(Received for publication, June 19, 1995; and in revised form, July 27, 1995)
From the
The carbene precursor
3-azi-1-[([6-H]-2-acetamido-2-deoxy-1-
-D-galactopyranosyl)thio]-butane
(also designated [
H]-1-ATB-GalNAc) has been used
as a photoaffinity label for human lysosomal
-hexosaminidase B
(Hex B, EC 3.2.1.52) purified to apparent homogeneity from postmortal
liver. [
H]-1-ATB-GalNAc behaved as an active
site-directed inhibitor, which bound covalently to Hex B upon
photolysis at 350 nm and resulted in 15% inactivation of enzyme
activity. Up to 75% of the inactivation of Hex B was prevented by
including the competitive inhibitor
2-acetamido-2-deoxy-D-glucono-1,5-lactone in the photoaffinity
experiment. Incubation of [
H]-1-ATB-GalNAc with
the enzyme followed by irradiation and subsequent separation of the
three polypeptides composing the
-subunit led mainly to labeling
of the
-polypeptide. Subsequent proteolysis of
with trypsin and separation of the resulting peptides
by high pressure liquid chromatography yielded one prominently labeled
peptide fraction. Edman degradation resulted in the sequence
E
ISEVFPDQFIHLGGDEVEFK
. However, no modified
amino acid was detected, indicating that the photoaffinity label was
presumably bound to the peptide by a labile ester linkage. This was
proven when the radiolabel was almost completely released from the
peptide by treatment with aqueous ammonium hydroxide. Simultaneously,
Glu-355 was converted into Gln-355, which is located within a region of
Hex B that shows considerable homology with the
-subunit of human
hexosaminidase A and other hexosaminidases from various species.
Human lysosomal hexosaminidases (EC 3.2.1.52) release terminal
-glycosidically linked N-acetylglucosamine and N-acetylgalactosamine residues from a number of
glycoconjugates. They are involved in the intracellular degradation of
glycolipids, like ganglioside G
, (
)its asialo
derivative G
, and globoside, as well as in the degradation
of carbohydrate chains of glycoproteins, glycosaminoglycans, and
oligosaccharides (for review see (1) and (2) ).
The
major isoenzymes -hexosaminidase A and
-hexosaminidase B (Hex
B) (
)are composed of two noncovalently linked subunits of
nearly equal molecular mass.
-Hexosaminidase A is a heterodimer
(
), whereas Hex B is a homodimer of two
-chains
(
)(3) . The homodimer
, also called
-hexosaminidase S, has been detected in patients with
Sandhoff's disease and seems to be unstable under normal
conditions(4, 5) . The mature
-chain is composed
of a major
-polypeptide (M
=
54,000) and a minor polypeptide
(M
=
6,000). The mature
-subunit originates from its precursor by two proteolytic cleavage
events forming a small
-polypeptide (M
=
11,000), the
-chain (M
=
24,000), and the
-chain (M
=
28,000).
In the mature
-subunit all 3 chain components are linked by
disulfide
bonds(6, 7, 8, 9, 10) .
Dimerization of the subunits
and
or
and
is
essential for catalytic activity of human
-hexosaminidase A and B,
respectively(11) . Each subunit possesses an active site that
differs in its substrate specificity. The active site of the
-chain hydrolyzes uncharged substrates, whereas the
-subunit,
in addition, slowly cleaves negatively charged substrates (12) . However, only
-hexosaminidase A is able to
hydrolyze the most important natural lipid-substrate, ganglioside
G
, in presence of the G
activator protein at
significant rates (13) .
Mutations in any of the three genes
encoding for the - or
-subunit or the G
activator protein can give rise to neuronal storage of
ganglioside G
in one of the three variants of G
gangliosidoses. In variant B (Tay-Sachs disease), deficiency of
the
-subunit caused by mutations of the HEXA gene brings
about a lack of
-hexosaminidase A and
-hexosaminidase S
activities, whereas the activity of Hex B is normal or increased.
Variant 0 (Sandhoff's disease) is characterized by
-hexosaminidase A and B deficiency due to mutations of the HEXB gene encoding the
-subunit. In variant AB the
G
activator protein is deficient due to mutations of the GM2A gene, whereas
-hexosaminidase A and B activity
levels appear to be normal (for review see (14) ).
Although
human hexosaminidases have been studied intensively, including their
gene structures, peptide sequences, biosynthesis, and
post-translational modifications, their active sites remain unknown.
Moreover, although Hex B has been crystallized(15) , no
crystallographic structure of a hexosaminidase is available from which
sequence alignments and modeling might be possible. Given the related
substrate specificities of the - and
-subunits and the
considerable extent of sequence homology between them, it seems likely
that their active sites have a high degree of structural similarity.
Site-directed mutagenesis techniques and naturally occurring mutations
yield useful information for the identification of amino acids
involved, but interpretation of such studies is difficult in the
absence of either crystallographic or affinity labeling information.
Therefore, we studied the binding site of Hex B using active
site-directed labeling techniques employing
[H]-1-ATB-GalNAc as a photoaffinity label for the
purified enzyme. The chemical structure of this ligand is shown in Fig. 1. Because glycoside hydrolases usually show a high
glycon-specificity and a relatively low aglycon-specificity, the
photolabile group was introduced into the aglycon of the
enzyme-resistant thioglycoside so that recognition of the compound as a
substrate analogue was not impaired(16) . After photolysis at
350 nm, the diazirine group located in the aglycon forms a highly
reactive carbene, which can react covalently with amino acid side
chains. [
H]-1-ATB-GalNAc has been previously
shown to specifically bind at the substrate binding site of
-hexosaminidase A (17) . The K
-values of this compound were estimated
to be 1.8 mM and 3.0 mM for the
-subunit of
-hexosaminidase A and B, respectively.
Figure 1:
Structure of
[H]-1-ATB-GalNAc. The position of the tritium
label [
H] is indicated by a star. See
``Experimental Procedures'' for further
details.
The present study
includes specific photoincorporation of
[H]-1-ATB-GalNAc into purified human lysosomal
Hex B, tryptic digestion of the covalently labeled enzyme, and
isolation and sequencing of the labeled peptide.
The
photoaffinity label 1-ATB-GalNAc was synthesized and tritiated as
previously reported(17) . The resulting
[H]-1-ATB-GalNAc had a specific radioactivity of
0.92 Ci/mmol. It was homogenous as judged by TLC, NMR spectroscopy, and
fast atom bombardment-mass spectrometry. All other reagents were of the
highest purity available.
Inactivation was
measured immediately after irradiation with 3 2 µl of the
[
H]-1-ATB-GalNAc-labeled enzyme solution. The
aliquots were diluted 1:20,000 with water containing 0.02% bovine serum
albumin (w/v) and assayed for enzyme activity using MUG as a substrate.
Under these conditions the concentration of photolyzed
[
H]-1-ATB-GalNAc was lowered to approximately 1
µM. Protein concentration of additional aliquots (4
1 µl) were measured to determine the specific activity of
the labeled enzyme. As control, a photolabeling experiment under the
same conditions but in the absence of
[
H]-1-ATB-GalNAc was performed and treated as
above.
H radioactivity was measured by liquid scintillation
counting using Ultima Gold from Packard as scintillant. For SDS-PAGE,
protein samples dissolved in 6 M guanidinium-HCl were dialyzed
against Tris-HCl buffer (50 mM, pH 6.0, containing 4% SDS and
12% glycerol) before being applied to the gel. Denaturing SDS-PAGE was
performed on 12% polyacrylamide gels according to
Schägger and von Jagow(23) .
Subsequent size exclusion chromatography of the
reduced and alkylated protein under denaturing conditions led to the
isolation of two protein-containing peaks. The first major peak
contained the main portion of protein-bound radioactivity (97%) and was
found by SDS-gel electrophoresis to comprise both the
- and the
-polypeptide. The second
minor peak contained only
3% of protein-bound radioactivity and
corresponded to the
-polypeptide, as shown by SDS-PAGE (Fig. 2). Additionally, a group of low molecular weight peaks
was detected containing the reducing and alkylating reagents together
with photolysis byproducts of [
H]-1-ATB-GalNAc.
Figure 2:
SDS-PAGE analysis of Hex B polypeptides
obtained after photoaffinity labeling. Hex B was irradiated in the
presence of [H]-1-ATB-GalNAc, denatured, reduced,
and alkylated as described under ``Experimental Procedures.''
Gel filtration of the radiolabeled enzyme was performed as described
under ``Experimental Procedures.'' Protein containing
fractions 8-10 and 11-12 of the gel filtration eluate were
pooled, and aliquots (
20 µg) were dialyzed against 50 mM Tris-HCl, pH 6.0, containing 4% SDS and 12% glycerol. Separation
by SDS-PAGE was performed under reducing conditions using a 12% tricine
gel. Protein bands were visualized with Coomassie Blue. Lane
1, molecular mass standards; lane 2, Hex B irradiated in
the presence of [
H]-1-ATB-GalNAc; lane
3, fractions 11-12 of the gel filtration eluate; lane
4, fractions 8-10.
Figure 3:
Separation of - and
-polypeptides of labeled Hex B by reverse phase HPLC.
Photoaffinity labeling of Hex B with
[
H]-1-ATB-GalNAc and separation of
- and
-chains by HPLC were conducted
as described under ``Experimental Procedures.'' Aliquots of
each fraction were assayed for radioactivity. A, the
chromatogram was obtained by HPLC separation of
- and
-polypeptides derived from labeled Hex B and the
corresponding radioactivity profile. B, fractions designated a, b, c, and d in A were
further analyzed by SDS-PAGE. Aliquots with equal amounts of
radioactivity (about 0.1 µCi) of fractions a-d were
freeze-dried and separated by SDS- PAGE. The radioactive spots were
identified by fluorography. Lane 1,
C-methylated
molecular mass markers; lane 2, polypeptide mixture of
and
obtained by gel filtration of
[
H]-1-ATB-GalNAc labeled Hex B; lanes
a-d, fractions designated a-d (see A).
Figure 4:
Specific radioactivities of the
[H]-1-ATB-GalNAc-labeled polypeptides in the
presence and the absence of the
-lactone. Human lysosomal Hex B
(50 µM) was labeled with
[
H]-1-ATB-GalNAc (12 mM) in the presence (shaded bars) or the absence (open bars) of
-lactone (1 mM). The
- and
-polypeptides were separated by reverse phase HPLC as
described under ``Experimental
Procedures.''
Figure 5:
Separation of the tryptic digest of the
-polypeptide after labeling with
[
H]-1-ATB-GalNAc by reverse phase HPLC. A, HPLC separation of peptides derived from the labeled
-polypeptide. The arrow indicates the labeled
peptide that was further purified by narrow bore HPLC and subjected to
sequence analysis. Photoaffinity labeling of Hex B with
[
H]-1-ATB-GalNAc, separation of
- and
-chains, digestion of labeled
-polypeptide with trypsin, and HPLC analysis were
conducted as described under ``Experimental Procedures.'' B, the corresponding radioactivity profile to A as
obtained by liquid scintillation counting. C, control to B radioactivity profile obtained after labeling of Hex B in the
presence of the competitive inhibitor
-lactone (1 mM) to
demonstrate the specificity of the reaction by reduced label
incorporation into the peak at 52 min. Experimental details are
provided under ``Experimental
Procedures.''
An aliquot (50 pmol) of this peptide
fraction was directly subjected to automated sequence analysis. The
effluent stream from the Edman degradation was split before it entered
the phenylthiohydantoin analyzer, so that radioactivity of the effluent
from each sequencing cycle could be correlated with analysis of
PTH-derivatives. Automated Edman degradation of the labeled peptide
yielded the sequence EISEVFPDQFIHLGGDEVEFK (peptide I), corresponding
to amino acid positions 339-359, within the preprosequence of
human lysosomal
-hexosaminidase B. The sequence assignment was
unambiguous, no secondary sequences were observed. However, contrary to
our expectations, no significant burst of radioactivity and no severely
reduced PTH-derivative signal were observed in any of the cycles during
sequence analysis. All of the radioactivity was recovered in the
sequencer waste. This result strongly indicated the presence of a
labile ester bond between an acidic side chain of either a glutamic or
an aspartic acid and the photoaffinity label, which was then cleaved
during sequencing under the conditions of Edman degradation.
For
further analysis, the radiolabeled peptide (50 pmol) was
rechromatographed by narrow bore HPLC (Fig. 6) at pH 2.0 using
the standard solvent system (0.1% trifluoroacetic acid in water versus 0.085% trifluoroacetic acid and 84% acetonitrile in
water). As shown in Fig. 6A, rechromatography yielded
one single peak comprising all of the radioactivity. However, its
calculated specific radioactivity (0.066 Ci/mmol) was rather low and
suggested a contamination by an unlabeled peptide. Therefore, the peak
was rechromatographed in the solvent system of Serwe et al.(21) (0.2% hexafluoroacetone in water, pH 7.0, versus 0.03% hexafluoroacetone and 84% acetonitrile in water). As shown
in Fig. 6B, the apparently homogenous peak (Fig. 6A) separated into three peaks designated peaks
1, 2, and 3, which were further analyzed by MALDI-TOF-MS and sequence
analysis. The majority of radioactivity was found in peak 3. The
specific radioactivity calculated for this peptide of 0.91 Ci/mmol is
close to the specific radioactivity of
[
H]-1-ATB-GalNAc indicating a 1:1 stoichiometry
of labeling.
Figure 6:
Rechromatography of
[H]-1-ATB-GalNAc most labeled peptide. A, the chromatogram obtained by rechromatography of an aliquot
(50 pmol) of the prominently labeled peak and the corresponding
radioactivity profile using 0.085% trifluoroacetic acid in 84%
acetonitrile as solvent B. Tryptic digestion of the labeled
-chain, separation of the resulting peptides, and
rechromatography of the prominently labeled peak on narrow bore HPLC
were conducted as described under ``Experimental Procedure.'' B, rechromatography of another sample (3 nmol) performed as
described above but now using 0.03% hexafluoroacetone in 84%
acetonitrile (pH 7.0) as solvent B. The apparently homogenous peak (see A) separated into three peaks, designated peaks 1, 2, and 3,
which were further analyzed by MALDI-TOF-MS and sequence
analysis.
MALDI-TOF-MS gave a (M+H) ion at m/z 2438.7 in the case of peak 1, which is close to
the calculated mass of the peptide I,
E
ISEVFPDQFIHLGGDEVEFK
with 2436.68. Peak 2
gave rise to a single molecular (M+H)
ion at m/z 3342.3, a value close to the calculated mass of
an elongated peptide I (positions 339-366; 3338.7), plus
iodoacetamide. In this minor cleavage product, the potential tryptic
site at lysine 359 has apparently been skipped. In case of peak 3, a
(M+H)
ion at m/z 2729.9 was
found matching the calculated mass for peptide I (positions
339-359) with a single affinity ligand attached (2728.04).
An
aliquot (30 pmol) of peak 3 containing the photoaffinity labeled
peptide I was subjected to sequence analysis. Again, no modified amino
acid and no burst of radioactivity were detected. Obviously, the bond
between the peptide and the ligand was cleaved again by the harsh
conditions of Edman chemistry. Therefore, following the strategy of
Gebler et al.(22) , we decided to choose an indirect
approach in order to identify the labeled amino acid. Assuming that an
acid-labile isobutyl ester had been formed between the
C-aglycon of [
H]-1-ATB-GalNAc and the
carboxyl group of an amino acid with an acidic side chain, then this
ester should be cleavable by base treatment. Aqueous ammonia should
liberate tritiated butanol-thio-GalNAc from the peptide and
simultaneously convert a significant proportion of the carboxyl group
of the former attachment site to the corresponding amide.
Consequently, 100 pmol of labeled peptide from peak 3 (Fig. 6B) were reacted with 25% aqueous NH at 37 °C for 8 h and rechromatographed under the same
separation conditions that had led to segregation of peaks 1, 2, and 3
(see Fig. 6B). All of the radioactivity now came off
the column with the void volume, as had been expected for the highly
polar GalNAc derivative. Within the time window where peaks 1 and 3
normally eluted, two peaks of equal intensity were observed. The
retention time of the earlier eluting peak corresponded to the
retention time of peak 1, the unlabeled peptide I. The second peak was
intermediate in retention between peaks 1 and 3. None of the peaks bore
any residual radioactivity.
Both peptides were subjected to sequence
analysis. The earlier eluting peptide was found to be peptide I
(positions 339-359). The later eluting peptide had the same
sequence, with the notable exception of cycle 17, where PTH-glutamine
instead of PTH-glutamic acid was unambiguously identified. This result
strongly suggests that [H]-1-ATB-GalNAc was
covalently attached with its aglycon to Glu-355 via an ester bond,
which could be cleaved by aminolysis reverting one-half of the
derivatized Glu-355 back to glutamic acid and converting the other half
to glutamine.
Our attempts to probe the binding sites of hexosaminidases
were initially based on the pyrrolidine alkaloid
2-acetamido-1,4-imino-1,2,4-trideoxy-D-galactitol, which was
shown to act as a competitive inhibitor of enzyme activity on both the
- (K
=
220 µM) and
the
-subunit (K
=
18
µM) of
-hexosaminidase A(28) . In order to
achieve covalent photoincorporation of this compound into
hexosaminidases, we introduced a photolabile diazirine group via a
butyl spacer at the nitrogen ring atom. Unexpectedly, incubation of
-hexosaminidase A with this photolabel even in concentrations up
to 1 mM and subsequent irradiation did not result in any
reduction of enzyme activity, although the inhibitory potential (K
=
14 µM,
-subunit
of
-hexosaminidase A) of the galactitol derivative was not
affected by attachment of the photoaffinity label (data not shown). The
ineffectiveness of certain carbohydrate-linked diazirines to react
covalently with their receptors has been reported by Lehmann and Petry (29) . However, 1-ATB-GalNAc, although showing only a moderate K
value (
3 mM) is able to reduce
enzyme activity after binding and therefore was used as a photoaffinity
label for purified Hex B.
Our results provide strong evidence that
the amino acid sequence EISEVFPDQFIHLGGDEVEFK
is involved in the substrate binding site of human lysosomal
-hexosaminidase B. This conclusion is supported by the ability of
-lactone to compete effectively for the active sites of the enzyme
and to reduce labeling of the
-polypeptide and peptide
I with [
H]-1-ATB-GalNAc. The fact that
photoincorporation of the label was not totally prevented was somewhat
surprising, because addition of 1 mM
-lactone into the
labeling experiment should completely protect the active sites of Hex B
from covalent reaction with [
H]-1-ATB-GalNAc. On
the other hand, it is known that the
-lactone is relatively labile
and in equilibrium with
2-acetamido-2-deoxy-D-glucono-1,4-lactone and
2-acetamido-2-deoxy-D-gluconic acid(30) . Therefore we
compared the inhibitory potential of a freshly prepared aqueous
solution of
-lactone (1 mM) with the activity of a
-lactone solution (1 mM) treated under the labeling
conditions. It turned out that a freshly prepared aqueous solution of
-lactone showed a K
value (
25
nM) that was about 20 times lower than the solution of
-lactone that ran through the whole labeling procedure (K
=
500 nM) (data not
shown). Referring this result to the competition experiment, it is
likely that the initially applied amount of
-lactone (1
mM) is severely reduced during the labeling experiment, so
that the
-lactone concentration appears to be in the same order of
magnitude as the enzyme concentration (50 µM) or even
below the active site concentration (100 µM). Under these
conditions, complete protection of labeling by the
-lactone cannot
be expected anymore.
Laser desorption mass spectrometry of the
predominantly labeled peptide identified peptide I with a single
affinity ligand attached. During the isolation and purification
procedures of peptide I, it was noticed that the label was partially
lost if the peptide was treated with strongly acidic or basic
solutions. Similarly, identification of the modified amino acid within
the sequence of peptide I by Edman degradation failed, presumably
because the protocols used for automated sequencing include treatment
with 25% aqueous trifluoroacetic acid for 20 min at 64 °C. These
observations led to the assumption that the photolabel was bound to the
peptide via an ester bond that would be hydrolyzed under strongly
acidic or basic conditions(31) . Therefore, we treated the
labeled peptide I with 25% aqueous ammonium hydroxide(22) ,
which resulted in quantitative cleavage of the label from the peptide
and simultaneously, conversion of Glu-355 into Gln-355, as shown by
Edman degradation. Exactly the same results were obtained when we
performed the entire photoaffinity labeling experiment with
-hexosaminidase B partially purified from human placenta (specific
activity 130 units/mg) instead of Hex B from human liver (data not
shown).
The mechanisms of enzymatic glycoside hydrolysis and the
approaches to identify active site residues by labeling techniques have
been recently reviewed by Withers and
co-workers(32, 33) . In case of retaining
glycosidases, such as hexosaminidases(34) , it is generally
known that hydrolysis of a glycoside bond is accomplished by a pair of
acidic side chains, which participate simultaneously in this reaction.
The carboxyl group of one of these side chains functions as a general
acid and base, whereas the other one acts as a nucleophile and a
leaving group. In this context, it is interesting that in our
experiments only one acidic amino acid is exclusively labeled by an
active site-directed affinity ligand. The labeling is highly specific,
because peptide I is the only peptide carrying significant amounts of
radioactivity, and furthermore the labeling reaction can be suppressed
to a significant extent by the addition of a competitive but
nonreactive inhibitor. Moreover, the reaction happens to the side chain
of Glu-355 exclusively and extends to neither Asp-354 nor Glu-357,
making it unlikely that the carbene just picks out the best nucleophile
around. On the other hand, the photoreactive site within the butyl
residue is rather remote from the bound sugar and furthermore possesses
an inherent flexibility, making it unlikely to assign labeled Glu-355
to one of the amino acids involved in catalysis. With the help of
naturally occurring mutations of the B1 variant (reviewed in (35) ), Brown and Mahuran (36) reported that Arg-178 of
the -subunit (corresponding to Arg-211 on the
-subunit) could
be implicated in the active site of human hexosaminidases. Although the
data strongly suggest that these residues are important for the
activity of hexosaminidase, the assignment that they participate in the
cleavage of the glycosidic bond is not compelling. Moreover, as shown
by molecular modeling, mutations of these basic amino acids result in a
severe disruption of the three-dimensional conformation of the subunits (35) and therefore do not prove that
-subunit Arg-178 or
-subunit Arg-211 are involved catalytically in the active site.
Although our results of photoaffinity labeling of the binding site of
Hex B with 1-ATB-GalNAc focus attention on Glu-355, full understanding
of the functional aspects of this amino acid will undoubtedly await
elucidation of the tertiary structure of Hex B.
Because this is, to
our knowledge, the first identification of a specific sequence at the
binding site of the -subunits from human hexosaminidase B, we were
interested in the extent to which this sequence is conserved among
hexosaminidases from other species and human
-chain in particular.
Alignment of amino acid sequences for hexosaminidases from various
species revealed that Glu-355 is highly conserved between the
-subunit of human
-hexosaminidase A, the
- and
-subunit of mouse, and the
-subunit of Dictyostelium
discoideum (Fig. 7). Moreover, there are several highly
conserved amino acids in the vicinity of Glu-355, being potential
targets for future studies using site-directed mutagenesis to probe the
binding site in hexosaminidases. Interestingly, Glu-355 is located near
Cys-360, which is bridged with Cys-309, connecting the
- and the
-polypeptides in Hex B.
These cysteine residues and also the size of the loop they enclose are
conserved in all known hexosaminidase subunit sequences(37) .
Figure 7:
Homologous regions near Glu-355 for
subunits of hexosaminidases following alignment of the full amino acid
sequences. Regions of sequence identity following the alignment of
entire protein sequences are denoted by gray shading. Peptide
I obtained from tryptic digestion of -polypeptide is boxed. The arrow indicates the site of
[
H]-1-ATB-GalNAc attachment in human lysosomal
Hex B. The abbreviations used for the proteins, the regions of
sequence, and the accession numbers for SWISS-PROT protein sequence
data base are as follows:
-human, amino acids
339-365 of the preprosequence of the
-subunit from human Hex
B, P07686;
-mouse, amino acids 318-344 of the
-subunit from mouse Hex B, P20060;
-human, amino
acids 307-333 of the
-subunit from human
-hexosaminidase A, P06865;
-mouse, amino acids
307-333 of the
-subunit from mouse
-hexosaminidase A,
P29416;
-DD, amino acids 292-318 of the
-subunit from D. discoideum
-hexosaminidase A,
P13723.
It is clear from previous attempts to locate the active sites of
hexosaminidases that there are several regions of homology among them.
One was found using the predicted amino acid sequence of the bacterial
-N-acetylhexosaminidase gene from Vibrio
vulnificus. Sequence alignments identified a region of 19 amino
acids largely conserved around the central portion of the amino acid
sequence among known hexosaminidases but not including
Glu-355(38) . Although regions of homology that have been
identified by sequence alignments may prove to be important in the
structure of the active sites of hexosaminidases, our results with
photoaffinity labeling and peptide sequencing provide the first
chemical evidence identifying a specific amino acid at the binding site
of Hex B.
This paper is dedicated to Prof. Heinz Egge on the occasion of his 65th birthday.