(Received for publication, April 22, 1996, and in revised form, October 9, 1996)
From McGill University-Montreal Children's
Hospital Research Institute, Departments of § Biology,
Human Genetics, and
§§ Pediatrics, McGill University, Montreal,
Canada H3H 1P3, ¶ Centre de Recherches sur les
Macromolécules Végétales, CNRS, Grenoble F-38041,
Cedex 9, France, and
European Molecular Biology Laboratory
(EMBL), 22603, Hamburg, Germany
The -hexosaminidases (Hex)
catalyze the cleavage of terminal amino sugars on a broad spectrum of
glycoconjugates. The major Hex isozymes in humans, Hex A, a heterodimer
of
and
subunits (
), and Hex B, a homodimer of
subunits (
), have different substrate specificities. The
subunit (HEXB gene product), hydrolyzes neutral substrates.
The
subunit (HEXA gene product), hydrolyzes both
neutral and charged substrates. Only Hex A is able to hydrolyze the
most important natural substrate, the acidic glycolipid GM2 ganglioside. Mutations in the HEXA gene cause Tay-Sachs
disease (TSD), a GM2 ganglioside storage disorder. We
investigated the role of putative active site residues Asp-
258,
Glu-
307, Glu-
323, and Glu-
462 in the
subunit of Hex A. A
mutation at codon 258 which we described was associated with the TSD B1
phenotype, characterized by the presence of normal amounts of mature
but catalytically inactive enzyme. TSD-B1 mutations are believed to
involve substitutions of residues at the enzyme active site.
Glu-
307, Glu-
323, and Glu-
462 were predicted to be active site
residues by homology studies and hydrophobic cluster analysis. We used
site-directed mutagenesis and expression in a novel transformed human
fetal TSD neuroglial (TSD-NG) cell line (with very low levels of
endogenous Hex A activity), to study the effects of mutation at
candidate active site residues. Mutant HEXA cDNAs
carrying conservative or isofunctional substitutions at these positions
were expressed in TSD-NG cells.
E323D,
E462D, and
D258N
cDNAs produced normally processed peptide chains with drastically
reduced activity toward the
subunit-specific substrate 4MUGS. The
E307D cDNA produced a precursor peptide with significant
catalytic activity. Kinetic analysis of enzymes carrying mutations at
Glu-
323 and Asp-
258 (reported earlier by Bayleran, J., Hechtman,
P., Kolodny, E., and Kaback, M. (1987) Am. J. Hum.
Genet. 41, 532-548) indicated no significant change in substrate
binding properties. Our data, viewed in the context of homology studies
and modeling, and studies with suicide substrates, suggest that
Glu-
323 and Asp-
258 are active site residues and that Glu-
323
is involved in catalysis.
The -hexosaminidases (Hex1, EC
3.2.1.52) are lysosomal hydrolases that catalyze the cleavage of
terminal
-N-acetylglucosamine or
-N-acetylgalactosamine residues on a broad spectrum of
glycoconjugates. The major Hex isozymes in humans are: Hex A, a
heterodimer composed of one
and one
subunit and Hex B, a
homodimer of two
subunits. A third isozyme, Hex S, is composed of
two
subunits, which are unstable and not normally found in most
tissues. The
and
subunits are structurally related, sharing
60% amino acid identity in the mature form (1, 2). Both subunits are
catalytically active with different but overlapping substrate
specificities (3). The
subunit, in Hex A and Hex B, hydrolyzes
neutral substrates, whereas the
subunit, in Hex A and Hex S,
hydrolyzes neutral substrates as well as substrates bearing a negative
charge either on the terminal sugar (e.g.
GlcNac-SO4) or on a distinct residue (e.g.
sialic acid) (4). The latter includes the most important natural
substrate, the sialic-acid containing glycosphingiolipid, GM2 ganglioside, found mainly in neuronal tissue. Only Hex
A catalyzes cleavage of the terminal
-N-acetylgalactosamine on GM2 ganglioside in
the presence of the substrate-specific protein cofactor, the GM2-activator protein (5).
Mutations in the HEXA, HEXB, and GM2A
genes, encoding the and
subunits of Hex A and the
GM2 activator, respectively, lead to a group of inherited
neurodegenerative diseases, collectively known as the GM2
gangliosidoses, that are characterized by lysosomal accumulation of
GM2 ganglioside mainly in neuronal tissue. These disorders
range in severity from Tay-Sachs disease (TSD), a progressive and fatal
neurodegenerative disorder of infancy, to clinically milder or later
onset forms of GM2 gangliosidosis occurring in patients
with some residual enzyme activity (reviewed in Gravel et
al. (6)).
Although more than 70 mutations at the human HEXA locus, and
14 at the HEXB locus have been described (6), few have
revealed information about the location or properties of the and
subunit active sites. A subset of HEXA mutations, known
as the B1 mutations, lead to production of normal amounts of mature Hex
A, which is deficient in
subunit catalytic activity without
affecting
subunit activity (7). The B1 mutations are thus
compatible with normal maturation of Hex subunits and delivery of
structurally intact Hex A to the lysosome. The presence of a mature Hex
A, unable to hydrolyze charged substrates, suggested that the B1 biochemical phenotype might be associated with mutations at or near the
subunit active site (4, 7, 8). The first B1 mutations described
were
R178H and
R178C (7, 8, 9). In vitro generated
mutation at the homologous site to Arg-
178 in the
subunit,
Arg-
211, resulted in production of mature Hex B devoid of catalytic
activity (10). More recently, we identified a third B1 mutation,
D258H (11). Both Arg-
178 and Asp-
258 have been proposed as
candidates for participation at or near the active site of the
subunit.
The mechanism of cleavage of glycosidic bonds by Hex remains unclear.
Sinnott (12) has proposed that glycosyl hydrolases employ an
acid-catalysis mechanism involving the participation of two acidic
residues (a proton donor and a nucleophile) at the active site, as
identified in glycoside cleavage by -glucosidases (13, 14, 15, 16),
-glucosidases (17),
-glucanases (18, 19, 20),
-galactosidases
(21, 22, 23), and chitinases (24, 25) and the
-N-acetylglucosaminidase (chitobiase) (26). In contrast,
based on inhibition studies using nitrogen-containing substrate
inhibitors, Legler and Boothagen (27) and Legler et al. (28)
proposed that the mechanism of Hex catalysis involves both an acidic
and a basic residue and depends on a transition state in which the
glycosidic O of the substrate is joined with the acetyl group of the
pyranose ring, a mechanism fundamentally different from that of other
glycosidases.
In this study, we investigated candidate active site residues in the
subunit of Hex A through analysis of mutations that affect
catalytic activity without disrupting maturation of the enzyme. We
introduced conservative mutations at residue Asp-
258, as well as at
three glutamic acid residues (Glu-
307, Glu-
323, and Glu-
462)
which are evolutionarily invariant in family 20 of glycosyl hydrolases
using the classification system of Henrissat (29). We then studied the
impact of these substitutions on enzyme activity and maturation after
transfection of mutant cDNAs using an SV40-transformed neuroglial
(NG) cell line established from a fetus with Tay-Sachs disease which is
devoid of endogenously expressed
subunits but produces functional
subunits.
NG141 (NG) and NG125 (TSD-NG),
SV40-transformed fetal TSD and normal neuroglial cell lines,
respectively, were provided by L. Hoffman and S. Brooks (Kingsbrook
Jewish Medical Center, Brooklyn, NY). Cells were cultured in
-minimum essential medium with 15% fetal calf serum and
antibiotics.
The Escherichia coli
-galactosidase gene, in pSVL
gal (Clontech), was used as a
reporter gene to control for transfection efficiency. The cloned human
HEXA cDNA (1) was inserted into pRCCMV and pREP4
(Invitrogen) to produce pRCCMV
and pREP4
. Preparation of
PRCCMV
involved: 1) subcloning the HEXA cDNA from
PSVL
into pCRII (Invitrogen) as an XhoI/BamHI
fragment, 2) subcloning of an NsiI HEXA
cDNA-containing fragment PCRII subclone into the PstI
site of pBluescript (Stratagene), 3) subcloning an
XhoI/XbaI fragment from pBluescript into a PCRII
intermediate, and 4) cloning a NotI/XbaI
HEXA cDNA-containing fragment from the PCRII subclone into PRCCMV. Preparation of pREP4
involved cloning the
XhoI/BamHI HEXA cDNA-containing
fragment from PSVL
into pREP4. Construction of pCMV
required
creating a HEXA cDNA flanked by NotI sites. The HEXA cDNA insert in pRCCMV
has a NotI
site at the 5
end of the gene. This insert was subcloned into the
BamHI site of pBluescript (KS
, Stratagene) introducing a
second NotI site downstream from the 3
end of the gene.
This step permitted the cloning of the insert into the unique
NotI site of pCMV
. All plasmids were purified on Qiagen
columns prior to transfection.
Mutations were introduced into
the HEXA cDNA using a modified protocol of the Clontech
Transformer Mutagenesis Kit. The second screening step for mutant
clones was omitted. Mutant clones were identified after the first
screen by PCR amplification of samples of isolated disrupted bacterial
colonies followed by restriction enzyme digestion or allele-specific
hybridization of amplified product to identify mutant genotypes.
Mutations were introduced into pSVL, a cassette containing the
altered sequence was subcloned into pBS(KS)HEXA, and the
full-length mutant cDNA was subsequently subcloned into pCMV.
Mutant pCMV
inserts were sequenced (Pharmacia T7 sequencing kit) and
the plasmids purified on Qiagen columns prior to transfection.
For transient expression in cell line NG 125, subconfluent T175s (175 cm2, Sarstedt) were harvested by
trypsinization and washed twice with 1 × phosphate-buffered
saline. The cell pellet was resuspended in Optimem medium (Life
Technologies, Inc., containing 5% fetal bovine serum) to obtain a
final concentration of 6 × 106 cells/ml. Cell
suspension (800 µl), 20 µg of pCMV, and 2 µg of pSVL
gal
were placed in a 0.4-cm cuvette, mixed, placed on ice for 5 min, and
pulsed (500 microfarads, 400 V) using a Bio-Rad electroporation
apparatus. The time constant was between 16 and 18 ms. After 15 min on
ice, 800 µl of
-minimum essential medium (without antibiotics) was
added to the suspension. For the
-galactosidase qualitative assay,
250 µl of transfected cell suspension were plated on 12 multiwell
plates. The remainder of the transfected cell suspension was grown for
48 h in a T75 (75 cm2).
A qualitative -galactosidase
assay was performed to determine the percentage of surviving cells
which expressed bacterial enzyme. Multiwell-plated cells were incubated
as described by Lake (30). After 24-h incubations the number of blue
cells was estimated microscopically. Harvested cells were lysed by
freeze-thawing in 0.25 M Tris-HCl (pH 7.4) and protein
determined by the Bradford method (Bio-Rad). A fluorescent assay (31)
was adapted for quantitation of
-galactosidase activity in
transfected cell lysates. The reaction mixture contained 3 µl of
100 × magnesium solution (4.5 M 2-mercaptoethanol, 0.1 M MgCl2), 100 µl of 0.5 mM 4 methyl-umbelliferyl
-D-galactoside, and approximately
2-5 µg of lysate protein in 0.1 M sodium phosphate buffer (pH 7.5) in a volume of 334 ml. After incubation at 37 °C for
15 min, fluorescence, due to release of 4MU, was determined using a
Perkin-Elmer spectrofluorimeter (excitation wavelength, 360 nm;
emission, 447 nm). Hexosaminidase activity was also determined fluorometrically using either 4MUGS (
subunit substrate) (32) or the
4MUG (
subunit) substrate.
The enhanced chemiluminescence (ECL)
Western blotting kit from Amersham Corp. was used to detect the
presence of the Hex A subunit with a polyclonal rabbit anti-human
Hex A antibody. Both the primary and secondary (rabbit Ig, horseradish
peroxidase-labeled antibody) antibodies were used at a 1:5000 dilution.
The hexosaminidase isoenzyme profile in cell lysates was determined by chromatofocusing using the Pharmacia Polybuffer Exchanger (PEB) system according to a modified protocol of O'Dowd et al. (33). Transfected cell extracts were freeze-thawed (three times) in 0.025 M imidazole buffer (pH 7.4), and the protein concentration was determined by the Bradford method. All steps were carried out at 7 °C. Approximately 1 ml of PEB74 slurry was used to prepare a column in a 1-ml syringe. The column was washed with the equivalent of 1.5-2 × bed volume with 0.025 M imidazole. The protein extract was added after passing 2 × 100 µl of Polybuffer (pH 4.0) through the column. Four hundred-microliter fractions were collected. Sodium citrate buffer (0.13 M, pH 3.46) was used to elute Hex S after a pH of 4.0 was reached using a pH gradient.
Kinetic AnalysisThe Km and Ki for wild type and mutant Hex A proteins were determined using 4MUGS substrate at concentrations from 1.0 to 7.5 mM. For inhibition studies, the competitive inhibitor N-aetylglucosamine-6-phosphate (Sigma) was added to a final concentration of 10 mM.
The endogenous
activity of NG cells toward the -specific substrate 4MUGS was
715 ± 13 nmol/mg/h. In untransfected TSD-NG cells, the rate of
4MUGS hydrolysis is <1% of that in NG cells. This trace activity is
probably due to the residual action of Hex B on 4MUGS (32).
In order to maximize subunit expression through transfection,
several vectors carrying the HEXA gene cDNA were
assessed for their ability to drive expression of enzymatic activity
when transfected into TSD-NG cells. Transfection efficiency (percent surviving cells catalyzing 5-bromo-4-chloro-3-indoyl
-D-galactoside hydrolysis) was determined to be
10-20%. Hexosaminidase activity in cells transfected with 20 µg of
plasmids pSVL
, pRCCMV
, pREP4
, or pCMV
was at least 10-fold
higher than activity in mock-transfected cells (Table
I). The highest level of expression (approximately 1000 × mock-transfected cells) was achieved when cells were
transfected with pCMV
. Further analysis of Hex activity in TSD-NG
cells at 24, 48, and 72 h post-transfection with pCMV
(Table I)
showed that activity continued to increase throughout the 72-h period. The pCMV
plasmid expressed over a 48-h incubation period
post-transfection was selected for all subsequent experiments.
|
Western blot analysis of transfected (pCMV and pSVL
gal) and
mock-transfected (pSVL
gal) cell extracts confirmed that the increase
in 4MUGS activity in TSD-NG cells is associated with
subunit
expression (Fig. 1, lane N). Both precursor
and mature
subunit are absent in mock-transfected TSD-NG cell
extracts (Fig. 1, lane M) and present in cells transfected
with pCMV
.
In order to determine whether the subunits encoded by the
HEXA cDNA were expressed as the heterodimeric enzyme Hex
A (
) or the homodimeric species Hex S (
), the Hex isozyme
forms in transfected TSD-NG cell lysates were resolved by
chromatofocusing. Fig. 2a illustrates the
chromatofocusing profile of TSD-NG cells transfected with pCMV
and
assayed with 4MUG (all Hex) and 4MUGS (Hex A and Hex S) to detect all
Hex isozyme species. All three hexosaminidase isoenzymes were present,
and eluted at their expected pIs. Hex S was the isoenzyme present in
greatest abundance followed by Hex A and Hex B respectively. This high
expression of the nonphysiological Hex S in TSD-NG cells is the most
likely due to the massive overexpression of
subunits in the face of
limiting, endogenously produced
subunits. Total Hex activity in
pCMV
transfected TSD-NG cells (harvested at 48 h
post-transfection) is 5-6-fold greater than endogenous Hex activity in
untransfected normal NG cells (Table I). Given an efficiency of
transfection of 10-20%, the transfected cells express up to 60-fold
greater Hex activity than normal untransfected NG cells. Despite
overwhelming
subunit synthesis, a significant proportion (22%) of
the transfected gene product is expressed as the Hex A heterodimer as
shown by the chromatofocusing profile (Fig. 2).
Expression of HEXA Mutations
In order to evaluate the
expression of mutant subunits in TSD-NG cells, we initially
examined mutations known to cause the infantile acute (
R170W) (11)
subacute (
G250D) (35) and chronic (
G269S) (37) forms of
GM2 gangliosidosis. The mutant cDNAs were expressed in
TSD-NG cells (Table II) and cell lysates assayed for Hex
A specific activity using 4MUGS. Hex A activity in lysates of
G269S-
and
G250D-transfected TSD-NG cells was <4% and 1.5%, respectively, of the activity measured after transfection with wild
type pCMV
. These Hex A activities were 9- and 4-fold greater than
mock-transfected activity measured with the 4MUGS substrate. In
contrast, expression of
R170W exhibited no significant 4MUGS activity above that of mock transfected cells. Western blot analysis showed that the
subunit synthesized by all three mutant cDNAs appeared in the precursor form (Fig. 1, lanes A,
J, and I). Only the
G269S mutation appeared to
be associated with expression of a small amount of mature
subunit
(Fig. 1, lane A). These results are compatible with previous
findings on
G269S (34) and
G250D (35) in COS cell expression
studies and demonstrate complete inactivation of Hex A by the
R170W
mutation consistent with the infantile TSD phenotype. In the latter
case, the R170W substitution is associated with expression of an
subunit precursor (Fig. 1, lane I) but not with its
maturation and targeting to the lysosome.
|
We next examined
the expression in TSD-NG cells of mutations at amino acid residues
which are candidates for participation in the Hex A subunit
catalytic site. To assess the role of mutation at residue Asp-
258
(
D258H), first described in association with the B1 biochemical
phenotype of TSD, HEXA cDNA constructs carrying the
conservative or isosteric substitutions
D258H,
D258E, or
D258N
were expressed in TSD-NG cells and Hex A activity measured in
transfected cell lysates. All three mutations resulted in negligible or
trace amounts of Hex A activity, with
D258N exhibiting 2-fold background and the others the same activity as measured in
mock-transfected cells (Table III, Experiment 1).
Western blot analysis of transfected cell extracts (Fig.
3), revealed that maturation of pro-
chains carrying
the
D258H mutation was reduced. In contrast, expression
D258N and
D258E mutant cDNAs resulted in production of mature
subunit.
Chromatofocusing of
D258N cell extracts revealed that this mutation
significantly reduced 4MUGS hydrolysis by Hex A and Hex S (data not
shown). The 4MUG hydrolysis of Hex A was reduced to a lesser extent.
This result was expected since 4MUG hydrolysis is catalyzed by both the
and
subunits of Hex A.
|
We then studied the effects of mutation at conserved residues
Glu-307, Glu-
323, and Glu-
462, predicted as candidate active site residues by homology studies and hydrophobic cluster analysis of
family 20 glycosyl hydrolases. Fig. 4, a and
b, illustrate multiple alignment of the two regions of
family 20 enzymes which produce significant alignment.
Asterisks indicate the three evolutionarily invariant
glutamate residues identified as candidate active site residues using
the classification system of Henrissat. Other invariant amino acids are
also indicated.
HEXA cDNA constructs carrying the conservative
substitutions E307D,
E323D, or
E462D were expressed in TSD-NG
cells and Hex activity measured in transfected cell lysates. All three
substitutions had dramatic effects on catalytic activity (Table III,
Experiment 2).
E323D did not exhibit any 4MUGS activity above
background whereas
E462D and
E307D exhibited 14- and 60-fold
4MUGS activity above the background activity obtained in mock
transfected cells respectively. Western blot analysis of
transfected cell extracts showed that only the
E323D and
E462D
mutations were compatible with synthesis of significant amounts of
precursor and mature
subunit (Fig. 5).
Chromatofocusing of
E323D and
E462D cell extracts confirmed that
the Hex A isoenzyme is processed normally but has a catalytically
defective
subunit that is unable to hydrolyze 4MUG or 4MUGS (Fig.
2, b and c). The
E307D mutation, however,
dramatically reduces
subunit maturation.
Kinetic experiments were performed to evaluate the impact of the
E323D mutation on the binding affinity of Hex A for the substrates
4MUGS and for the competitive inhibitor
N-acetylglucosamine-6-PO4 (Fig.
6). The Lineweaver-Burke plots for the wild type enzyme are monophasic, representing the contribution of a single active site
to the hydrolysis of 4MUGS. In contrast, the kinetics of hydrolysis of
this substrate by the Hex A
E323D enzyme are biphasic revealing a
significant contribution of a second active site (the
subunit) at
high substrate concentrations. When the kinetic parameters of the high
affinity site are evaluated the Km for 4MUGS is 2.5 mM for Hex A
E323D compared to 1.7 mM for
the wild type enzyme. Similiarly Ki values for the
competitive inhibitor are 15.6 mM for the
E323D-substituted Hex A and 11.0 mM for wild type Hex A. We showed, previously, that the Km for Hex A in
fibroblasts from a patient carrying the D258H allele (and a null
allele) was identical to that of wild type enzyme (7).
-Hexosaminidase catalyzes the hydrolysis of its glycoside
substrates by a "retaining" mechanism in which the anomeric
configuration of the substrate remains unchanged (12, 36). Most
retaining glycosidases employ an acid catalysis mechanism in which two
amino acids function respectively as proton donor and nucleophile (12). Alternatively, glycosaminidases may employ a second mechanism, termed
substrate-assisted catalysis, in which an acidic residue and a basic
residue participate in substrate cleavage. In this study, we attempted
to identify acidic residues in the Hex A
subunit which may
participate in the active site of Hex A.
Since our studies involved the use of a novel TSD-NG expression system,
we first evaluated expression in this system of wild type and mutant
HEXA cDNAs. Transient expression of mutant
HEXA cDNAs in COS1 cells (reviewed in Brown and Mahuran
(34)) has several disadvantages which are overcome in the TSD-NG
expression system. COS1 cells exhibit a high level of endogenous Hex A
and Hex B activity which compromises the analysis of low levels of Hex
activity expressed by mutant HEXA cDNAs. Furthermore,
because the cells are of heterologous origin, human HEXA
gene mutations are expressed in COS1 cells as the non-physiological
isozyme Hex S (7, 37, 38), an homodimer which differs from Hex A both in stability and substrate specificity. As a result, the activity
of mutant
subunits produced following transfection of mutant
HEXA cDNAs, does not correlate well with clinical
severity of disease or biochemical phenotypes observed in patients (34, 35).
In contrast, TSD-NG cells, produce no endogenous subunits.
Endogenous expression of human
subunits in these cells,
however, permits dimerization with transfected human
subunits, to
form active Hex A. When wild type
subunits are overexpressed in
TSD-NG cells,
subunit concentration limits the formation of Hex A. As the
subunits become depleted, excess
subunits dimerize to
form Hex S, distinguishable from Hex A by chromatofocusing. The
presence of both the precursor (67 kDa) and mature (54 kDa) forms of
subunit following transfection confirms that newly synthesized
subunit can be normally processed and targeted to the lysosome, but
also indicates that excess
subunits are poorly processed in the
absence of
subunits.
We evaluated the expression in TSD-NG cells of subunits carrying
mutations associated with acute (
R170W), subacute (
G250D), and
chronic (
G269S) forms of GM2 gangliosidosis,
respectively, and demonstrated that residual enzyme activity was
inversely correlated with disease severity. Results for the
R170W
mutation confirmed our previous report (11), that this mutation causes
the classical infantile form of TSD. We proceeded to use this
expression system to evaluate the role of candidate active site
residues.
The four acidic amino acids we examined were chosen on the basis of
(a) occurrence in the B1 variant of Tay-Sachs disease (Asp-258) or (b) conservation in family 20, a sequence
related family of glycosyl hydrolases (
307,
323, and
462). The
TSD-B1 phenotype is believed to be caused by mutations which lead to substitution of residues with catalytic function (4, 7, 10). These
include the basic residue Arg-
178 (4, 8, 9, 10) (which is invariant in
family 20 enzymes) and the acidic Asp-
258 (7, 10). Conservative
substitutions at these positions have little affect on
Km but dramatically reduce the Vmax of Hex A (7). The residues Glu-
307,
Glu-
323, and Glu-
462 were selected on the basis of their
evolutionary invariance in family 20 glycosyl hydrolases (39), a
classification system (29) based on amino acid sequence similarity.
Members of homology families generally share folding characteristics
and catalytic residues within families are strictly conserved (40). The
three glutamate residues are invariant at homologous positions in all members of family 20. The position corresponding to Asp-
258 shows variation in a single enzyme which carries the functionally related acidic residue glutamate at this position.
Analysis of residual enzymatic activity and subunit maturation in
cells transfected with HEXA cDNAs carrying conservative or isosteric substitutions at residues Glu-
307 and Glu-
462 led us
to conclude that these residues are unlikely to have catalytic functions. Residual Hex A activity associated with both mutations was
too high to be compatible with the loss of either an acid catalyst or a
proton donor. Furthermore, the
E307D substitution prevented
subunit maturation (Fig. 5). In contrast, both the isosteric
substitution
D258N and the conservative substitution
E323D were
associated with production of mature Hex A with drastically reduced
enzyme activity. Since maturation of the
subunit is dependent on
dimerization with the
subunit, we were also able to conclude that
the
D258H and
E323D mutations are compatible with production of
normal amounts of
heterodimer and that loss of activity is not
the result of compromised dimerization. Furthermore, kinetic analysis
of residual Hex A activity in
E323D transfected cells (Fig. 6) or
D258H in patient fibroblasts (7) demonstrates that loss of enzymatic
activity is the consequence of decrease in kcat
rather than a change in Km.
Our proposal that Asp-258 and Glu-
323 are catalytic residues is
in accord with results of other structure/function studies. The x-ray
crystallographic structure for Serratia marcescens
chitobiase, the first member of family 20 glycosyl hydrolases to be
crystallized, was recently reported (26, 40, 42). Residues Glu-519 and Glu-740 of chitobiase are not located at or near the active site of the
bacterial enzyme suggesting, by analogy, that the homologous residues,
Glu-
307 and Glu-
462, in the
subunit of Hex A would also not
be present at the the
subunit active site. Most significantly, the
x-ray crystallographic data (26) identify Glu-540, the residue corresponding to Glu-
323, as the acid catalyst in the S. marcescens enzyme.
Members of family 20 glycosyl hydrolases share a conserved central
region which aligns in all members of the family (Fig. 4). According to
the crystal structure of chitobiase, this region comprises the
catalytic domain which has an /
barrel fold (26). The modeling of
the catalytic domain of Hex A (Fig. 7) (26), brings Arg-
178,
Asp-
258, and Glu-
323 in proximity within the substrate binding
cleft (facing toward the center of the
/
barrel) and spatially
arranged to facilitate catalysis. In contrast, Glu-
307, Glu-
462,
and Asp-
163 which corresponds to Asp-
196, a residue recently
proposed as an active site residue in the
subunit (44), appear to
be remote from the catalytic domain. While it is likely that the model,
illustrated in Fig. 7, will contain some errors in its
proposed structure of Hex A, the high degree of conservation of
residues in and around the active site suggest that the model is much
less prone to errors in the active site pocket of the enzyme.
A role for Glu-323 at the Hex A active site is also supported by the
studies of Liessem et al. (43). Using a mechanism-based pyrrolidine substrate analog, this group identified residue Glu-
355, which corresponds to Glu-
323, as the only reactive residue at or
near the
subunit active site. The role of Arg-
178 is less clear.
Given the high pKa of arginine (12.0) it is unlikely that this residue functions as a nucleophile at the pH optimum of Hex
(3.9-4.2). The role of active-site arginine may be to maintain the
acid catalyst in its protonated state at a pH which is significantly higher than the pKa for dicarboxylic amino
acids.
The extensive sequence similarity of the and
subunits
underscores the structural and catalytic similarity of two subunits of
Hex A. Chimeric Hex enzymes, generated by the fusion of different segments of the
and
subunits have recently been used to
identify domains required for substrate specificity (41, 45). The
structural modelling of Hex should now permit the more precise
localization of other residues involved in the active site as well as
in other functions of the enzyme, such as dimerization, substrate
binding and activator recognition.
We thank Franelli Yadeo and Daniel Phaneuf for advice and technical assistance.