From the Medical Research Council Group in Protein
Structure and Function, Department of Biochemistry, University of
Alberta, Edmonton, Alberta T6G 2H7, Canada, the ¶ Department of
Chemistry, University of British Columbia, Vancouver, British Columbia
V6T 1Z1, Canada, the ** Department of Chemistry, Rutgers University,
New Brunswick, New Jersey 08903, and the
Department of Biochemistry and Medical
Genetics, University of Manitoba,
Winnipeg, Manitoba R3E 0W3, Canada
Received for publication, December 8, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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Carbohydrates are involved in many diverse biological functions
including cell structural integrity, energy storage, pathogen defense
and invasion mechanisms, viral penetration, and cellular signaling.
Therefore, a large number of enzymes dedicated to carbohydrate metabolism have evolved. Enzymes specifically responsible for carbohydrate catabolism are collectively referred to as glycosyl hydrolases and have been classified into 77 families based on amino
acid sequence similarity (1-3). Three-dimensional structures are known
for representatives of 30 of the families. Although there are
differences in chain length and domain structure between proteins of a
single family, all proteins of a family hydrolyze the glycosidic bond
with the same stereochemical outcome (4).
Family 20 includes the Stereochemical outcome studies on the family 20 chitobiase from
Serratia marcescens (13) and human -Hexosaminidase, a family 20 glycosyl
hydrolase, catalyzes the removal of
-1,4-linked
N-acetylhexosamine residues from oligosaccharides and their
conjugates. Heritable deficiency of this enzyme results in various
forms of GalNAc-
(1,4)-[N-acetylneuraminic acid
(2,3)]-Gal-
(1,4)-Glc-ceramide gangliosidosis, including Tay-Sachs
disease. We have determined the x-ray crystal structure of a
-hexosaminidase from Streptomyces plicatus to 2.2 Å resolution (Protein Data Bank code 1HP4).
-Hexosaminidases are
believed to use a substrate-assisted catalytic mechanism that generates
a cyclic oxazolinium ion intermediate. We have solved and refined a
complex between the cyclic intermediate analogue
N-acetylglucosamine-thiazoline and
-hexosaminidase from S. plicatus to 2.1 Å resolution (Protein Data Bank code
1HP5). Difference Fourier analysis revealed the pyranose ring of
N-acetylglucosamine-thiazoline bound in the enzyme active
site with a conformation close to that of a 4C1
chair. A tryptophan-lined hydrophobic pocket envelops the thiazoline ring, protecting it from solvolysis at the iminium ion carbon. Within
this pocket, Tyr393 and Asp313 appear important
for positioning the 2-acetamido group of the substrate for nucleophilic
attack at the anomeric center and for dispersing the positive charge
distributed into the oxazolinium ring upon cyclization. This complex
provides decisive structural evidence for substrate-assisted catalysis
and the formation of a covalent, cyclic intermediate in family 20
-hexosaminidases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-N-acetylhexosaminidases
(
-hexosaminidases)1 (EC
3.2.1.52), enzymes that catalyze the removal of terminal
-1,4 linked
N-acetylhexosamine residues from the nonreducing ends of
oligosaccharides and their conjugates. In humans, there are two major
-hexosaminidase isoforms: HexA and HexB. HexA is a heterodimer of
subunits
(encoded by HEXA) and
(encoded by HEXB), whereas HexB is a homodimer of
subunits. HexA is
essential for degrading GalNAc-
(1,4)-[N-acetylneuraminic
acid (2,3)]-Gal-
(1,4)-Glc-ceramide ganglioside; the biological
importance of HexA activity is illustrated by the fatal
neurodegenerative disorders that result from its heritable deficiency
(5). Mutations in HEXA or HEXB cause Tay-Sachs and Sandhoff disease, respectively. These genetic diseases have made
the human
-hexosaminidase isoforms the subject of much research. A
substantial amount of genetic and biochemical information is available
for these isozymes (5), but detailed information about their catalytic
mechanism is limited. Mechanistic studies have been primarily limited
by the difficulties in producing sufficient amounts of recombinant
enzyme needed for kinetic analysis (6, 7); however, recent improvements
in expression and purification procedures have allowed more accurate
kinetic measurements to be made (8). Crystals of human HexB have been
grown (9); however, attempts at solving its three-dimensional structure
have not been successful. Nonetheless, much insight into the mechanism of human HexA and HexB has been provided by structural and functional studies carried out on related family 20 glycosyl hydrolases
(10-12).
-hexosaminidase (14) demonstrated that this family operates via a retaining mechanism. The
consensus view for the mechanism of
-retaining glycosyl hydrolases involves general acid catalyzed cleavage of the
-(1,4)-glycosidic linkage via a transition state with substantial oxacarbenium ion character to form a glycosyl-enzyme intermediate with an active site
carboxylate. General base-catalyzed attack of water at the anomeric
center of this intermediate yields a product with the same anomeric
stereochemistry as the substrate. This mechanism is commonly referred
to as the double displacement mechanism of hydrolysis (1, 15, 16).
Unexpectedly, family 20
-hexosaminidases and chitobiases, as well as
the functionally related chitinases of family 18, were found to lack a
carboxylate group suitably disposed to stabilize the oxacarbenium ion
transition state (10, 11, 17, 18). Instead, x-ray structural analysis
of S. marcescens chitobiase (SmCHB) and kinetic
studies with inhibitors have provided strong evidence for catalysis
involving participation of the neighboring C2-acetamido group on the
substrate (Fig. 1A and Refs.
10, 13, 19, and 20). The 2-acetamido group of the substrate acts in place of the enzyme nucleophile to generate an enzyme-stabilized oxazolinium ion intermediate. The cyclic intermediate is then hydrolyzed by general base catalyzed attack of water at the anomeric center in a manner analogous to the double displacement mechanism described above.
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Fig. 1.
Proposed catalytic mechanism for
-hexosaminidase. A, detailed
SpHEX catalytic mechanism. The general acid/base
(Glu314) and the residue (Asp313) primarily
responsible for stabilizing positive charge on the oxazolinium ion
intermediate are shown, although no attempt is made to indicate their
true locations. Hydroxyl groups and C6 have been removed from the
pyranose ring for clarity. B, chemical structure of the
cyclic intermediate analogue NAG-thiazoline.
We have determined the three-dimensional crystal structure of a family
20 -hexosaminidase cloned from Streptomyces plicatus (21). The 55-kDa enzyme, referred to as SpHEX, is a highly
active and stable glycosyl hydrolase that functions over a broad pH
range. Co-crystallization of SpHEX with the cyclic
intermediate analogue N-acetylglucosamine (NAG)-thiazoline
(Fig. 1B and Ref. 19), and subsequent crystallographic
analysis has provided decisive structural evidence for a
substrate-assisted catalytic mechanism involving 2-acetamido group
participation, resulting in the formation of a covalent, cyclic
intermediate (Fig. 1).
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EXPERIMENTAL PROCEDURES |
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Protein Expression and Purification-- Escherichia coli strain JM109 was used for plasmid amplification, and plasmid purification was carried out using Qiagen purification systems. Restriction enzymes and Vent DNA polymerase were from New England Biolabs. T4 DNA ligase was from Roche Molecular Biochemicals. All cloning procedures are described in Ref. 22. SpHEX is a 506-amino acid protein having a predicted molecular mass of 55010 Da (GenBankTM accession number AF063001). It was expressed as a recombinant, N-terminal His7-tagged fusion protein. Briefly, the plasmid psHEX-1.8 (11) contained the SpHEX open reading frame. The first 100 base pairs of the 5'-end of the SpHEX open reading frame was amplified by the polymerase chain reaction using the sense primer (5'-GGAATTCCATATGCATCATCATCATCATCATCACACCGGCGCCGCCCCGGACCGGAAG-3') and the antisense primer (5'-TGGCGCGCCGCCGGGGTCGACCGAGGCGGG-3'). This polymerase chain reaction product was restriction digested with AscI and NdeI for ligation into the final expression plasmid. To obtain the remaining 1.7-kilobase pair fragment of the SpHEX open reading frame, a further aliquot of psHEX-1.8 was restriction digested with AscI and BamHI. The 100-base pair (NdeI/AscI) and 1.7-kilobase pair (AscI/BamHI) fragments were then ligated into the T7 expression plasmid pET-3a (Novagen) that had been linearized by digestion with NdeI and BamHI. The ligation product resulted in the expression plasmid p3AHEX-1.8 whose sequence was verified prior to use in fusion protein expression.
The His7-SpHEX fusion protein was expressed in
E. coli strain BL21 (DE3). Transformed cells were grown at
37 °C to an A600 = ~0.5 and then
induced with 0.4 mM
isopropyl-1-thio--D-galactopyranoside for 3 h at
25 °C. Cells were pelleted by centrifugation, resuspended in a lysis
buffer (20 mM Tris-Cl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10 mM
-mercaptoethanol) and
lysed by French press. After centrifugation at 20,000 × g for 1 h, the supernatant was loaded onto a
nickel-nitrilotriacetic acid superflow (Qiagen) column pre-equilibrated
with lysis buffer. Once loaded, the column was washed with the lysis
buffer supplemented with 80 mM imidazole (pH 8.0). The
fusion protein was eluted from the column using lysis buffer
supplemented with 250 mM imidazole, pH 8.0, and
precipitated with 55% ammonium sulfate for storage at 4 °C.
Aliquots of the precipitated protein were routinely resuspended and
dialyzed twice against 50 mM trisodium citrate, pH 6.0, 300 mM NaCl, and 0.5 mM dithiothreitol and then
concentrated to ~10 mg/ml with a Millipore concentrator.
Approximately 40-60 mg of pure fusion protein was routinely obtained
per liter of culture. Electrospray ionization mass spectrometric
analysis using a VG Quattro triple quadrupole mass spectrometer (VG
Biotech, Altringham, UK) determined the mass of the purified fusion
protein to be 56,054 Da, in good agreement with the theoretical mass of
56,049 Da.
Seleno-Met-substituted His7-SpHEX was expressed
in E. coli strain BL21 (DE3) pLys S using the method
described in Ref. 23. Transformed cells were grown at 37 °C in M9
minimal medium until mid-log phase growth was reached. The culture was
then supplemented with 0.5 mM Lys, 0.8 mM Thr,
0.6 mM Phe, 0.8 mM Leu, 0.8 mM Ile, and 0.8 mM Val to inhibit endogenous Met biosynthesis.
After a 30-min incubation, the culture was further supplemented with
0.25 mM seleno-Met and induced with 0.5 mM
isopropyl-1-thio--D-galactopyranoside for 10 h.
Seleno-Met substituted His7-SpHEX was purified
in the same manner as native His7-SpHEX except
that the protein was dialyzed against 3 mM dithiothreitol
before concentrating to avoid selenium oxidation. Electrospray mass
spectrometric analysis verified that all 6 Met residues in the
512-amino acid SpHEX protein had been substituted with
seleno-Met. All purified fusion protein was visualized for purity by
SDS-polyacrylamide gel electrophoresis.
Crystallization and Data Collection-- Both native and seleno-Met substituted His7-SpHEX crystallized in the hexagonal space group P6122 within 2 weeks by vapor diffusion at room temperature. The mother liquor consisted of 2.2 M ammonium sulfate, 100 mM trisodium citrate, pH 6.0, and 20-25% glycerol. Hanging drops were set up by mixing an aliquot of SpHEX (concentrated to 10 mg/ml) with an equal amount of the mother liquor. Crystals of His7-SpHEX in complex with NAG-thiazoline were obtained by co-crystallization of the native fusion protein (from which dithiothreitol had been removed by dialysis) with 2-5 mM NAG-thiazoline. Diffraction data for a MAD phasing experiment were collected at the Advanced Photon Source, BioCARS sector beamline BM-14-C and BM-14-D on native and seleno-Met substituted His7-SpHEX crystals flash cooled to 100 K, respectively (see Table I). Diffraction data from crystals of the complex between His7-SpHEX and NAG-thiazoline were collected at Stanford Synchrotron Radiation Laboratory, beamline 9-2 (see Table I). All diffraction data were processed using DENZO and SCALEPACK (24).
Structure Determination and Refinement--
A solution to the
crystal structure of the protein was obtained by a MAD phasing
experiment performed on seleno-Met-substituted protein crystals (25). A
combination of data derived from the MAD phasing experiment at beamline
BM-14-D with data collected from native SpHEX crystals at
beamline BM-14-C allowed for the determination of the three-dimensional
structure of S. plicatus -hexosaminidase to 2.2 Å resolution. Although the SpHEX crystals diffracted to
slightly higher resolution than 2.2 Å, data collection was restricted
to this resolution to avoid excessive data rejection caused by spot
overlap. The program SOLVE (26) was used for local scaling of the data
and to calculate the anomalous and dispersive differences needed to
find selenium sites and to determine phase probability distributions.
Patterson maps, calculated from the anomalous and dispersive
differences, allowed us to find clearly five of the six selenium atoms
present in the SpHEX structure. The missing selenium atom
was part of the initiation Met whose position could not be determined
because of disorder of the first 14 residues of the
His7-tagged N terminus.
Electron density maps, generated using structure factor phases obtained
from the MAD phasing experiment (initial figure of merit 0.8),
were improved only slightly by solvent flattening using Density
Modification (Fig. 2 and Ref. 27). Map
boundaries were extended beyond the CCP4 asymmetric unit using EXTEND
(28) and skeletonized using MAPMAN (29). A molecular model of the enzyme was built from the skeletonized map using O (30). Residues 8-512 were readily fit into the density as one continuous chain. Coordinates for the small molecules glycerol and
SO
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The molecular model of native SpHEX was refined using a
maximum likelihood target function during both simulated annealing and
conjugate gradient minimization as implemented in Crystallography and
Nuclear Magnetic Resonance System (32). Prior to refinement, 10% of the diffraction data was randomly flagged for cross-validation using the free R factor. After each round of refinement, the
model was manually inspected with O using 2Fo Fc and Fo
Fc maps. The final refinement statistics for the
model reflect the high quality data (see Table I).
NAG-thiazoline Complex--
An Fo Fc map, used to visualize NAG-thiazoline in the
active site, was obtained using structure factor phases calculated from
the native SpHEX model that had been positioned into the unit cell of the NAG-thiazoline complex using rigid body refinement followed by conjugate gradient minimization. Solvent molecules were
removed from the model before placing it into the new cell and were
relocated during later rounds of refinement. Any waters found in the
active site were deleted until the NAG-thiazoline had been modeled into
the electron density ascribed to it. The initial NAG-thiazoline model
and its geometrical parameters were based on the x-ray crystal
structure of N-acetylgalactosamine-thiazoline (GalNAc-thiazoline).2
Refinement of the NAG-thiazoline complex was carried out using Crystallography and Nuclear Magnetic Resonance System as
described for the native SpHEX model above. The final
refinement statistics are presented in Table I.
Coordinates--
The coordinates and structure factors have been
deposited into the Protein Data Bank (native SpHEX Protein
Data Bank code 1HP4; SpHEX/NAG-thiazoline complex Protein
Data Bank code 1HP5).
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RESULTS AND DISCUSSION |
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Structure of -Hexosaminidase--
Excellent crystallographic
data (Table I) produced easily
interpretable electron density maps into which a model of
SpHEX was built (Fig. 2). The enzyme is a kidney shaped,
two-domain protein having overall dimensions of ~68 × 58 × 56 Å (Fig. 3). The two domains of
SpHEX have a similar fold to domains II (residues 214-335)
and III (residues 336-818) of SmCHB (Fig.
4); however, significant deviations
between the two structures exist. The most striking structural
difference between SpHEX and SmCHB is the absence
in SpHEX of two of the four domains that compose
SmCHB (Fig. 4). This results in a solvent-exposed active
site at the C-terminal end of the (
/
)8 barrel forming
domain II. Such a solvent-exposed active site appears to explain why
-hexosaminidases, such as human
-hexosaminidase A, can
accommodate large glycoconjugates like
GalNAc-
(1,4)-[N-acetylneuraminic acid
(2,3)]-Gal-
(1,4)-Glc-ceramide ganglioside.
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Domain I of SpHEX is composed of residues 1-151. As in
SmCHB, this domain has an /
topology consisting of a
solvent exposed, seven-stranded anti-parallel
-sheet that buries
two, roughly parallel,
-helices (Fig. 3). Similar topologies have
been found in matrix metalloproteinases (10) and collagenases. The
amino acid sequence identity between SpHEX and
SmCHB is lowest throughout this domain. A structure-based
alignment using SwissPDBviewer (33) indicated only a 16.1% amino acid
identity. Nonetheless, the fold is well conserved, with 87 C
atoms of the two homologous domains having a rms
difference of only 1.34 Å. A multiple sequence alignment of all family
20 glycosyl hydrolases indicates that domain I is conserved throughout
the entire family. Such conservation suggests a functional requirement for this domain by family 20 glycosyl hydrolases; ironically, however,
its function remains unknown.
Domain II of SpHEX is composed of residues 151-512 and is
folded into a (/
)8 barrel with the active site of the
enzyme residing at the C termini of the 8
-strands of the barrel.
This domain is homologous to domain III in SmCHB, and a
structure-based sequence alignment demonstrated there to be a 29.5%
sequence identity between the two domains, where 236 of the
C
atoms had a rms difference of 1.30 Å. What may indeed
be a common feature of this (
/
)8 barrel domain in
family 20 glycosyl hydrolases is the conspicuous absence of regular
helices at positions
5 and
7 in the (
/
)8
barrel. In both SpHEX and SmCHB helix
5 consists of only a single turn of a 3/10 helix, whereas helix
7 is
completely absent and is instead replaced by an extended loop. Overall,
this domain in SpHEX contains shorter surface loops and is
much more compact than its homologous counterpart, domain III, in
SmCHB. Multiple sequence alignments of family 20 glycosyl hydrolases suggest that such a compact (
/
)8 barrel
domain may be a common feature among many family 20
-hexosaminidases, including the human isoforms (10,
11).
Unlike the basic (/
)8 barrel motif, domain II of
SpHEX contains three major loop structures that extend from
the C termini of three of the 8
-strands of the barrel. First, loop
Lp7 replaces helix
7 as described above (Fig. 3). Second, a 36-amino
acid loop, Lp2, extends from the C terminus of strand
2 and contains a short helical segment that packs against and stabilizes the third
major loop Lp3. Lp3 is a 41-amino acid loop that extends from the C
terminus of strand
3 and contains a helical segment that is
complimentary to and packs against the helical segment found in Lp2
(Fig. 3). There is only one disulfide bond in SpHEX (Cys263-Cys282), and its presence close to the
base of Lp3 may help to stabilize the conformation of this loop. Lp3
and Lp7 act in concert to form the hydrophobic faces of sugar binding
site +1 described below (Fig. 5). There
are two homologous loops in SmCHB; however, they are longer
and perform an additional function by interacting with a domain not
present in SpHEX (SmCHB domain I) (Fig. 4).
Finally, an extra helix continues on from helix
8 of the
(
/
)8 barrel to complete the C terminus of
SpHEX. This extra helix stabilizes domains I and II with
respect to each other (Fig. 3). It is interesting to observe that the
relative orientation of domains I and II of SpHEX is the
same as the homologous domains II and III in SmCHB.
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The Complex with NAG-Thiazoline: Mechanistic
Implications--
According to our x-ray structure of SpHEX
and that of the SmCHB-chitobiose complex (10), family
20 glycosyl hydrolases do not appear to contain a side chain in a
position suitable to act as a catalytic nucleophile that would
stabilize developing oxacarbenium ion character. Instead, it has been
observed that, in the conformation bound by the enzyme, the C2
acetamido oxygen of the nonreducing sugar in subsite 1 is held within
3 Å of its C-1 anomeric carbon. When in this position, it is believed
that the acetamido oxygen can act as a nucleophile and attack the
anomeric center to form a cyclic NAG-oxazolinium ion intermediate
(10).
We have determined the three-dimensional structure of an analogue of
the proposed NAG-oxazolinium ion intermediate bound to SpHEX. Because NAG-oxazoline itself is too hydrolytically
unstable for use in structural studies, a relatively stable analogue,
NAG-thiazoline, has been synthesized and shown to be a potent
competitive inhibitor of jack bean -hexosaminidase
(Ki = 280 nM) (19). NAG-thiazoline also
acts as an excellent competitive inhibitor of both SpHEX and
human
-hexosaminidase B.
Fig. 6 shows NAG-thiazoline bound in the
SpHEX active site and the quality of the electron density
into which it was modeled. Excluding O-4 and O-6 because of
differences in C-4 chirality and enzyme packing effects, respectively,
the remaining atoms in NAG-thiazoline had an rms difference of only
0.071 Å compared with the equivalent atoms in the small molecule
structure of GalNAc-thiazoline. NAG-thiazoline was bound in the 1
subsite of SpHEX and adopts a conformation that is close to
a 4C1 chair, although the current data do not
exclude small distortions toward a sofa or skew boat conformation.
There are no significant changes in the SpHEX structure upon
binding NAG-thiazoline except for a slight opening of the active site
pocket. Fig. 7 clearly shows Trp residues
344, 361, and 442 of the
1 subsite of SpHEX and the
homologous residues in SmCHB (Trp 616, Trp 639, and Trp 737)
forming a tight hydrophobic pocket into which the nonreducing GlcNAc
residue binds. This pocket appears to help force the C2 acetamido
oxygen into close proximity with the anomeric carbon, and the tight
packing between the acetamido group and the enzyme helps ensure a
precise alignment of the acetamido oxygen with the anomeric carbon. The
hydrophobic pocket will also protect the otherwise reactive oxazolinium
ion intermediate from hydrolysis by attack at the oxazolinium carbon
atom originally derived from the amide. Indeed, such a protected
hydrophobic pocket is highly reminiscent of that found around the
catalytic nucleophile in the structures of glycosyl-enzyme
intermediates in "normal" retaining glycosidases. In both cases
such an environment would protect the intermediate from solvolysis via
unwanted pathways. Importantly, the conformation of the sugar in this
intermediate is a 4C1 chair in both covalent
glycosyl-enzyme and cyclic oxazoline.
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Numerous hydrogen-bonding interactions lock NAG-thiazoline into the active site of SpHEX and disperse the positive charge distributed into the thiazoline ring upon cyclization (Fig. 6B). These include at least one hydrogen bond to every hydroxyl group on the pyranose ring. However, no hydrogen bonds to the ring oxygen O-5 are evident; indeed, a hydrogen bond to O-5 would be counter-catalytic because it would decrease the extent of lone pair donation by O-5 to the antibonding orbital of the scissile bond (34, 35).
NAG-thiazoline is held in place particularly strongly by
Arg162, which forms hydrogen bonds to both O-3 and O-4 of
the inhibitor. The mutation R162H results in a 40-fold increase
in Km relative to wild type SpHEX and a
5-fold decrease in Vmax when assayed using
4-methylumbelliferyl--N-acetylglucosaminide (11). The
resultant 200-fold decrease in
Vmax/Km confirms that this
residue is involved in stabilization of the transition states occurring
along the reaction coordinate. The analogous mutation in the
-subunit of human HexA (R178H) is associated with the B1 variant
form of Tay-Sachs disease in which the enzyme appears to be normally
folded and processed but lacks sufficient enzymatic activity and thus
results in disease (36, 37). Recently, the mutation R211K (homologous
to Arg178 of the
-subunit of human HexA) was created in
human HexB (8). The mutation resulted in a 10-fold increase in
Km, paralleling the findings with SpHEX
(Arg162). Furthermore, the kcat
value for the R211K mutation was 500-fold less than that of the wild
type enzyme, suggesting that it may serve a more important role in
transition state stabilization than its counterpart in SpHEX
(8). Such bidentate hydrogen bonding from an Arg side chain to two
vicinal hydroxyl groups on the substrate has been seen previously in
other glycosyl hydrolases. For example, functionally equivalent
hydrogen bonding is seen in SmCHB (Arg349) (10)
and in Bacillus circulans xylanase, where Arg112
hydrogen bonds to both O-2 and O-3 (34), and mutation of this residue
to Asn results in a 35-fold decrease in
kcat/Km.3
Two particularly important hydrogen-bonding interactions are formed with the thiazoline ring of NAG-thiazoline when it binds to SpHEX. First, the OH of Tyr393 donates a hydrogen bond to the sulfur atom of the thiazoline ring. In the substrate complex such a hydrogen bond would orient the carbonyl oxygen into position for nucleophilic attack on the anomeric carbon C-1. A similar role is envisioned for Tyr669 of SmCHB (10). Second, upon formation of the cyclic intermediate, the nitrogen atom N-2 develops a positive charge and SpHEX appears to stabilize this positive charge by delocalizing it through a hydrogen-bonding network between Asp313, Asp246, and the main chain NH group of Met247. This is seen in the two short hydrogen bonds of 2.5 and 2.4 Å from the nitrogen N-2 of the thiazoline ring and the carboxylate oxygens of Asp313 and Asp246, respectively (Fig. 6). These short hydrogen bond distances indicate that the carboxylate of Asp313 is likely deprotonated and possesses a delocalized negative charge during catalysis.
The other key residue in the active site of retaining glycosidases is
the acid/base catalyst, which adopts a dual role, functioning to
protonate the departing aglycone in the first step and then to
deprotonate the incoming water in the second step. In the structure of
the complex of SmCHB with chitobiose, a 2.9 Å hydrogen bond was seen between the glycosidic oxygen of chitobiose and
Glu540, leading to the assignment of Glu540 as
the acid catalyst (10). Comparative molecular modeling combined with
site-directed mutagenesis and kinetic studies of SpHEX and human -hexosaminidase subunits
and
have shown
Glu314, Glu323, and Glu355 to be
homologous to SmCHB Glu540, respectively (10,
11, 38, 39). The mutation E314Q in SpHex decreases both
Vmax and Km for
4-methylumbelliferyl
-N-acetylglucosaminide by
296- and 7-fold, respectively, confirming an important role for this
residue in catalysis (11). Superposition of the crystal structures of
SpHEX and SmCHB confirms that Glu314
of SpHEX is indeed positioned within the active site such
that it too would make a hydrogen bond to the glycosidic oxygen of the
superimposed chitobiose model (Fig. 7).
The second and final step in the double displacement mechanism is the
hydrolysis of the intermediate by general base-catalyzed attack of
water at the anomeric center C-1, resulting in overall retention of the
anomeric configuration. Figs. 5 and 8
show the position of a glycerol molecule bound in the +1 subsite. This glycerol superimposes onto half of the pyranose ring of chitobiose and
suggests that subsite +1 in SpHEX causes the sugar in this subsite to be twisted ~90° relative to the sugar bound in subsite 1 (Fig. 5). Furthermore, one of the hydroxyl groups of this glycerol is within 3.4 Å of the anomeric C-1 of NAG-thiazoline and forms a
hydrogen-bonding interaction with the carboxylate of the general acid/base Glu314. We postulate that this hydroxyl group
occupies the position that an incoming water molecule would take to
nucleophilically attack C-1, thereby hydrolyzing the oxazolinium ion
intermediate, with release of
-N-acetylglucosamine.
Abstraction of the proton from water by Glu314 is assisted
by a hydrogen-bonding network formed between its carboxylate group, the
imidazole nitrogens of His250, the carboxylate of
Asp191 and the main chain NH group of Asp192
(Fig. 8). The active site water molecule seen in the SmCHB
structure, and proposed to be the reactant species (10), is indeed
conserved in the SpHEX structure and is indicated in Figs. 6
and 8 as WAT. However, this water molecule is buried within
the active site of both structures, and it seems more plausible that
the incoming water enters directly from the bulk solvent after
departure of the aglycone rather than occupying this site first. The
role of buried water is unclear, but structured waters that mediate the binding of sugars with proteins are quite common and may provide some
of the flexibility required to accommodate substrates of both
gluco and galacto configuration.
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A -retaining mechanism utilizing acetamido group participation in
family 20
-hexosaminidases and chitobiases is consistent with
observations from glycosyl hydrolases from the functionally related
family 18 (17). In this family, there is also no apparent enzyme
nucleophile, and crystallographic analysis of the family 18 plant
chitinase hevamine in complex with the chitinase inhibitor allosamidin
suggests that a similar cyclic reaction intermediate is formed in
chitinases by C2-acetamido group participation (18, 40). Further
examples of enzymes possibly utilizing substrate assisted catalysis
include soluble lytic transglycosylase (41), and goose lysozyme (42).
Hence, it appears that substrate-assisted catalysis is a common feature
between glycosyl hydrolase families 18 and 20 and potentially other families.
Mechanistic Conclusion-- A combination of the results from this study, in which the structure of a complex with an intermediate analogue is presented, with those from a previous study of the structure of the substrate (chitobiose) complex with SmCHB allows interesting insights into the reaction mechanism and particularly into the substrate conformational changes that occur along the reaction coordinate.
The substrate binds to the enzyme with the sugar in the 1 subsite in
a distorted sofa/boat conformation, as seen in the bound chitobiose
structure (Fig. 7). This places the scissile bond in a pseudo-axial
orientation similar to that seem for the complex of lysozyme with
NAM-NAG-NAM bound as a product (43). Such a conformation allows atoms
C-5, O-5, C-1, and C-2 of the sugar in the
1 site to adopt the
coplanar configuration required for effective overlap of the nonbonding
lone pair of electrons on O-5 with the antibonding orbital at the
electron-deficient anomeric center of the oxacarbenium ion. This
conformation not only satisfies the requirements of stereoelectronic
theory, it also obeys the principle of least nuclear motion and the
need to minimize 1,3-diaxial repulsive interactions between the
approaching nucleophile and H3 and H5 of the substrate (15, 35, 44). A
similar conformational distortion of the analogous sugar has been
observed in a nonhydrolyzable thiooligosaccharide mimic of cellulose
bound to endoglucanase I from family 7 (45). Upon cleavage of the
glycosidic bond, with concerted proton donation from
Glu314, the sugar ring relaxes to the
4C1 chair conformation, as evidenced by the
structure of the spHEX-thiazoline complex. Hydrolysis of this
intermediate then follows a similar conformational itinerary, with
formation of a product complex in a skew boat conformation, and finally
product release. A very similar conformational itinerary has been shown
for a normal retaining
-glycosidase in which a covalent
glycosyl-enzyme intermediate is formed.
Interestingly, these crystal structures reveal that as the bound
substrate proceeds along the reaction coordinate to yield the
enzyme-bound product, the greatest nuclear motion of heavy atoms occurs
at C-1, as shown in Fig. 9. As the
reaction proceeds, the C-1 atom scribes an arc from its initial
position (position 1) as it breaks a covalent bond to the
glycosidic oxygen to form a new bond with the acetamido oxygen
(position 3). Approximately halfway along this arc is the
transition state where C-1, C-2, C-5, and O-5 are coplanar
(position 2). During hydrolysis of the intermediate, C-1
traces the reverse path as it breaks the bond with the oxazolinium ion
ring oxygen, proceeds through the transition state, and forms a
covalent bond with a suitably positioned water molecule. Thus the
motion of C-1 through the catalytic cycle can be described as a
"wagging" back and forth from positions below and above the plane
of the sugar ring. Very little motion of protein atoms is required; all
changes occur within a site that has been optimized for this minimized
motion. This behavior is entirely consistent with both the
antiperiplanar lone pair hypothesis and the principle of least nuclear
motion and appears to be general for retaining glycosyl hydrolases (44,
46).
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ACKNOWLEDGEMENTS |
---|
We thank K. Ng, M. Fraser, W. Wolodko, E. Bergman, and the staff of BioCARS and Stanford Synchrotron Radiation Laboratory for assistance with x-ray data collection; L. Burke for performing mass spectrometric analyses; and Dr. Brian Patrick for performing the X-ray structure determination of GalNAc-thiazoline.
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FOOTNOTES |
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* This work was supported in part by the United States Department of Energy, Basic Energy Sciences, Office of Science; the National Institutes of Health, National Center for Research Resources; the United States Department of Energy (Basic Energy Sciences, Bureau of Education and Research); the National Institutes of Health (National Center for Research Resources, National Institute of General Medical Sciences); the Medical Research Council of Canada; and the Natural Sciences and Engineering Research Council of Canada.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.
§ Supported by studentships from the Medical Research Council, the Alberta Heritage Foundation for Medical Research, and the University of Alberta.
Supported by studentships from the British Columbia Science
Council and Natural Sciences and Engineering Research Council of Canada.
§§ To whom correspondence should be addressed. Fax: 780-492-0886; E-mail: michael.james@ualberta.ca.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M011067200
2 D. J. Vocadlo and S. G. Withers, unpublished data.
3 M. Joshi, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
-hexosaminidase,
-N-acetylhexosaminidase;
HexA and HexB, human
-hexosaminidase A and B, respectively;
SpHEX, S.
plicatus
-hexosaminidase;
SmCHB. S. marcescens chitobiase, NAG, N-acetylglucosamine;
rms, root mean square;
MAD, multiwavelength anomalous diffraction.
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