regenerated.
The essential role of FGly in forming the transient enzyme-sulfate
ester has been shown indirectly (8). Mutants of ASA and ASB were
expressed in which the FGly had been replaced by a serine. These
mutants were able to cleave sulfate ester and to generate a sulfated
enzyme intermediate with a serine sulfate ester in position 69 (ASA) or
91 (ASB). However, due to the lack of a second hydroxyl group, the
release of the sulfate group regenerating the aldehyde did not
occur.
-formylglycine that is post-translationally
generated by oxidation of a conserved cysteine or serine residue. The
formylglycine acts as an aldehyde hydrate with two geminal hydroxyls
being involved in catalysis of sulfate ester cleavage. In arylsulfatase
A and N-acetylgalactosamine 4-sulfatase this formylglycine
was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved
among the eukaryotic and prokaryotic members of the sulfatase family.
To analyze their function in binding and cleaving sulfate esters, we
substituted a total of nine putative active site residues of human ASA
by alanine (Asp29, Asp30, Asp281,
Asn282, His125, His229,
Lys123, Lys302, and Ser150). In
addition the Mg2+-complexing residues (Asp29,
Asp30, Asp281, and Asn282) were
substituted conservatively by either asparagine or aspartate. In all
mutants Vmax was decreased to 1-26% of wild
type activity. The Km was more than 10-fold
increased in K123A and K302A and up to 5-fold in the other mutants. In
all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units.
These results indicate that each of the nine residues examined is
critical for catalytic activity, Lys123 and
Lys302 by binding the substrate and the others by direct
(His125 and Asp281) or indirect participation
in catalysis. The shift in the pH optimum is explained by two
deprotonation steps that have been proposed for sulfate ester cleavage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-formylglycine (FGly;
Refs. 1 and 2),1 which is
post-translationally generated from a cysteine in the endoplasmic
reticulum (3). Failure of this amino acid modification leaves newly
synthesized sulfatases inactive and is the cause of multiple sulfatase
deficiency, a rare but fatal lysosomal storage disorder (Ref. 1;
reviewed in Refs. 4 and 5). X-ray determination of the
three-dimensional structures of two human sulfatases, arylsulfatase A
(ASA; Ref. 6) and N-acetylgalactosamine 4-sulfatase
(arylsulfatase B, ASB; Ref. 7), revealed that the FGly residue is
buried at the bottom of a cavity that is formed by positively and
negatively charged amino acids. This cavity proved to be the active
site by two lines of evidence. Cocrystallization of ASB with vanadate, a potent inhibitor of ASB, revealed that vanadate was covalently bound
to FGly (7). In addition, an ASA mutant containing serine instead of
FGly was found to be covalently sulfated at the position of
FGly69 after incubation with sulfate ester (see below and
Ref. 8). The 2-fold disordered electron density of FGly was interpreted to be an aldehyde hydrate with two geminal hydroxyls protruding into
the lumen of the cavity (6). In close vicinity to the aldehyde hydrate
a metal ion (Mg2+ in ASA and Ca2+ in ASB) is
complexed by three aspartates and one asparagine. Two lysines, two
histidines, and a serine complete the arrangement of residues
potentially involved in binding and cleavage of the sulfate group
within the active site.
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Fig. 1.
Proposed scheme of sulfate ester cleavage by
sulfatases. In the first half-cycle (I) one of the
hydroxyls of the aldehyde hydrate attacks the sulfur of the sulfate
ester forming an enzyme-sulfate ester, whereas the residual alcohol is
released. Sulfate is eliminated in the second half-cycle
(II) by an intramolecular rearrangement cleaving the ester
bond and forming an aldehyde group at the enzyme. Finally, the aldehyde
hydrate is regenerated by the entry of water (III). This
scheme is based on the reaction mechanism described by Lukatela
et al. (6).
The function of the other residues forming the active site remained
speculative. In ASA one oxygen of the aldehyde hydrate is in close
vicinity to the Mg2+ ion, which is complexed by residues
Asp29, Asp30, Asp281, and
Asn282, and the other oxygen is close to the imidazole ring
of His125. Whereas Mg2+ is thought to
coordinate the oxygen that attacks the sulfur in the first step of
sulfate cleavage, His125 is a candidate to deprotonate the
second hydroxyl of the aldehyde hydrate, which is involved in the
release of the sulfate and the regeneration of the aldehyde. By
modeling a sulfate anion into the active site, the positively charged
residues Lys123, Lys302, His229,
and residue Ser150 were proposed to fix the sulfate group
(6). To obtain experimental evidence for the function of the putative
active site residues, we replaced residues Asp29,
Asp30, Asp281, Asn282,
His125, His229, Lys123,
Lys302, and Ser150 by alanine in ASA and
analyzed Vmax, Km, and the pH optimum of the cleavage of p-nitrocatechol sulfate. The
change of enzymatic activity observed for all mutants was interpreted on the basis of the three-dimensional structure of the active site.
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EXPERIMENTAL PROCEDURES |
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Amino Acid Sequence Alignment--
The amino acid sequences of
the ten known human sulfatases, six lower eukaryotic, and three
bacterial sulfatases (listed in Fig. 2),
which are known to be active in cleaving sulfate esters, were retrieved
from the SWISSPROT data base (entry names: ARSA_HUMAN, ARSB_HUMAN,
STS_HUMAN, ARSD_HUMAN, ARSE_HUMAN, GA6S_HUMAN, GL6S_HUMAN, IDS_HUMAN,
SPHM_HUMAN, ARS_HEMPU, ARS_STRPU, ARS_VOLCA, ARS_CHLRE, ARS_PSEAE, and
ARS_KLEAE) and from the GenBankTM data base (accession
numbers HSARSF, AFO13158, and U89492). Sequence alignments were
performed using the computer programs SIMILARITY, CLUSTAL, and
BLAST-2-SEQUENCES (Genetic Computer Group, Madison, WI).
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Determination of Distances and Potential Hydrogen Bonds between
the Residues of the Active Site of ASA--
The atomic coordinates of
human ASA (identification code 1AUK, Protein Data Bank of the
Brookhaven National Laboratory) were analyzed for the distances between
the functional groups of the active site residues using the computer
program XtalView (9). Interatomic distances in the range of 2.5-3.2 Å between potential proton donors and acceptors were considered to be
bridged by hydrogen bonds (10). All these potential hydrogen bonds are indicated in Fig. 3. The coordinates for
the aldehyde hydrate residue of FGly69 were obtained from
G. Lukatela (Berlin, Germany).
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Site-directed Mutagenesis-- Single amino acid substitutions were performed replacing the codons for the putative active site residues Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150 of human ASA by codons for alanine. In a second series of mutations the codons for Asp29, Asp30, and Asp281 were replaced by codons for asparagine, and the codon for Asn282 was replaced by that of aspartate. Mutagenesis was performed using the pAlter cloning kit (Promega Corp.), the cDNA for human ASA (11) cloned into the pAlter mutagenesis vector (12), and appropriate primers. Following mutagenesis mutant cDNAs were cloned as EcoRI fragments into the pMPSVEH vector downstream of the myeloproliferative sarcoma virus promoter.
Expression and Analysis of ASA Mutants--
The plasmids
carrying the mutated cDNA were stably transfected into mouse
embryonic fibroblasts deficient for both mannose 6-phosphate receptors
(mpr MEF cells; Ref. 13). After selection and
expansion of the clones stably expressing the mutants, sulfatases were
purified from cell secretions by affinity chromatography and dialyzed
against 10 mM Tris/HCl, 150 mM NaCl, pH 7.5. Vmax, Km, and the pH optimum
of sulfatase activity of the purified sulfatases were determined using
2, 5, 10, and 20 mM p-nitrocatechol sulfate as substrate as described earlier (12). All kinetic data were determined under initial rate conditions. Purity and amount of ASA proteins were
calculated from the integral of the ASA peak at 280 nm after reversed
phase HPLC as described (12). To determine the percentage of
Cys69 converted to FGly69, purified mutants
were digested with trypsin. The peptides containing Cys69
and FGly69 were purified by reversed phase HPLC and
quantified by Edman sequencing (12).
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RESULTS AND DISCUSSION |
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Putative Active Site Residues of Arylsulfatase A--
The amino
acid residues supposed to form the active site of ASA are shown in Fig.
3 in a three-dimensional view. FGly69 is shown as an
aldehyde hydrate with two geminal hydroxyl groups. The Mg2+
ion is octahedrically coordinated by one hydroxyl oxygen of
FGly69 and by five -oxygens of Asp29,
Asp30, Asp281, and Asn282. The same
hydroxyl oxygen of FGly69 is in close vicinity to one of
the oxygens of Asp281. The atomic distance of 3.4 Å between FGly69 and Asp281, derived from the
data of Lukatela et al. (Ref. 6 and see "Experimental
Procedures"), may be smaller considering the weak resolution of the
electron density in this area and the free rotatability of the
C
-C
bond in FGly69. Thus, a hydrogen bond may be
formed between the neighboring oxygens of FGly69 and
Asp281 leading to an at least partial deprotonation of this
hydroxyl of FGly69. The free oxygen would then be able to
start a nucleophilic attack on the sulfur of the sulfated substrate.
Coordination to Mg2+ may help to stabilize this
catalytically active form of FGly69. On the other side
His125 has an atomic distance of 2.7 Å to the second
oxygen of the FGly hydrate. Therefore, His125 is in an
appropriate position to de-protonate and thereby activate this
second oxygen of the FGly hydrate leading to the regeneration of the
oxogroup of the aldehyde and the cleavage of the enzyme-sulfate intermediate. Lys123, Lys302,
His229, and Ser150 have been proposed to
contribute to the binding of the sulfate group and to the activation of
the sulfur atom by increasing its electrophilia (6). In addition, these
residues may form hydrogen bonds with other residues of the active
site: Lys123 with Asp29 and Ser150
as well as Lys302 with Asp30. All residues
supposed to be involved in sulfate binding or in catalysis are able to
form hydrogen bonds to neighboring residues, resulting in a net-like
connection of the residues to a functional catalytic system (Fig.
3).
The putative active site residues are highly conserved among 19 eukaryotic and prokaryotic sulfatases, which are known to exhibit sulfatase activity (Fig. 2). Most of these residues are part of short sequences of conserved amino acids. Cys69 in ASA and its equivalents in the other sulfatases are converted to FGly. The sulfatase of Klebsiella pneumoniae is the only biochemically characterized sulfatase where a serine rather than a cysteine is encoded by the DNA. However, this serine is also converted to FGly (14). Asp29, Asp30, Lys123, His125, His229, Asp281, Asn282, and Lys302 are conserved or conservatively replaced in 14-19 sulfatases. Ser150 is only found in ASA but replaced by threonine in 6 sulfatases. The high conservation of these residues indicates that the structure of the active site and the catalytic mechanism of sulfate cleavage have been conserved in the sulfatase family.
Expression of Active Site Mutants--
To obtain experimental
evidence for their role in sulfate ester cleavage and sulfate binding,
nine of the putative active site residues of ASA were individually
substituted by alanine: Asp29, Asp30,
Asp281, and Asn282 as the metal coordinating
residues, His125 as one of the two FGly69
activating residues (the other one being Asp281), and
Lys123, Lys302, His229, and
Ser150 as possible sulfate binding residues.
Cys69 has been substituted by alanine earlier (12), and the
mutant C69A proved to be completely inactive. Arg73 that is
interacting with Asp281 (Fig. 3) is another putative active
site residue. However, the R73A mutant has been expressed earlier (12)
and was found to be unstable, thus preventing its further
characterization. The cDNAs encoding the mutant forms of ASA were
stably expressed in mpr MEF cells. Due to
deficiency in mannose 6-phosphate receptors, these cells secrete the
newly synthesized lysosomal enzymes such as ASA (13). Clones stably
expressing the mutant sulfatases were selected and expanded. The
sulfatases were purified from the medium by affinity chromatography and
analyzed for purity by analytical reversed phase chromatography and
SDS-polyacrylamide gel electrophoresis. In all preparations ASA
polypeptides represented 80-90% of total protein. The major
contamination (5-10%) was bovine serum albumin originating from the
culture medium (data not shown).
Kinetic Properties of Active Site Mutants-- We analyzed Vmax, Km, and pH optimum for the cleavage of p-nitrocatechol sulfate by the different substitution mutants (Table I). To control whether a loss of activity could be accounted for by a diminished conversion of Cys69 to FGly, all mutant proteins were analyzed for the presence of either FGly or Cys in position 69. In all mutants FGly was found to account for 49-95% of residue 69. In wild type control ASA 94% of residue 69 were converted to FGly (Table I). Earlier experiments have shown that this modification efficiency depends on the expression level, and values of 60-90% have been observed (1). Thus, the analysis of the modification efficiency of the ASA mutants indicates that a decrease of FGly formation can account for a not more than 50% decrease of Vmax in the mutants.
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Substitution of three of the Mg2+ complexing residues
(Asp29, Asp30, and Asp281) by
alanine led to a strong decrease of Vmax to
1-5% compared with wild type ASA, whereas substitution of
Asn282 by alanine decreased Vmax to
24%. This reflects the importance of the negatively charged aspartates
and, to a lower degree, of the polar asparagine for enzyme activity by
ensuring the exact positioning of a catalytically active metal ion or
the general three-dimensional structure of the active site. Because of
their close vicinity to the first oxygen of FGly69, a
direct involvement of the Mg2+ and the oxygen of
Asp281 in catalysis by stabilizing the deprotonated form of
this FGly oxygen is likely. The importance of the metal complexing
residues (Asp29, Asp30, Asp281, and
Asn282) for the general structure of the active site is
supported by the fact that in all of these mutants the
Km was increased 3-8-fold. It should be noted,
however, that modeling of the substrate into the cavity did not predict
a direct contact to one of the substituted residues. Asp29
and Asp30 may influence substrate binding by their
coordination of Lys123 and Lys302,
respectively, which are strong candidates for binding the sulfate group
(see below).
Whereas the Mg2+ and Asp281 are thought to be involved in the activation of FGly69 during the cleavage of the substrate-sulfate ester, His125 is proposed to catalyze the desulfation of the enzyme-sulfate ester leading to the elimination of sulfate and to the regeneration of the aldehyde. Substitution of His125 by alanine led to a decrease of Vmax to 11% and to a 7-fold increase of the Km, indicating a participation in the catalytic process but also pointing to an influence on the binding of the substrate (see below).
Sulfated substrates are expected to be bound by positively charged residues, thereby orientating the sulfate group next to the catalytically active oxygen of FGly69 and increasing the electrophilic property of the sulfur. When the potentially positively charged residues in the vicinity of FGly69 (His125, His229, Lys123, and Lys302) were substituted by alanine, 5-fold (His125 and His229) and more than 10-fold (Lys123 and Lys302) increases of the Km were observed. Because a 3-5-fold increase of Km was also observed in other mutants, which are unlikely to interact directly with the substrate molecule, only the two lysines (Lys123 and Lys302) remain as strong candidates for binding the sulfate group. Additionally, the Vmax of all of these four mutants was decreased to 6-11% of wild type ASA. In the H125A mutant this is obviously due to the interference with FGly coordination (see above). For K123A and K302A it may be ascribed to their direct vicinity to Asp29 and Asp30, which are essential for the coordination of the Mg2+ ion (see above).
Substitution of Ser150, a residue close to Lys123, led to a strong decrease of Vmax (2% of wild type) without affecting the Km. A direct participation of Ser150 in catalysis cannot be expected, because of the large distance to FGly69 (4.9 Å). At least part of the loss of activity may be explained by the close vicinity to Lys123, which coordinates Asp29, one of the metal complexing residues. The Km of the S150A mutant was in the range of the Km of wild type ASA, indicating that Ser150 is not essential for binding of p-nitrocatechol sulfate as substrate.
The strong decrease of activity in mutants containing alanine instead of the metal complexing aspartates may be explained by the loss of negative charges coordinating the Mg2+ ion and for Asp281 additionally by the loss of direct FGly69 activation. Substitution of aspartate by asparagine replaces one oxygen with a nitrogen. By its free pair of electrons the latter may partly take over the function of the negative charge of the oxygen. To analyze the effect of a reciprocal exchange of aspartate and asparagine in the metal binding site, we substituted Asp29, Asp30, and Asp281 by asparagine and Asn282 by aspartate. Surprisingly the kinetic parameters of all of these mutants were similar to those of the respective alanine substitution mutants (Table I). Only the D30N mutant showed a higher Vmax as compared with D30A, indicating that one oxygen of Asp30 could partly be replaced with a nitrogen. Taken together, these results demonstrate that even relatively small changes in the metal binding complex, induced by aspartate/asparagine exchanges, affect catalytic activity and substrate binding. In line with this, the metal complexing residues are highly conserved in the sulfatase family (Fig. 2).
The pH optimum of all mutants was shifted to a more alkaline pH by
0.2-0.8 units as compared with wild type ASA (Table I and Fig.
4). This change indicates that proton
abstraction is a crucial aspect in the catalytic mechanism of sulfate
ester cleavage. In wild type ASA both oxygens of the aldehyde hydrate
have to be activated by abstraction of a proton to facilitate the
cleavage of the sulfated substrate and the release of the sulfate group from the enzyme (see above and Fig. 1). Proton abstraction may be
impaired by substituting either His125 and
Asp281, which are directly involved in these steps, with
alanine or by point mutations that alter the overall structure of the
active site. An increase in pH promotes the deprotonation and could
partially compensate for the loss of catalytic deprotonation. The
latter appears to depend on the exact positioning of
Asp281, Mg2+, and His125 next to
FGly69. In line with this, the decrease in
Vmax observed in all of these mutants can at
least partly be ascribed to an impaired deprotonation of the aldehyde
hydrate.
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Conclusions--
The amino acid residues neighboring
FGly69 in the active site of ASA are highly conserved among
eukaryotic and prokaryotic members of the sulfatase family, indicating
a similar structure of the reactive center and a common reaction
mechanism. Crystal structure analysis revealed that these residues are
probably interconnected by a net of hydrogen bonds, thus forming the
active site. The structural data are consistent with the biochemical
finding that all ASA mutants, in which putative residues of the active
site were replaced by alanine, have a markedly decreased
Vmax. The increased pH optimum observed for all
mutants is in good agreement with the decreasing
Vmax, because in both of the two half-reactions of sulfate ester cleavage a deprotonation step is involved. Among the
residues that have been proposed to bind the sulfate group, Lys123 and Lys302 were shown to be most
critical for substrate binding. Their substitution by alanine led to a
marked increase of Km. The moderate 3-5-fold
increase of Km in the other mutants, except for
S150A, supports the view of the active site of ASA (and other sulfatases) as a highly interconnected network of polar residues, easily disturbed by the replacement of one of its elements.
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ACKNOWLEDGEMENTS |
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We thank Martina Balleininger and Katja Unthan-Hermeling for technical assistance, Klaus Neifer for protein sequencing, and Isabel Usón for critically reading the manuscript.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Abt. Biochemie II,
Gosslerstr. 12d, D-37073 Göttingen, Germany. Tel.: 49-551-395951; Fax: 49-551-395979; E-mail: bschmidt{at}uni-bc2.gwdg.de.
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ABBREVIATIONS |
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The abbreviations used are:
FGly, C-formylglycine;
ASA, arylsulfatase A;
ASB, N-acetylgalactosamine 4-sulfatase;
HPLC, high pressure
liquid chromatography.
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REFERENCES |
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