Mechanism of Human Nucleocytoplasmic Hexosaminidase D

Article abstract of DOI:10.1021/acs.biochem.5b01285

Mammalian β-hexosaminidases have been shown to play essential roles in cellular physiology and health. These enzymes are responsible for the cleavage of the monosaccharides N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) from cellular subs

Full text of DOI:10.1021/acs.biochem.5b01285

Article
pubs.acs.org/biochemistry
Mechanism of Human Nucleocytoplasmic Hexosaminidase D
Matthew G. Alteen,†,‡ Verena Oehler,‡ Ivana Nemcovicova,§,∥ Iain B. H. Wilson,§ David J. Vocadlo,*,†
̌
̌
́
and Tracey M. Gloster*,‡
†Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada
‡Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, U.K.
§Department fur Chemie, Universitat fur Bodenkultur, Muthgasse 18, A-1190 Wien, Austria
̈
̈
̈
S
* Supporting Information
ABSTRACT: Mammalian β-hexosaminidases have been
shown to play essential roles in cellular physiology and health.
These enzymes are responsible for the cleavage of the
monosaccharides N-acetylglucosamine (GlcNAc) and N-
acetylgalactosamine (GalNAc) from cellular substrates. One
of these β-hexosaminidases, hexosaminidase D (HexD),
encoded by the HEXDC gene, has received little attention.
No mechanistic studies have focused on the role of this
unusual nucleocytoplasmically localized β-hexosaminidase, and its cellular function remains unknown. Using a series of kinetic
and mechanistic investigations into HexD, we define the precise catalytic mechanism of this enzyme and establish the identities of
key enzymic residues. The preparation of synthetic aryl N-acetylgalactosaminide substrates for HexD in combination with
measurements of kinetic parameters for wild-type and mutant enzymes, linear free energy analyses of the enzyme-catalyzed
hydrolysis of these substrates, evaluation of the reaction by nuclear magnetic resonance, and inhibition studies collectively reveal
the detailed mechanism of action employed by HexD. HexD is a retaining glycosidase that operates using a substrate-assisted
catalytic mechanism, has a preference for galactosaminide over glucosaminide substrates, and shows a pH optimum in its second-
order rate constant at pH 6.5−7.0. The catalytically important residues are Asp148 and Glu149, with Glu149 serving as the
general acid/base residue and Asp148 as the polarizing residue. HexD is inhibited by Gal-NAG-thiazoline (Ki = 420 nM). The
fundamental insights gained from this study will aid in the development of potent and selective probes for HexD, which will serve
as useful tools to improve our understanding of the physiological role played by this unusual enzyme.
studies have indicated a link between this enzyme and
ammals possess four genes encoding β-hexosaminidases.
MThese enzymes are responsible for the cleavage of the rheumatoid arthritis.10 The intracellular and extracellular roles
played by this enzyme within cells are therefore topics of great
interest.
terminal monosaccharides N-acetylglucosamine (GlcNAc) and
N-acetylgalactosamine (GalNAc) from cellular substrates. The
HEXA and HEXB genes encode an α subunit and a β subunit,
respectively, that dimerize to form three different isozymes:
hexosaminidase A (HexA; comprising an α subunit and a β
subunit), hexosaminidase B (HexB; comprising two β
subunits), and hexosaminidase S (HexS; comprising two α
subunits).1−3 All three isozymes localize to the lysosome, where
they hydrolyze terminal GlcNAc and GalNAc residues from
glycoconjugates and oligosaccharides to aid in the disposal of
unneeded glycoconjugates. Deficiencies in either of these genes
lead to the genetic diseases known as Sandhoff and Tay-Sachs
disease.4 O-GlcNAcase (OGA), encoded by the HEXC gene, is
a nucleocytoplasmic β-hexosaminidase.5 This enzyme removes
GlcNAc residues from proteins bearing N-acetylglucosamine
O-linked to serine and threonine residues of nucleocytoplasmic
proteins (O-GlcNAc) and has been implicated in an array of
physiological processes and also found to be essential for
development of mammals.6−8 The fourth gene encoding a
mammalian β-hexosaminidase (HEXDC) was identified re-
cently.9 While the physiological substrate and function of the
gene product, hexosaminidase D (HexD), remain unknown,
Preliminary characterization of recombinantly expressed
murine HexD, which is 80% similar to human HexD, shows
it can hydrolyze substrates containing both GalNAc and
GlcNAc but with a preference for the former.9 This differs from
OGA, which exclusively hydrolyses GlcNAc-containing sub-
strates,5 whereas HexA, HexB, and HexS can hydrolyze
substrates containing both GlcNAc and GalNAc. HexD was
shown, by reverse transcriptase polymerase chain reaction (RT-
PCR) experiments, to be ubiquitously expressed in all murine
tissues examined and localized to the nucleus and cytoplasm
upon being overexpressed in mammalian cells.9 Interestingly,
the biochemical properties of recombinantly expressed murine
HexD are in accord with the characteristics of an enzyme
observed in bovine brain tissue isolates decades earlier;11,12
however, the gene encoding it was unknown, and this
observation received limited interest.
Received: November 30, 2015
Revised: April 13, 2016
© XXXX American Chemical Society
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Figure 1. Substrate-assisted catalytic mechanism employed by GH20 enzymes. Binding of substrate to the enzyme leads to formation of a Michaelis
complex in which the pyranose ring of the substrate adopts a conformation placing the leaving group in a pseudoaxial orientation. This allows
nucleophilic attack of the anomeric center by the C2 acetamido group and departure of the leaving group. The resulting transient bicyclic oxazoline
intermediate is then hydrolyzed by attack of a molecule of water at the anomeric center, releasing the sugar hemiacetal product. Two conserved
enzymic carboxyl groups provide general acid and general base catalysis during each step of the reaction.
Glycoside hydrolases (GHs) are classified by sequence
homology into more than 100 families [Carbohydrate Active
enZyme (CAZy) database].13 In general, the enzyme
mechanism and three-dimensional fold are conserved among
members within any family. The CAZy database classifies
HexD, along with the lysosomal β-hexosaminidases encoded by
the HEXA and HEXB genes, into family GH20. Conversely,
OGA bears no sequence similarity to the other β-
hexosaminidases and falls into family GH84. Although there
is a low degree of sequence conservation between these two
families of mammalian β-hexosaminidases, studies have shown
they share a similar, but unusual, substrate-assisted catalytic
mechanism.14−16 The three-dimensional structures of human
HexA,17 human HexB,16 and bacterial homologues of OGA18,19
have also provided insights into the active site architecture,
revealing how these enzymes facilitate catalysis using key
enzymic residues. Together, these mechanistic and structural
studies have guided the development of potent and specific
inhibitors against β-hexosaminidases,14,16,18−20 which have
acted as powerful tools for probing the cellular function of
the enzymes and the roles they play in disease.21−24
Investigation into the mechanism of the family GH20
lysosomal β-hexosaminidases and their homologues has shown
they catalyze hydrolysis with retention of stereochemistry using
a substrate-assisted catalytic mechanism (Figure 1).25−27 This
mechanism involves the 2-acetamido group of the N-
acetylhexosamine residue of the substrate playing an essential
role during catalysis. In the first step of the mechanism, a
conserved active site carboxyl residue polarizes this acetamido
group, increasing its nucleophilicity and facilitating its attack at
the anomeric position, which results in the formation of an
oxazoline intermediate. Concomitantly, a second conserved
aspartate or glutamate residue, the general acid/base residue,
aids glycosidic bond cleavage and leaving group departure by
providing general acid catalysis. During the second step of the
reaction, the acid/base residue acts as a general base to facilitate
attack of a water molecule at the anomeric center of the
oxazoline intermediate. The overall result is the net retention of
stereochemistry of the resulting N-acetylglycopyranose product.
To gain insights into HexD at the molecular level, we
chemically synthesized a series of aryl N-acetylgalactosaminide
substrates and, using these, undertook a series of kinetic and
mechanistic investigations to elucidate the detailed mechanism
of action of this enzyme. While chromogenic substrates such as
p-nitrophenyl glycosides will differ in structure at the anomeric
center compared to the physiological substrate for HexD, these
compounds serve as effective tools for probing the binding
affinities, kinetic properties, and mechanistic features of
glycoside hydrolases.14,28 We envisage that by understanding
the subtleties of HexD-catalyzed hydrolysis of its substrates, the
design of useful tools will be enabled, including potent and
selective inhibitors that could be used to probe the
physiological function of HexD. While our knowledge of the
cellular function of HexD lags behind that of the other
mammalian β-hexosaminidases, given the increasing important
roles played by β-hexosaminidases in health and disease,
gaining a clear fundamental understanding of this unusual
nucleocytoplasmically localized enzyme is important.
MATERIALS AND METHODS
■
HexD Protein Expression and Purification. The plasmid
containing the gene encoding human HexD in a pET30a
vector, which has been reported previously,9 was transformed
into Escherichia coli BL21(DE3) cells. Successful transformants
were cultured in Luria-Bertani broth supplemented with 50 μg/
mL kanamycin at 37 °C until an optical density of 0.6
absorbance units was reached. Protein expression was induced
with 0.5 mM isopropyl β-D-thiogalactoside at 15 °C for 20 h.
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Cells were harvested and resuspended in 20 mM HEPES (pH
7.4), 150 mM NaCl, and 5 mM imidazole and incubated in the
presence of 1 mg/mL lysozyme, 0.02 mg/mL DNase, and an
EDTA-free protease inhibitor tablet (Roche) for 20 min at 4
°C. Cells were lysed by high-pressure cell disruption (Constant
Systems). Following clarification, the supernatant was applied
to a 5 mL HisTrap nickel column (GE Healthcare), pre-
equilibrated in the same buffer, and the protein was eluted from
an imidazole gradient. The elution was concentrated and
applied to a HiPrep 26/10 desalting column equilibrated in 20
mM HEPES (pH 7.4), 150 mM NaCl buffer to remove the
imidazole. The elution was subsequently concentrated and
applied to a S200 16/60 gel filtration column, pre-equilibrated
in the same buffer, and fractions judged to be pure were pooled
for subsequent kinetic studies.
reaction, the solvent was removed under vacuum, and then
CH2Cl2 (50 mL) and NaHCO3 (saturated aqueous, 50 mL)
were added to the residue. The organic layer was collected, and
the aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The
combined organic fractions were washed with a NaHCO3
solution (50 mL) and brine (50 mL), dried with MgSO4,
filtered, and concentrated under vacuum. Purification by flash
column silica gel chromatography (97.5:2:0.5 DCM/MeOH/
TEA) afforded the title compound as a yellow gum (105 mg,
1
44%): H NMR (400 MHz, CDCl3) δH 8.21 (d, J = 9.2 Hz,
2H), 7.12 (d, J = 9.2 Hz, 2H), 5.43 (d, J = 3.3 Hz, 1H), 4.98 (d,
J = 7.9 Hz, 1H), 4.89 (dd, J = 10.8, 3.2 Hz, 1H), 4.23−4.08 (m,
3H), 3.52 (dd, J = 10.8, 7.9 Hz, 1H), 2.15 (s, 3H), 2.07 (s, 3H),
2.06 (s, 3H); 13C NMR (100 MHz, CDCl3) δC 170.44, 170.37,
170.24, 161.53, 143.23, 125.88, 116.66, 101.66, 73.24, 71.65,
66.11, 61.71, 51.41, 20.87, 20.81, 20.75; LRMS (ESI+) [M +
H]+ calcd for C18H24N2O10 m/z 427.14, found m/z 427.13.
4-Nitrophenyl 3,4,6-Tri-O-acetyl-2-deoxy-2-fluoroacetami-
do-β-D-galactopyranoside (S3b). Sodium fluoroacetate (24
mg, 0.235 mmol) was added to a mixture of Amberlite IR-120
resin (H+ form) in anhydrous DMF. After the mixture had been
stirred for 1 h, the fluoroacetic acid solution was added to a
stirred solution of compound S2 (100 mg, 0.235 mmol) and
triethylamine (0.12 mL, 0.823 mmol) in anhydrous DMF, and
the resulting mixture was stirred overnight at room temper-
ature. Upon completion, the solvent was removed under
vacuum, and then CH2Cl2 (50 mL) and NaHCO3 (saturated
aqueous, 50 mL) were added to the residue. The organic layer
was collected, and the aqueous layer was extracted with CH2Cl2
(2 × 50 mL). The combined organic fractions were washed
with water (50 mL) and brine (50 mL), dried with MgSO4,
filtered, and concentrated under vacuum. The crude product
was purified by flash column silica gel chromatography (40:60
EtOAc/hexanes) to afford the product as white crystals (81 mg,
HexD Mutagenesis. The primers listed in Table S1 were
used to amplify the plasmid encoding the HexD gene with the
desired mutation. Reaction mixtures were subjected to
digestion with DpnI for 2 h at 37 °C and subsequently
transformed into E. coli DH5α cells. Plasmid DNA was
extracted from cells using standard procedures and sub-
sequently sequenced (GATC sequencing) to ensure the
mutation was successfully incorporated. Protein expression
and purification were performed as described for wild-type
HexD, and the enzymes were obtained in similar yields.
General Procedures for Synthesis of Compounds. All
synthetic reagents used in this study were obtained from Sigma-
Aldrich (Oakville, ON), Carbosynth (San Diego, CA), Alfa
Aesar (Ward Hill, MA), or Acros Organics (Geel, Belgium).
Anhydrous reactions were conducted in flame-dried glassware
under a positive pressure of dry argon. Air- or moisture-
sensitive reagents and anhydrous solvents were transferred with
oven-dried syringes or cannulae. Flash chromatography was
performed using E. Merck silica gel (230−400 mesh). Solution-
phase reactions were monitored using analytical thin layer
chromatography (TLC) with E. Merck 0.2 mm precoated silica
gel aluminum plates 60 F254; compounds were visualized by
illumination with short-wavelength (254 nm) ultraviolet light
and/or staining with a ceric ammonium molybdate or
potassium permanganate staining solution. Pyridine was dried
extensively over activated 4 Å molecular sieves under argon. 1H
NMR (400 or 500 MHz) and 13C NMR (100 or 125 MHz)
spectra were recorded at ambient temperature on a Bruker
Avance III 400 or 500 NMR spectrometer. Deuterated
chloroform (CDCl3), acetone [(CD3)2CO], dimethyl sulfoxide
(DMSO-d6), methanol (CD3OD), and water (D2O) were used
as NMR solvents, unless otherwise stated. Chemical shifts are
reported in parts per million downfield from tetramethylsilane
(TMS) and corrected using the solvent residual signal as a
reference. Splitting patterns are designated as follows: s, singlet;
d, doublet; t, triplet; q, quartet; quin, quintet; hex, hextet; m,
multiplet; br, broad. Low- and high-resolution mass spectrom-
etry was performed on a Micromass Quattro-LC electrospray
spectrometer with a pump rate of 20 μL/min using electrospray
ionization (ESI).
1
71%): H NMR (500 MHz, CDCl3) δH 8.20 (d, J = 9.3 Hz,
2H), 7.10 (d, J = 9.3 Hz, 2H), 6.50 (dd, J = 8.6, 3.4 Hz, 1H),
5.56 (d, J = 8.4 Hz, 1H), 5.52−5.47 (m, 2H), 4.76 (d, J = 47.3
Hz, 2H), 4.33 (m, 1H), 4.22−4.16 (m, 2H), 2.19 (s, 3H), 2.08
(s, 3H), 2.04 (s, 3H); 13C NMR (125 MHz, CDCl3) δC 170.52,
170.47, 170.23, 168.46, 161.41, 143.25, 126.87, 116.71, 97.87,
80.16, 71.63, 69.29, 66.57, 61.74, 51.22, 20.79, 20.77, 20.76; 19
F
NMR (500 MHz, CDCl3) δF −225.54 (t, J = 47.1 Hz); LRMS
(ESI+) [M + Na]+ calcd for C20H23FN2O11Na m/z 509.12,
found m/z 509.11.
4-Nitrophenyl 3,4,6-Tri-O-acetyl-2-deoxy-2-difluoroaceta-
mido-β-D-galactopyranoside (S3c). Compound S2 (95 mg,
0.223 mmol) was dissolved in anhydrous DMF (5 mL) in a 50
mL flame-dried round-bottom flask charged with argon.
Triethylamine (0.11 mL, 0.781 mmol) and difluoroacetic acid
(15 μL, 0.223 mmol) were added; a solution of HBTU (296
mg, 0.781 mmol) in 5 mL of DMF was transferred via syringe,
and the reaction mixture was stirred overnight at room
temperature. Upon completion, the solvent was removed
under vacuum, and then CH2Cl2 (50 mL) and NaHCO3
(saturated aqueous, 50 mL) were added to the residue. The
organic layer was collected, and the aqueous layer was extracted
with CH2Cl2 (3 × 50 mL). The combined organic fractions
were washed with NaHCO3 (saturated aqueous, 50 mL) and
brine (50 mL), dried with MgSO4, filtered, and concentrated
under vacuum. The crude residue was purified by silica gel flash
column chromatography (40:60 EtOAc/hexanes) to yield the
4-Nitrophenyl 2-Amino-3,4,6-tri-O-acetyl-2-deoxy-β-D-gal-
actopyranoside (S2). 3,4,6-Tri-O-acetyl-2-amino-2-deoxy-β-D-
galactopyranosyl bromide hydrobromide (S1, 250 mg, 0.557
mmol) was added to a 50 mL flame-dried round-bottom flask
under an atmosphere of argon. The compound was dissolved in
anhydrous CH3CN (10 mL); sodium p-nitrophenolate (180
mg, 1.11 mmol) was added, and the reaction mixture was
stirred overnight at room temperature. Upon completion of the
1
product as white crystals (73 mg, 62%): H NMR (400 MHz,
CDCl3) δH 8.19 (d, J = 9.3 Hz, 2H), 7.09 (d, J = 9.3 Hz, 2H),
C
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6.76 (d, J = 8.7 Hz, 1H), 5.86 (t, J = 54.3 Hz, 1H), 5.47 (dd, J =
7.2, 3.4 Hz, 1H), 5.45 (d, J = 8.4 Hz, 1H), 5.43 (m, 1H), 4.41
(dd, J = 8.5, 10.9 Hz, 1H), 4.22−4.16 (m, 3H), 2.19 (s, 3H),
2.07 (s, 3H), 2.02 (s, 3H); 13C NMR (125 MHz, CDCl3) δC
170.57, 170.51, 170.19, 163.43, 161.37, 143.39, 125.92, 116.82,
108.37, 98.14, 71.71, 69.19, 66.49, 61.64, 51.37, 20.80, 20.76,
20.63; 19F NMR (400 MHz, CDCl3) δF −127.57 (d, J = 48.5
Hz), −127.68 (d, J = 48.5 Hz); LRMS (ESI+) [M + Na]+ calcd
for C20H22F2N2O11Na m/z 527.11, found m/z 527.09.
112.42, 97.92, 72.15, 69.16, 66.76, 62.32, 51.39, 23.64, 20.77,
20.63; HRMS (ESI+) [M + H]+ calcd for C20H24N3O13 m/z
514.1304, found m/z 514.1296.
3-Fluoro-4-nitrophenyl 2-Acetamido-3,4,6-tri-O-acetyl-2-
deoxy-β-D-galactopyranoside (S5f). The compound was
synthesized according to general procedure A and purified by
column chromatography (60:40 to 70:30 EtOAc/hexanes, off-
white crystals, 43% yield): 1H NMR (500 MHz, CDCl3) δ 8.05
(t, J = 8.8 Hz, 1H), 6.98 (dd, J = 12.5, 2.5 Hz, 1H), 6.89 (dd, J
= 9.3, 2.5 Hz, 1H), 6.00 (d, J = 8.4 Hz, 1H), 5.56−5.43 (m,
3H), 4.26−4.19 (m, 3H), 4.13 (m, 1H), 2.17 (s, 3H), 2.08 (s,
3H), 2.03 (s, 4H), 1.95 (s, 3H); 13C NMR (126 MHz, CDCl3)
δ 171.11, 170.63, 170.15, 162.09, 158.30, 155.67, 131.82,
127.53, 112.90, 105.68, 97.71, 71.66, 69.37, 66.67, 62.05, 50.95,
23.40, 20.63, 20.61, 20.54; HRMS (ESI+) [M + H]+ calcd for
C20H24FN2O11 m/z 487.1364, found m/z 487.1349.
4-Nitrophenyl 3,4,6-Tri-O-acetyl-2-deoxy-2-trifluoroaceta-
mido-β-D-galactopyranoside (S3d). Compound S2 (100 mg,
0.235 mmol) and triethylamine (0.1 mL, 0.705 mmol) were
dissolved in anhydrous CH2Cl2 (10 mL) in a 50 mL round-
bottom flask. The mixture was cooled to 0 °C, and
trifluoroacetic anhydride (0.1 mL, 0.705 mmol) was added
dropwise. The contents were then stirred overnight at room
temperature. Upon completion of the reaction, NaHCO3
(saturated aqueous, 50 mL) was added, the organic layer was
collected, and the aqueous layer was extracted with CH2Cl2 (3
× 50 mL). The combined organic fractions were washed with
additional NaHCO3 solution (50 mL) and brine (50 mL), dried
with MgSO4, filtered, and concentrated. The crude residue was
purified by silica gel flash column chromatography using
gradient elution (30:70 to 40:60 EtOAc/hexanes) to furnish
3-Nitrophenyl 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-
D-galactopyranoside (S5g). The compound was synthesized
according to general procedure A and purified by column
chromatography (60:40 to 70:30 EtOAc/hexanes, off-white
1
crystals, 53% yield): H NMR (500 MHz, CDCl3) δ 7.93 (d, J
= 8.2 Hz, 1H), 7.89 (s, 1H), 7.45 (t, J = 8.2 Hz, 1H), 7.34 (d, J
= 8.2 Hz, 1H), 5.64 (d, J = 8.7 Hz, 1H), 5.48−5.43 (m, 2H),
5.44 (d, J = 8.3 Hz, 1H), 4.32−4.10 (m, 4H), 2.18 (s, 3H), 2.09
(s, 3H), 2.04 (s, 3H), 1.97 (s, 3H); 13C NMR (126 MHz,
CDCl3) δ 170.81, 170.79, 170.57, 170.30, 157.39, 149.16,
130.22, 123.81, 118.01, 111.36, 98.79, 71.70, 69.49, 66.94,
62.19, 51.60, 23.58, 20.79, 20.73; HRMS (ESI+) [M + H]+
calcd for C20H25N2O11 m/z 469.1453, found m/z 469.1471.
4-Chlorophenyl 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
β-D-galactopyranoside (S5h). The compound was synthesized
according to general procedure A and purified by column
chromatography (60:40 EtOAc/hexanes, white crystals, 40%
yield): 1H NMR (500 MHz, CDCl3) δ 7.23 (d, J = 8.9 Hz, 2H),
6.95 (d, J = 8.9 Hz, 2H), 5.61 (d, J = 8.6 Hz, 1H), 5.44−5.41
(m, 2H), 5.28 (d, J = 8.4 Hz, 1H), 4.22−4.14 (m, 3H), 4.07
(dd, J = 6.6, 6.6 Hz, 1H), 2.17 (s, 3H), 2.05 (s, 3H), 2.02 (s,
3H), 1.95 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 170.81,
170.53, 170.50, 170.37, 155.74, 129.50 (2C), 128.19, 118.42
(2C), 99.32, 71.09, 69.66, 66.78, 61.74, 51.61, 23.48, 20.78,
20.75 (2C); HRMS (ESI+) [M + H]+ calcd for C20H25NO9Cl
m/z 458.1237, found m/z 458.1242.
Phenyl 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galac-
topyranoside (S5i). The compound was synthesized according
to general procedure A and purified by column chromatog-
raphy (60:40−70:30 EtOAc/hexanes, yellow solid, 37% yield):
1H NMR (500 MHz, CDCl3) δ 7.29 (t, J = 7.9 Hz, 2H), 7.06 (t,
J = 7.4 Hz, 1H), 7.01 (d, J = 8.0 Hz, 2H), 5.50 (d, J = 8.5 Hz,
1H), 5.46−5.39 (m, 2H), 5.30 (d, J = 8.4 Hz, 1H), 4.29−4.12
(m, 3H), 4.08 (t, J = 6.6 Hz, 1H), 2.17 (s, 3H), 2.05 (s, 3H),
2.03 (s, 3H), 1.97 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
170.82, 170.48, 170.45, 170.37, 157.14, 129.48, 123.03, 116.94,
99.21, 70.85, 69.78, 66.75, 61.66, 51.48, 23.34, 20.68, 20.65;
HRMS (ESI+) [M + Na]+ calcd for C20H25NO9Na m/z
446.1432, found m/z 446.1422.
1
the product as a white amorphous solid (49 mg, 40%): H
NMR (500 MHz, CDCl3) δH 8.16 (d, J = 9.2 Hz, 2H), 7.14 (d,
J = 9.0 Hz, 1H), 7.08 (d, J = 9.2 Hz, 2H), 5.47 (d, J = 3.3 Hz,
1H), 5.40 (dd, J = 11.3, 3.3 Hz, 1H), 5.37 (d, J = 8.4 Hz, 1H),
4.50 (dd, J = 11.1, 8.7 Hz, 1H), 4.21−4.16 (m, 3H), 2.19 (s,
3H), 2.06 (s, 3H), 2.02 (s, 3H); 13C NMR (125 MHz, CDCl3)
δC 170.77, 170.61, 170.26, 161.34, 143.34, 125.92, 116.76,
98.16, 71.72, 69.28, 66.41, 60.64, 51.51, 20.77, 20.71, 20.55; 19
F
NMR (470 MHz, CDCl3) δF −72.21 (br); LRMS (ESI+) [M +
Na]+ calcd for C20H21F3N2O11Na m/z 545.10, found m/z
545.09.
General Procedure A: Synthesis of Aryl 2-Acetamido-
3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosides
(S5e−j). To a mixture of 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-α-D-galactopyranosyl chloride (S4, 1 equiv), benzyltrie-
thylammonium chloride (1 equiv), and acceptor phenol (2
equiv) was added sufficient dichloromethane (1 volume) to
yield a solution of 200 mM chlorosugar. An equal volume of 1
M sodium hydroxide was added, and the resulting biphasic
mixture was stirred vigorously at room temperature for 1−3 h.
Upon completion, ethyl acetate (5 volumes) was added, and
the organic phase was washed successively with 1 M NaOH (1
volume), water (2 × 1 volume), and brine (1 volume). The
combined organic layers were dried (MgSO4), filtered, and
concentrated under vacuum. The crude product was purified by
column chromatography to produce aryl 2-acetamido-3,4,6-tri-
O-acetyl-2-deoxy-β-D-galactopyranosides as crystalline solids in
yields ranging from 39 to 68%.
3,4-Dinitrophenyl 2-Acetamido-3,4,6-tri-O-acetyl-2-
deoxy-β-D-galactopyranoside (S5e). The compound was
synthesized according to general procedure A and purified by
column chromatography (60:40 to 80:20 EtOAc/hexanes, off-
white crystals, 35% yield): 1H NMR (400 MHz, CDCl3) δ 7.99
(d, J = 9.1 Hz, 1H), 7.51 (d, J = 2.6 Hz, 1H), 7.30 (dd, J = 9.1,
2.6 Hz, 1H), 6.07 (d, J = 8.4 Hz, 1H), 5.59 (d, J = 8.3 Hz, 1H),
5.54−5.45 (m, 2H), 4.27 (dd, J = 11.4, 4.3 Hz, 1H), 4.25−4.18
(m, 2H), 4.09−4.04 (m, 1H), 2.17 (s, 3H), 2.09 (s, 3H), 2.03
(s, 3H), 1.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 171.10,
170.88, 170.80, 170.15, 160.36, 145.31, 136.26, 127.19, 120.36,
4-Methoxyphenyl 2-Acetamido-3,4,6-tri-O-acetyl-2-
deoxy-β-D-galactopyranoside (S5j). The compound was
synthesized according to general procedure A and purified by
column chromatography (60:40 EtOAc/hexanes, off-white
1
crystals, 39%): H NMR (500 MHz, CDCl3) δ 6.96 (d, J =
9.0 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 5.53 (d, J = 8.2 Hz, 1H),
5.43−5.37 (m, 2H), 5.18 (d, J = 8.4 Hz, 1H), 4.24−4.13 (m,
3H), 4.02 (t, J = 6.7 Hz, 1H), 2.17 (s, 3H), 2.05 (s, 3H), 2.03
D
DOI: 10.1021/acs.biochem.5b01285
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
(s, 3H), 1.98 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 170.58,
170.57, 170.53, 170.40, 155.76, 151.32, 118.69, 114.68, 100.51,
71.03, 69.78, 66.81, 61.64, 55.81, 23.64, 20.86, 20.83; HRMS
(ESI+) [M + H]+ calcd for C21H28NO10 m/z 454.1708, found
m/z 454.1702.
General Procedure B: Synthesis of Aryl 2-Acetamido-
2-deoxy-β-D-galactopyranosides (1e−j). To a solution of
aryl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyrano-
side (1 equiv) in anhydrous methanol was added a spatula
tip of anhydrous sodium methoxide. The mixture was stirred at
room temperature (2−3 h or overnight). Amberlite IR-120
resin (H+ form, pH 4) was prerinsed with MeOH and added to
the mixture, which was stirred for an additional 10 min until a
pH of 5−6 was reached. The mixture was filtered and
concentrated to yield aryl 2-acetamido-2-deoxy-β-D-galactopyr-
anoside as white crystals (93−99%). The products were
crystallized from a MeOH/Et2O solvent prior to use in
biological assays.
67.38, 60.32, 51.53, 23.05; HRMS (ESI+) [M + H]+ calcd for
C14H18N2O8F m/z 361.0968, found m/z 361.0969.
3-Nitrophenyl 2-Acetamido-2-deoxy-β-D-galactopyrano-
1
side (1g). H NMR (500 MHz, MeOD) δ 7.90 (m, 2H),
7.53 (t, J = 8.2 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 5.15 (d, J =
8.4 Hz, 1H), 4.3 (dd, J = 9.6, 9.2 Hz, 1H), 3.93 (m, 1H), 3.84−
8.72 (m, 4H), 1.99 (s, 3H); 13C NMR (126 MHz, MeOD) δ
174.24, 159.41, 131.44, 124.24, 118.12, 112.52, 101.00, 77.37,
72.78, 69.53, 62.41, 54.06, 23.00; HRMS (ESI+) [M + H]+
calcd for C14H19N2O8 m/z 343.1063, found m/z 343.1058.
4-Chlorophenyl 2-Acetamido-2-deoxy-β-D-galactopyrano-
side (1h). 1H NMR (500 MHz, MeOD) δ 7.25 (d, J = 8.9 Hz,
1H), 7.02 (d, J = 8.9 Hz, 1H), 4.99 (d, J = 8.4 Hz, 1H), 4.17
(dd, J = 10.7, 8.4 Hz, 1H), 3.90 (d, J = 3.2 Hz, 1H), 3.85−3.74
(m, 1H), 3.71 (dd, J = 10.7, 3.2 Hz, 1H), 3.66 (d, J = 6.0 Hz,
1H), 1.98 (s, 2H); 13C NMR (126 MHz, MeOD) δ 174.19,
157.88, 130.32 (2C), 128.37, 119.30 (2C), 101.31, 77.18, 72.92,
69.58, 62.47, 54.19, 22.98; HRMS (ESI+) [M + H]+ calcd for
C14H19NO6Cl m/z 332.0895, found m/z 332.0885.
Phenyl 2-Acetamido-2-deoxy-β-D-galactopyranoside (1i).
1H NMR (500 MHz, MeOD) δ 7.26 (t, J = 7.9 Hz, 2H), 7.03
(d, J = 8.0 Hz, 2H), 6.99 (t, J = 7.3 Hz, 1H), 5.01 (d, J = 8.4 Hz,
1H), 4.22−4.15 (m, 1H), 3.90 (d, J = 2.9 Hz, 1H), 3.79 (m,
2H), 3.72 (dd, J = 10.7, 3.2 Hz, 1H), 3.66 (t, J = 6.0 Hz, 1H),
1.98 (s, 3H); 13C NMR (126 MHz, MeOD) δ 174.20, 159.27,
130.43, 123.44, 117.74, 101.29, 77.07, 73.00, 69.60, 62.46,
54.33, 22.98; HRMS (ESI+) [M + H]+ calcd for C14H20NO6 m/
z 298.1285, found m/z 298.1294.
4-Methoxyphenyl 2-Acetamido-2-deoxy-β-D-galactopyra-
noside (1j). 1H NMR (500 MHz, MeOD) δ 6.98 (d, J = 9.0 Hz,
2H), 6.82 (d, J = 9.0 Hz, 2H), 4.88 (d, J = 8.5 Hz, 1H), 4.14
(dd, J = 10.4, 8.7 Hz, 1H), 3.89 (d, J = 2.9 Hz, 1H), 3.84−3.75
(m, 2H), 3.74 (s, 3H), 3.69 (dd, J = 10.7, 3.2 Hz, 1H), 3.62 (t, J
= 6.0 Hz, 1H), 1.99 (s, 3H); 13C NMR (126 MHz, MeOD) δ
174.18, 156.70, 153.32, 119.18, 115.48, 102.40, 77.00, 73.02,
69.60, 62.46, 56.04, 54.40, 23.00; HRMS (ESI+) [M + H]+
calcd for C15H22NO7 m/z 328.1391, found m/z 328.1389.
Taft-like Analyses. Kinetic analyses with 4-nitrophenyl N-
acetylgalactosamine (PNP-GalNAc) and derivatives with
mono-, di-, and trifluoroacetamido groups were conducted at
37 °C in phosphate-buffered saline (PBS) buffer (pH 7.4).
Reactions were performed in triplicate using 96-well plates
(Sarstedt) with final reaction volumes of 200 μL (100 μL of
substrate and 100 μL of enzyme). Final concentrations of
substrates ranged from 2 mM to 15.6 μM. Upon addition of
HexD (final concentration of 100 nM), reactions were
monitored continuously using a Molecular Devices SpectraMax
M5 spectrophotometer at 400 nm. Initial velocities were
calculated within the linear region of the reaction progress
curve and compared to a standard curve of varying
concentrations of 4-nitrophenol measured under identical
conditions. Michaelis−Menten kinetic parameters were calcu-
lated for all substrates through nonlinear regression of
Michaelian saturation curves using Graphpad Prism. Substrates
were predissolved in DMSO (final volume of 1−2%) to
facilitate solubility, followed by addition of PBS. Test reactions
with PNP-GalNAc revealed no significant difference in enzyme
activity in the presence or absence of DMSO.
4-Nitrophenyl 2-Deoxy-2-fluoroacetamido-β-D-galacto-
1
pyranoside (1b). H NMR (500 MHz,DMSO-d6) δH 8.22 (d,
J = 9.1 Hz, 2H), 8.06 (d, J = 9.5 Hz, 1H), 7.17 (d, J = 9.2 Hz,
2H), 5.25 (d, J = 8.4 Hz, 1H), 4.91 (d, J = 6.3 Hz, 1H), 4.81 (d,
J = 46.9 Hz, 2H), 4.78−4.71 (m, 2H), 4.21 (m, 1H), 3.78 (br s,
1H), 3.68 (br s, 1H), 3.62−3.53 (m, 3H); 13C NMR (125
MHz, DMSO-d6) δC 162.28, 141.80, 125.80, 116.60, 98.66,
80.83, 76.01, 70.68, 67.32, 60.28, 51.17; 19F NMR (500 MHz,
DMSO-d6) δF −226.05; HRMS (ESI+) [M + Na]+ calcd for
C14H17FN2O8Na m/z 383.0861, found m/z 383.0862.
4-Nitrophenyl 2-Deoxy-2-difluoroacetamido-β-D-galacto-
pyranoside (1c). 1H NMR (500 MHz, MeOD) δH 8.21 (d, J =
9.1 Hz, 2H), 7.19 (d, J = 9.2 Hz, 2H), 6.06 (t, J = 53.7 Hz, 1H),
5.23 (d, J = 8.4 Hz, 1H), 4.34 (dd, J = 10.5, 8.5 Hz, 1H), 3.95
(d, J = 3.1 Hz, 1H), 3.85−3.74 (m, 4H); 13C NMR (125 MHz,
MeOD) δC 163.72, 144.08, 126.64, 117.79, 110.09, 100.17,
77.55, 72.14, 69.51, 62.41, 53.91; 19F NMR (500 MHz, DMSO-
d6) δF −127.51 (d, J = 53.5 Hz); HRMS (ESI+) [M + Na]+
calcd for C14H16F2N2O8Na m/z 401.0767, found m/z
401.0768.
4-Nitrophenyl 2-Deoxy-2-trifluoroacetamido-β-D-galacto-
pyranoside (1d). 1H NMR (500 MHz, MeOD) δH 8.20 (d, J =
9.2 Hz, 2H), 7.18 (d, J = 9.2 Hz, 2H), 5.23 (d, J = 8.5 Hz, 1H),
4.35 (dd, J = 8.7, 10.3 Hz, 1H), 3.96 (d, J = 3.2 Hz, 1H), 3.86−
3.77 (m, 4H); 13C NMR (125 MHz, MeOD) δC 163.52,
144.13, 126.69, 117.72, 100.04, 77.58, 71.90, 69.51, 62.38,
54.39; 19F NMR (470 MHz, MeOD) δF −77.46 (br); HRMS
(ESI+) [M + Na]+ calcd for C14H15F3N2O8Na m/z 419.0678,
found m/z 419.0675.
3,4-Dinitrophenyl 2-Acetamido-2-deoxy-β-D-galactopyra-
1
noside (1e). H NMR (500 MHz, MeOD) δ 8.11 (d, J = 9.0
Hz, 1H), 7.62 (d, J = 2.6 Hz, 1H), 7.42 (dd, J = 9.1, 2.6 Hz,
1H), 5.23 (d, J = 8.4 Hz, 1H), 4.25 (dd, J = 10.7, 8.4 Hz, 1H),
3.92 (d, J = 3.2 Hz, 1H), 3.85−3.71 (m, 4H), 1.98 (s, 1H); 13
C
NMR (126 MHz, MeOD) δ 174.29, 162.45, 128.54, 120.96,
114.04, 100.64, 77.69, 72.57, 69.48, 62.45, 53.69, 22.99; HRMS
(ESI+) [M + H]+ calcd for C14H18N3O10 m/z 388.0987, found
m/z 388.0981.
3-Fluoro-4-nitrophenyl 2-Acetamido-2-deoxy-β-D-galac-
1
topyranoside (1f). H NMR (500 MHz, MeOD) δ 8.11 (t, J
= 8.9 Hz, 1H), 7.08 (dd, J = 12.9, 2.6 Hz, 1H), 6.99 (d, J = 9.3
Hz, 1H), 5.17 (d, J = 8.5 Hz, 1H), 4.23 (dd, J = 10.7, 8.3 Hz,
1H), 3.92 (d, J = 3.3 Hz, 1H), 3.87−3.69 (m, 3H), 1.98 (s,
3H); 13C NMR (126 MHz, DMSO-d6) δ 162.78, 157.42,
155.34, 130.95, 127.94, 112.94, 105.42, 98.88, 75.94, 70.98,
Kinetic Characterization of Wild-Type HexD and Its
Mutants. The kinetic parameters of wild-type HexD and its
mutants were determined essentially as described above using
PNP-GalNAc and 3F4NP-GalNAc as substrates. In addition,
kinetic parameters for wild-type HexD were determined with 4-
E
DOI: 10.1021/acs.biochem.5b01285
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Figure 2. Determination of substrate preference and stereochemical outcome for HexD hydrolysis. (A) Michaelis−Menten kinetics for HexD-
●
▲
catalyzed hydrolysis of PNP-GalNAc (1a, ) and PNP-GlcNAc (2a, ). (B) Michaelis−Menten kinetics for HexD-catalyzed hydrolysis of 3F4NP-
1
■
●
GalNAc (1f, ) and 3F4NP-GlcNAc (2b, ). Error bars represent the SEM. (C) H NMR spectrum showing the progress of HexD hydrolysis of
PNP-GalNAc. The initial product formed was β-GalNAc, which was subsequently mutarotated to α-GalNAc (500 MHz, D2O). (D) Structures of
PNP-GalNAc (1a), PNP-GlcNAc (2a), 3F4NP-GalNAc (1f) and 3F4NP-GlcNAc (2b).
nitrophenyl GlcNAc (PNP-GlcNAc) and 3-fluoro-4-nitro-
phenyl GlcNAc (3F4NP-GlcNAc). Final concentrations of
substrates ranged from 15.6 μM to 2 mM; final concentrations
of enzyme were 100 nM for the wild type and 100 nM to 2 μM
for the mutants depending on their activity. Reaction volumes
of 200 μL were used in all cases. The wavelengths at which
measurements were taken correspond to the absorbance
maxima of the aryl leaving groups, which have been previously
determined as shown in Table S2. Michaelis−Menten
parameters were calculated from nonlinear regression of
Michaelian saturation curves using GraphPad Prism. Relative
activities are reported as a ratio of the second-order rate
constants of wild-type HexD versus those of its mutants.
Brønsted Analyses. Brønsted analyses with N-acetylgalac-
tosamine-derived substrates with a range of leaving groups were
performed as described above with the exception that reactions
were conducted in quartz cuvettes (Sarstedt, volume of 200 μL,
path length of 1.0 cm). Substrate concentrations ranged from
15.6 μM to 2 mM; enzyme concentrations used for each
substrate are listed in Table S2. Reactions were performed in
duplicate and monitored using the wavelengths listed in Table
S2. Michaelian saturation kinetics were observed for all
substrates and were calculated as described above.
5−5.5; 50 mM phosphate, 100 mM NaCl buffer was used for
reactions in the pH range of 6.0−7.5, and 50 mM CHES, 100
mM NaCl buffer was used for reactions in the pH range of 8.0−
9.0. The stability of HexD at each pH was verified. Absorbance
values were corrected according to the proportion of
protonated and deprotonated 4-nitrophenol at each pH value.
A final enzyme concentration of 100 nM was used for each
reaction, and saturation kinetics were observed in all cases;
Michaelis−Menten parameters were calculated from saturation
curves as described above. A bell-shaped ionization curve,
described by eq 1, was fitted to the data.
kcat
KM
max × 10pH−pKa1
kcat
KM
=
102×pH−pK 1−pK 2 + 10pH−pK 1 + 1
a
a
a
(1)
Experimental kinetic pKa values were obtained from the pH
values corresponding to the half-maximal enzyme activity on
each limb of the curve.
NMR Experiments. NMR-based monitoring of HexD-
catalyzed hydrolysis was conducted using a Bruker AvanceII
500 MHz spectrometer equipped with a 5 mm TXI inverse
probe. 0.6 mg of PNP-GalNAc was dissolved in 600 μL of
deuterated phosphate-buffered saline [137 mM NaCl, 2.7 mM
KCl, and 10 mM Na2HPO4 and KH2PO4 in D2O (pH 7.4)],
and a t0 spectrum was recorded. HexD was exchanged into
deuterated PBS by repeated concentration using a 10 kDa
molecular mass cutoff centrifugation device and subsequent
pH Profiles. Reactions were conducted in 96-well plates as
described above, using PNP-GalNAc as the substrate (final
concentration between 15.6 μM and 2 mM). 50 mM citrate,
100 mM NaCl buffer was used for reactions in the pH range of
F
DOI: 10.1021/acs.biochem.5b01285
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Biochemistry
Article
resuspension; 120 μL of HexD was added to the substrate at a
final concentration of 1 μM. Reaction progress was monitored
at 5 min intervals for 1 h, with a final spectrum recorded 3 h
after enzyme addition. Spectra were processed using
MestReNova version 10.
Inhibition Studies. Inhibition studies of HexD with Gal-
NAG-thiazoline29,30 were performed in 96-well plates using
PNP-GalNAc as the substrate (62.5 μM to 1 mM) with 0.0063
mg/mL enzyme. Gal-NAG-thiazoline was tested at six
concentrations ranging from 5 times above to 5 times below
the apparent Ki. Data were plotted using GraphPad Prism, and
Ki values were obtained using nonlinear regression analysis with
the equation for competitive binding: KM(app) = KM(1 + [I]/Ki);
Y = VmaxX/[KM(app) + X].
each substrate were similar, ranging between 30.4 and 122
min−1, and the difference in catalytic efficiency between the
GalNAc- and GlcNAc-containing substrates is governed
primarily by differences in the Michaelis constant (KM).
Michaelis constant values were determined to be 72 μM for
PNP-GalNAc (1a) as compared to 607 μM for PNP-GlcNAc
(2a), and 172 μM for 3F4NP-GalNAc (1f) compared to 852
μM for 3F4NP-GlcNAc (2b). These studies show HexD has a
preference for galactosaminide substrates, although glucosami-
nides are processed surprisingly well, suggesting there is either
plasticity in the active site region around the C4 position or a
pocket that tolerates stereoisomers at this position.
Establishing the Stereochemical Outcome. Other β-
hexosaminidases have been shown to hydrolyze β-linked
substrates and release either β-GalNAc or β-GlcNAc products
upon hydrolysis, illustrating that they operate with retention of
stereochemistry.14,29 This outcome is enforced by participation
of the N-acetamido group, employed in the substrate-assisted
hydrolytic mechanism used by these β-hexosaminidases, which
limits nucleophilic attack to only the α face of the carbohydrate.
To determine whether HexD also operates with retention of
stereochemistry, we monitored the HexD-catalyzed hydrolysis
of 4-nitrophenyl GalNAc in deuterated phosphate-buffered
saline by NMR spectroscopy (Figure 2C). After 5 min, a new
doublet at 4.65 ppm emerges with a coupling constant of 8.4
Hz, consistent with the anomeric proton signal of the β-anomer
of GalNAc. After 15 min, an additional peak appears at 5.23
ppm with a J of 3.4 Hz, indicative of mutarotation from the β-
anomer to the thermodynamically preferred α-anomer. A final
equilibrium 55:45 α:β anomeric mixture is eventually reached
after consumption of PNP-GalNAc. The formation of the β-
anomer as the product that is observed first, prior to
mutarotation, provides direct experimental evidence that
HexD is a retaining glycosidase.
RESULTS
■
Substrate Selectivity. Gutternigg et al. qualitatively
showed previously that HexD was active on synthetic substrates
containing both GalNAc and GlcNAc β-linked to the leaving
group, while it was not active on those substrates containing an
α-linkage or on gluco- or galacto-derived substrates lacking an
N-acetyl group.9 We first set out to establish the detailed kinetic
parameters for HexD acting on both gluco- and galactosami-
nide substrates. Kinetic parameters were determined with
substrates containing 4-nitrophenyl (PNP) or 3-fluoro-4-
nitrophenyl (3F4NP) leaving groups (Figure 2A,B,D).
Measurements showed that the catalytic efficiency as reflected
in the second-order rate constant (kcat/KM) was higher for
substrates containing GalNAc than for substrates containing
GlcNAc: 21-fold higher for the 4-nitrophenyl leaving group and
only 7-fold higher for the 3-fluoro-4-nitrophenyl leaving group
(Table 1). Interestingly, the first-order rate constants (kcat) for
a
Table 1. Kinetic Parameters for HexD Hydrolysis of
Galactosaminides vs Glucosaminides
Assessing the Extent of the Involvement of the
Substrate Acetamido Group by Taft Analysis. To evaluate
the involvement of the substrate acetamido group in catalysis
and establish whether HexD employs a substrate-assisted
mechanism like other GH20 β-hexosaminidases, a series of
galacto-configured synthetic substrates were synthesized to
modulate the nucleophilicity of the carbonyl group. Similar
studies have been used to examine β-hexosaminidases that
hydrolyze glucosaminide substrates.14,28 Substrates that in-
corporated zero, one, two, or three electronegative fluorine
atoms at the N-acyl group were synthesized, and the kinetic
kcat/KM
substrate
KM (mM)
kcat (min−1
)
(min−1 mM−1
)
PNP-GalNAc (1a) 0.0721 0.0048
79.3 1.2
1090 75
50.1 4.6
707 81
PNP-GlcNAc (2a)
0.607 0.053
0.172 0.019
30.4 0.89
122 3.9
3F4NP-GalNAc
(1f)
3F4NP-GlcNAc
(2b)
0.852 0.054
92.4 2.7
109 7.6
a
Calculated kinetic parameters represent the mean SEM of triplicate
values.
Figure 3. HexD activity with N-fluoroacetyl PNP-GalNAc substrates. (A) Michaelis−Menten kinetics of N-fluoroacetyl PNP-GalNAc derivative
■
●
▲
▼
hydrolysis catalyzed by HexD: ( ) PNP-GalNAc (1a), ( ) PNP-GalNAc-F (1b), ( ) PNP-GalNAc-F2 (1c), and ( ) PNP-GalNAc-F3 (1d). Error
bars represent the SEM. (B) Taft-like linear free energy analysis plotting log(kcat/KM) for HexD-catalyzed hydrolysis of substrates 1a−1d vs the Taft
electronic parameter (σ*) of the N-fluoroacetyl groups. (C) Structures of N-fluoroacetyl PNP-GalNAc substrates (1b−1d).
G
DOI: 10.1021/acs.biochem.5b01285
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Biochemistry
Article
parameters governing their hydrolysis by HexD was deter-
mined. Panel A of Figure 3 show the Michaelis−Menten plots
of data for HexD acting on PNP-GalNAc (1a) and the series of
fluorinated derivatives (1b−1d) (Figure 3C). Incorporation of
each successive fluorine atom significantly impairs the rate of
hydrolysis, with a 34-fold reduction in kcat/KM for trifluorinated
substrate 1d relative to that of nonfluorinated PNP-GalNAc 1a
(Table 2). A plot of the log kcat/KM values against the Taft
7.2). The decrease in second-order rate constant kcat/KM is 870-
fold for the D148A mutant compared to that of WT HexD and
>4000-fold for the D148N mutant. While the KM for both
Asp148 variants increased 3−10-fold, the loss of overall
catalytic efficiency was due primarily to a decrease in the rate
of turnover as reflected by the lower kcat values. The activity of
the E149A mutant was affected to a lesser extent, with the
E149A and E149Q mutations leading to a 1.6−2.2-fold
decrease in kcat/KM when PNP-GalNAc was used as the
substrate.
a
Table 2. Kinetic Parameters for HexD Hydrolysis of PNP-
GalNAc and Fluorinated Derivatives
We also tested the importance of His92, which is conserved
on the basis of sequence alignments with the GH20 lysosomal
β-hexosaminidases (Figure S1) and is predicted to be an active
site residue on the basis of structural information from the
lysosomal β-hexosaminidases, for HexD activity. The kcat/KM
for the H92A mutant was 70-fold lower than that of the wild-
type enzyme, suggesting it makes an important contribution to
catalysis and may reside in the enzyme active site as seen for
His262 in HexA and His294 in HexB.16,17 The overall decrease
in kcat/KM is contributed solely by a decrease in the rate of
turnover, whereas the KM is consistent with the wild-type value.
Brønsted Linear Free Energy Analyses. To further
probe the importance of Asp148 and Glu149 as catalytic
residues, Brønsted linear free energy analyses were conducted
for WT HexD and the D148A and E149A mutants. A panel of
aryl galactosaminide substrates (1e−1j) (Figure 4C) was
synthesized, and Michaelis−Menten kinetic parameters were
determined for each enzyme using these substrates as well as 4-
methylumbelliferyl 2-acetamido-2-deoxy-β-D-glucopyranoside
(3). The logarithm of the second-order rate constant measured
for turnover of each substrate was plotted against the pKa of the
corresponding phenol leaving group (Figure 4A). A βlg (V/K)
value of −0.44 0.05 was obtained for WT HexD, indicating a
moderate dependence on leaving group ability. In contrast,
more negative βlg values of −0.89 and −0.85 were obtained for
the D148A and E149A mutant enzymes, respectively. The
steeper negative slopes of both mutants relative to the WT
enzyme are indicative of a stronger dependency on leaving
group ability. This perturbation may stem from the inability of
the mutants to stabilize the accumulation of charge in the
transition state as the anomeric substituent departs, leading to a
transition state in which there is greater charge development on
the departing glycosidic oxygen. Alternatively, the steeper
slopes observed for these βlg (V/K) values could be explained
by the mutations causing a change in the position of the
transition state along the reaction coordinate. In any event,
these data provide strong direct evidence that D148 and E149
are key active site residues involved in transition state
stabilization.
kcat/KM
b
substrate
σ*
KM (mM)
kcat (min−1
)
(min−1 mM−1
827 71
)
PNP-GalNAc
(1a)
0
0.176 0.014
145 3.5
PNP-GalNAc-F
0.8
0.533 0.017
0.102 0.016
0.103 0.027
156 1.9
299 11
159 25
24.5 6.5
(1b)
PNP-GalNAc-F2 2.0
(1c)
16.2 0.65
2.52 0.17
PNP-GalNAc-F3 2.8
(1d)
a
Calculated kinetic parameters represent the mean SEM of triplicate
b
values. Values of the Taft electronic parameters for N-fluoroacetyl
substituents were taken from ref 39.
electronic parameter (σ*) of the acyl groups reveals a Taft
reaction constant (ρ) of −0.49 0.1 (Figure 3B). The negative
slope of this correlation is consistent with the carbonyl group of
the N-acetamido group participating as a nucleophile in the
enzyme-catalyzed reaction. This provides strong evidence of
HexD employing a substrate-assisted hydrolysis mechanism,
similar to those of other mammalian β-hexosaminidases in GH
families 20 and 84.
Establishing the Importance of Residues Predicted To
Be Involved in Catalysis. We next sought to identify key
active site residues involved in the substrate-assisted mechanism
of HexD. By examining sequence homology across enzymes of
GH family 20 (Figure S1), we identified Asp148 and Glu149 as
the predicted candidate residues acting as the catalytic
polarizing and acid/base residues. Site-directed mutagenesis
was used to produce HexD variants in which these residues
were mutated to either alanine or asparagine/glutamine. The
activity of these mutants was assessed relative to that of wild-
type (WT) HexD using PNP-GalNAc or 3F4NP-GalNAc as
the substrate. Mutation of either residue to alanine or the
corresponding amide caused a loss of activity (Table 3). Full
Michaelis−Menten kinetics for the D148A mutant of HexD
could be determined only when 3F4NP-GalNAc was used as
the substrate, likely because it bears a leaving group (3F4NP;
pKa = 6.4) that is better than that of PNP-GalNAc (PNP; pKa =
a
Table 3. Kinetic Parameters for Hydrolysis by Wild-Type HexD and Various Mutants
substrate
KM (mM)
kcat (min−1
)
kcat/KM (min−1 mM−1
)
relative activity (WT/mutant)
WT
3F4NP-GalNAc
PNP-GalNAc
3F4NP-GalNAc
3F4NP-GalNAc
PNP-GalNAc
3F4NP-GalNAc
PNP-GalNAc
PNP-GalNAc
0.172 0.019
0.0721 0.0048
0.566 0.068
1.73 0.27
122 3.9
79.1 1.2
708 83
1090 77
0.815 0.11
0.169 0.030
691 52
1
1
WT
D148A
D148N
E149A
E149A
E149Q
H92A
0.461 0.023
0.293 0.027
36.1 0.52
176 4.8
868
4190
1.6
0.0525 0.0034
0.213 0.018
0.175 0.031
0.196 0.028
825 74
0.85
2.2
89.3 4.6
508 94
3.09 0.13
15.8 2.4
69
a
Calculated kinetic parameters represent the mean SEM of triplicate values.
H
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Figure 4. Investigation into the roles played by predicted catalytic residues for HexD. (A) Brønsted linear free energy plot of HexD-catalyzed
◆
□
●
hydrolysis of aryl GalNAc derivatives (1a and 1e−1j) as a function of aryl leaving group pKa: ( ) wild-type HexD, ( ) D148A mutant, and ( )
■
●
E149A mutant. (B) pH dependence of HexD hydrolysis of PNP-GalNAc (1a): ( ) wild-type HexD () and ( ) E149A mutant (---). Error bars
represent the SEM. (C) Structures of aryl GalNAc substrates (1e−1j) and 4-methylumbelliferyl-GalNAc (3) used in Brønsted analyses.
pH Dependence of HexD Activity. The pH dependence
of HexD activity was determined by measuring kcat/KM across a
pH range of 5.0−9.0. To perform these studies, we first
established that HexD was stable for at least 30 min over this
pH range under the conditions used for the assay. A plot of the
logarithm of the second-order rate constant versus pH (Figure
4B) displays a bell-shaped profile with a pH optimum of 6.5−
7.0, which is not unexpected given the subcellular localization
of HexD in the nucleus and cytoplasm. The pH profile for
HexD is consistent with that observed for the GH84 β-
hexosaminidase OGA,31,32 which is similarly localized to the
nucleus and cytoplasm. When a bell-shaped ionization curve
(eq 1) was fit to the data, the kinetic pKa values of the D148
and E149 residues can be estimated by identifying the pH
Figure 5. (A) Inhibition of HexD using Gal-NAG-thiazoline (4).
values corresponding to half of the maximum kcat/KM value on
Lineweaver−Burk plot of HexD-catalyzed hydrolysis of PNP-GalNAc
each limb of the curve. Using this technique, the pKa values of
(1a) in the presence of varying concentrations of Gal-NAG-thiazoline
the acidic and basic limbs were estimated to be 5.6 and 7.8,
respectively. The pH dependence of activity for the E149A
mutant displays a similar bell-shaped curve but has an alkaline
shift in comparison to wild-type HexD with a pH optimum of
7.5. This shift suggests E149 plays a key role in catalysis but
indicates at least two other residues contribute to the pH
profile. The kinetic pKa values of the E149A pH curve are 6.7
for the acidic limb and 7.8 for the basic limb. A small decrease
in activity and a small upward shift in pH optimum were also
observed for E149A and E149Q mutants of the murine HexD
(data not shown). Unfortunately, we were unable to determine
the pH dependence of activity for the D148A mutant as its
activity was extremely low even at the pH optimum.
Inhibition of HexD. NAG-thiazoline was first designed as a
mimic of the oxazoline intermediate that forms during
substrate-assisted hydrolysis for hexosaminidases,29 and this
thiazoline feature has been successfully incorporated into
inhibitors of a range of hexosaminidases that employ a
substrate-assisted catalytic mechanism.14,16,17,25,33 Given the
preference of HexD for galacto-configured substrates, we
decided to test the inhibition of HexD by galacto-configured
NAG-thiazoline (Gal-NAG-thiazoline, 4; Figure 5B). Com-
pound 4 is an effective inhibitor of HexD-catalyzed hydrolysis
of PNP-GalNAc, and the Lineweaver−Burk plot confirms an
expected competitive mode of inhibition (Figure 5A). Non-
linear regression reveals the Ki value to be 420 nM. A very
similar value (520 nM) has been determined in preliminary
studies with the murine enzyme (data not shown). Curiously,
we were unable to observe any inhibition with gluco-configured
◆
■
○
▼
▲
(4). Concentrations of 4 of ( ) 20, ( ) 10, ( ) 5, ( ) 2.5, ( )
●
1.25, and ( ) 0 μM. Error bars represent the SEM. (B) Structure of
Gal-NAG-thiazoline (4).
NAG-thiazoline at a concentration of 1 mM despite the
significant activity we observe for this enzyme toward β-N-
acetylglucosaminide substrates. Inhibition of HexD by 4
provides further evidence that it employs a catalytic mechanism
involving anchimeric assistance with formation of a transient
oxazoline intermediate.
DISCUSSION
■
Glycoside hydrolases from GH families 18, 20, 56, 84, and 85
have been shown to operate through a substrate-assisted
catalytic mechanism (Figure 1) with some mechanistic
variations but usually involving two carboxyl residues
positioned within the active site.14,15,25,32−34 One of these
residues acts as a polarizing residue, hydrogen bonding with the
N−H bond of the acetamido group to both appropriately orient
and promote attack of the carbonyl oxygen at the anomeric
center. Concurrently, a general acid/base residue, acting as a
general acid in the first step of the reaction, stabilizes the
developing charge on the departing glycosidic oxygen to
facilitate its departure, leading to formation of the bicyclic
oxazoline intermediate. The oxazoline is hydrolyzed through
the near microscopic reverse of the first step where the acid/
base residue acts as a base; it is considered a “near” microscopic
reverse because in the first step of the mechanism the leaving
I
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Table 4. Comparison of the Kinetic and Inhibition Properties of Hexosaminidases HexA and HexB (GH20), OGA (GH84), and
HexD (GH20)
HexD
HexA
HexB
OGA
a
b
b
b
GlcNAc/GalNAc selectivity
Taft reaction constant (ρ)
βlg (V/K)
0.048
−0.49 0.1
8.6
8.6
>50
a
j
c
c
e
ND
−1.0 0.1
−0.29
0.19
−0.42 0.08
−0.11 0.01
0.070
a
j
d
−0.44 0.05
ND
a
f
f
c
Ki for NAG-thiazoline (μM)
>1000
0.27
c
0.070
a
g
h
i
Ki for Gal-NAG-thiazoline (4) (μM)
0.42
0.82
0.32
poor
a
b
c
d
e
f
g
h
i
j
From this work. From ref 40. From ref 14. From ref 28. From ref 41. From ref 42. From ref 43. From ref 44. From ref 21. Not determined.
group/nucleophile is an alcohol whereas in the second step it is
a water molecule.
which the leaving group is departing. Accordingly, the relatively
constant KM values observed among these fluorinated
substrates suggest that binding ability is not substantially
affected by substitution with fluorine and imply that the
electronic effects of the fluorine atoms are the major
contributor to the reduction in hydrolysis rate for HexD.
Sequence alignments of enzymes within a CAZy family have
demonstrated that the catalytic residues, most commonly two
carboxylic acid residues, are highly conserved. Structural and
kinetic studies have demonstrated that GH families 18 and 56
possess a DXE motif, where the aspartate and glutamate
residues are the critical residues used to effect catalysis.32,34 The
lysosomal β-hexosaminidases HexA and HexB, members of GH
family 20, use a conserved DE catalytic pair with two carboxylic
acid residues adjacent to each other25 (Figure S1), whereas
GH84 enzymes, such as OGA, use a DD catalytic pair.31
Unusually, GH85 enzymes possess an asparagine residue in
place of the aspartate in an NXE motif; the asparagine has been
proposed to exist as the imidic acid tautomer to accept a
hydrogen bond from the substrate amide.33
The data obtained in this study through kinetic analyses of
HexD mutants strongly support the assignment of Asp148 and
Glu149 as the key catalytic residues. Mutation of Asp148 to
either alanine or asparagine significantly impairs the activity of
the enzyme even in the presence of substrates with activated
leaving groups. Notably, mutation of Glu149 to alanine or
glutamine has a much less pronounced effect on the rate of
hydrolysis of activated substrates such as PNP-GalNAc. These
data suggest Glu149 functions as the catalytic general acid/base
during catalysis, because the modest reductions in activity
observed for the E149 mutants with activated substrates can be
rationalized by the low pKa values of the phenolic leaving
groups of PNP-GalNAc and 3F4NP-GalNAc (7.18 and 6.42,
respectively). These leaving groups cannot benefit greatly from
general acid catalysis when the microscopic pKa of the general
acid is greater than the pKa of the leaving group. These changes
in activity in association with mutating the general acid/base
catalytic residue are consistent with analysis of the correspond-
ing mutants of OGA.31 Analysis of the pH dependence of wild-
type HexD activity reveals a bell-shaped profile (Figure 4B),
with an acidic limb pKa of 5.6 and a basic limb pKa of 7.8. Such
pH profiles are notoriously hard to dissect fully,35 but the
simplest interpretation is the bell shape stems from titration of
the two catalytic residues. In such a case, the acidic limb would
reflect titration of the polarizing residue and the basic limb
titration of the acid/base residue. If this interpretation is
correct, the pKa values of the activated substrates are lower than
the kinetic pKa of the general acid/base catalytic residue.
Accordingly, the general acid catalysis afforded by Glu149 is
inefficient because it cannot donate a proton to the departing
phenol. When tested against substrates bearing worse leaving
A distinguishing feature of the substrate-assisted catalytic
mechanism employed by β-hexosaminidases is the participation
of the 2-acetamido group of the substrate in which the carbonyl
oxygen acts as a nucleophile. The presence of electron-
withdrawing fluorine atoms on the acetamido group of a
substrate is therefore expected to decrease the level of catalysis
by reducing the electron density of the carbonyl oxygen. HexD
activity with substrates possessing zero, one, two, or three
fluorine atoms on the acetamido group was tested, and a trend
(Figure 3A) is observed whereby the rate of hydrolysis is
reduced as the degree of fluorination increases. The data were
subjected to Taft-like linear free energy analysis, which
accounts for the influence of polar effects on a reaction rate
and can be used to determine whether the acetamido group is
responsible for the variability in reaction rate observed between
substrates. By plotting the log kcat/KM of each substrate against
the Taft electronic parameter (σ*) of the corresponding N-acyl
substituent, we obtained the Taft reaction constant (ρ) (Figure
3B), revealing a negative linear correlation with an increasing
degree of fluorine substitution. The negative slope of the
reaction constant (−0.49 0.1) suggests this residue interacts
with a positively charged center in the transition state,
consistent with the hypothesis that the carbonyl oxygen acts
in a nucleophilic capacity to intercept the electrophilic
anomeric center. The magnitude of the reaction constant is
similar to that calculated for OGA (−0.42 0.08) (Table 4), in
which the acetamido group has been unambiguously shown to
participate in catalysis.14 In contrast, the value observed for
HexD is smaller than the reaction constant obtained for HexB
(−1.0
0.1), which also operates using a substrate-assisted
mechanism.14 An important consideration, however, is that the
Taft reaction constant is governed by both a steric and
electronic component according to
ρ = ρ* + δ
(2)
where ρ* represents the sensitivity of the reaction to the
electronic character of the substituents (σ*) and δ represents
the sensitivity of the reaction to the Taft steric coefficients of
the substrates (Es). It is, therefore, conceivable that the decrease
in reaction rate with an increasing degree of fluorine
substitution is due in part to an increased number of steric
interactions caused by the difference in size between fluorine
(atomic radius of 147 pm) and hydrogen atoms (atomic radius
of 120 pm). However, though KM is a kinetic parameter and
might not always reflect substrate dissociation, particularly in
cases where an enzyme-bound intermediate accumulates, the
clear dependence of the first-order rate constants (kcat) on
leaving group ability suggests that the rate-determining step for
these substrates with WT HexD is the ring-forming step in
J
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groups, the catalytic ability of the E149A mutant is significantly
impaired relative to that of wild-type HexD, as demonstrated by
the steeper negative correlation observed in the Brønsted
analyses (Figure 4A). It should be pointed out here, however,
that the pH dependence for the E149A mutant also gave a bell-
shaped profile, with an alkaline shift relative to the profile for
wild-type HexD. This indicates there is titration of at least two
residues other than Glu149 contributing to the pH depend-
ence; whether this is true only in the case of the Glu149 mutant
HexD, where the chemistry of the active site environment will
have been perturbed by removal of the charged residue or is
also true for the wild-type enzyme is difficult to know in the
absence of structural data. The low activity observed for the
D148A HexD mutant, even with activated substrates, as well as
the steep negative correlation observed in the Brønsted
analyses, is consistent with this residue playing a role in
polarizing the N−H group on the acetamido group of the
substrate to aid nucleophilic attack by the carbonyl oxygen at
the anomeric position. The impact of this mutation is also
reminiscent of the effect of analogous mutations made in OGA
and HexB, where impairments of 6700- and 2400-fold have
been observed, respectively.27,31 The assignment of Asp148 as
the polarizing residue and Glu149 as the general acid/base is
consistent with previous conclusions for analogous residues in
the DE motif of GH20 enzymes, such as the lysosomal β-
hexosaminidases,27 and the DD motif of GH84 enzymes, such
as OGA.31
The inhibitor NAG-thiazoline was originally designed to
mimic the transient oxazoline intermediate that is formed
during substrate-assisted catalysis (Figure 1).29 Interestingly,
however, Whitworth and colleagues have since demonstrated
using linear free energy relationships that NAG-thiazoline is a
mimic of the transition state for human OGA.36 Incorporation
of the thiazoline motif during design of inhibitors has proven to
lead to effective inhibitors of a range of hexosaminidases that
employ a substrate-assisted mechanism.14,16,17,25,33 On the basis
of the mechanistic evidence obtained from kinetic analyses in
this study, we expected the galacto-configured derivative of
NAG-thiazoline, Gal-NAG-thiazoline (4), would inhibit HexD.
We observe this to be the case, with Gal-NAG-thiazoline
displaying potent competitive inhibition of HexD with a Ki
value of 420 nM, which provided further experimental support
for HexD using substrate-assisted catalysis.
Notably, functionalized derivatives of NAG-thiazoline have
been developed as potent and selective inhibitors of the
lysosomal β-hexosaminidases HexA/B and OGA, which have
proven to be useful tools for interrogating the biological role of
these enzymes.14,23,37,38 Unfortunately, Gal-NAG-thiazoline has
limited use as a chemical tool for studying the physiological role
played by HexD because it also inhibits the lysosomal β-
hexosaminidases (Table 4). These studies, however, lay
important groundwork for the rational design of selective
inhibitors of HexD. The inhibition of HexD by Gal-NAG-
thiazoline suggests this inhibitor scaffold may be suitable for
adaptation and may allow creation of compounds that will
selectively inhibit HexD without impacting the activity of
lysosomal β-hexosaminidases. Given the limited tolerance of
HexD toward glucose-based substrates, combined with its
localization in the nucleus and cytoplasm, the enzyme likely
serves a biological role that is distinct from that of the
lysosomal β-hexosaminidases and OGA. Accordingly, the
development of potent and specific probes will be useful for
investigating the potential physiological function of HexD.
CONCLUSIONS
■
In summary, we have performed a detailed kinetic character-
ization of HexD using a combination of kinetic studies with
wild-type and mutant enzymes, linear free energy analyses,
NMR experiments, and inhibition studies. HexD is a retaining
glycosidase, which operates through a substrate-assisted
hydrolysis mechanism consistent with that of other mammalian
β-hexosaminidases in GH families 20 and 84. The enzyme
shows a moderate preference for galactosaminides over
glucosaminides and operates optimally in the pH range of
6.5−7.0. We have dissected the catalytic roles played by Asp148
and Glu149, which we propose act as the polarizing residue and
general acid/base, respectively. Furthermore, HexD is inhibited
by Gal-NAG-thiazoline, which is consistent with the use of a
substrate-assisted mechanism, and this observation will provide
a starting point for further inhibitor development. Despite the
mechanistic similarities it shares with the lysosomal β-
hexosaminidases, which belong to family GH20, HexD is
distinct as it localizes to the nucleus and cytoplasm and shows
only weak activity against glucosaminides. We anticipate the
insights gained from this study will assist in the development of
potent and selective inhibitors of HexD as well as other
research tools, which will serve to improve our understanding
of the physiological role played by HexD in human health and
disease.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
chem.5b01285.
■
S
Primers used for HexD mutagenesis, spectrophotometric
parameters used for Brønsted analyses, synthetic schemes
for galactosaminide substrates, and a sequence alignment
for HexD with the lysosomal β-hexosaminidases (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: tmg@st-andrews.ac.uk.
*E-mail: dvocadlo@sfu.ca.
Present Address
∥I.N.: Virologicky ustav, Slovenska
́
akademia vied, Dubravska
́
́
́
́
́
cesta 9, 845 38 Bratislava, Slovakia.
Author Contributions
M.G.A., V.O., and I.N. conducted experimental research. I.N.
and I.B.H.W. contributed unique material to the project.
M.G.A., T.M.G., and D.J.V. analyzed data and wrote the
manuscript.
Funding
This project work was supported by a Royal Society
International Exchanges Scheme grant to T.M.G. and D.J.V.
This work was supported by an NSERC Discovery and a CIHR
Operating grant (MOP-123341). V.O. and T.M.G. are funded
by a Wellcome Trust Career Development Fellowship (Grant
095828). I.N. was supported by the Slovak Research and
Development Agency under Contract APVV-14−0839 and by
Science Grant Agency Project VEGA 2/0103/15. D.J.V. thanks
the Canada Research Chair program for support as a Canada
Research Chair in Chemical Glycobiology. M.G.A. thanks the
NSERC CREATE Collaborative Medicinal Chemistry Network
in Epigenetics Training (ChemNET) for support. I.N.
K
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Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine;
HexA, hexosaminidase A; HexB, hexosaminidase B; HexS,
hexosaminidase S; OGA, O-GlcNAcase; HexD, hexosaminidase
D; GH, glycoside hydrolase; CAZy, Carbohydrate Active
enZyme; PBS, phosphate-buffered saline; PNP-GalNAc, 4-
nitrophenyl GalNAc; 3F4NP-GalNAc, 3-fluoro-4-nitrophenyl
GalNAc; PNP-GlcNAc, 4-nitrophenyl GlcNAc; 3F4NP-
GlcNAc, 3-fluoro-4-nitrophenyl GlcNAc; WT, wild type;
NMR, nuclear magnetic resonance; SEM, standard error of
the mean.
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