(Received for publication, March 21, 1996, and in revised form, November 20, 1996)
From the Department of Biochemistry and Molecular
Biology and the § Complex Carbohydrate Research Center,
University of Georgia, Athens, Georgia 30602
-1,6-N-Acetylglucosaminyltransferase
V (EC 2.4.1.155) catalyzes the transfer of
N-acetylglucosamine (GlcNAc) from UDP-GlcNAc in
(1,6)-linkage to the
(1,6)-linked mannose of N-linked
oligosaccharides. Circular dichroism (CD) was used to investigate the
secondary structure of a recombinant, soluble form of the enzyme and
its interaction with UDP-GlcNAc and an inhibitory substrate analog. The
CD spectrum of the apoenzyme indicated the presence of small amounts of
-structure and substantial amounts (>50%) of
-helicity. The CD
spectra of solutions containing UDP-GlcNAc and different ratios of
UDP-GlcNAc:enzyme were measured. Interestingly, the spectrum of each
mixture could not be accounted for by simple additivity of the two
individual spectra, indicating a change in environment of the
chromophores and/or a conformational change of the substrate or protein
concomitant with binding. Similar results were obtained with mixtures
of UDP and the enzyme. Analysis of the CD difference spectra at three
wavelengths yielded an estimated average Kd of 4.4 mM for UDP-GlcNAc and 3.8 mM for UDP. By
contrast, addition of the CD spectrum of an inhibitory substrate analog
of its oligosaccharide acceptor substrate and the CD spectrum of the
enzyme could account for that observed of an inhibitor-enzyme mixture;
moreover, addition of the inhibitor to a mixture of UDP-GlcNAc and
enzyme did not alter the Kd associated with
UDP-GlcNAc binding to the enzyme. These results and kinetic studies
reported herein suggest an ordered reaction in which UDP-GlcNAc binds
first to the enzyme, followed by the sequential binding of the
trisaccharide substrate.
Glycosyltransferases transfer sugars from sugar-donor substrates to acceptor substrates and synthesize complex carbohydrates (1). In animal cells, these enzymes are normally membrane-bound and present in relatively low copy number per cell, although many of the enzymes have been detected in soluble form in serum and cerebrospinal fluid after release from Golgi membranes by proteolysis (2). The development of affinity chromatography techniques (3-5) has allowed many glycosyltransferases to be purified and the cDNAs, which encode them, to be isolated and characterized, although several glycosyltransferase cDNAs have been isolated by expression cloning techniques (6-8). Although the amino acid sequences of many of these enzymes are now known, none of the enzymes has as yet been studied by x-ray crystallography techniques; consequently, very little is known of their secondary and tertiary structure. Several studies, however, have used modified substrates or differential labeling techniques to localize regions in the primary sequence of particular glycosyltransferases, which are in proximity to their active sites or are involved in catalysis (9, 10). Although short sequence motifs are shared between a few enzymes in this family (cf. Refs. 11 and 12), little information is available concerning the physical interaction between any of these enzymes and its donor and acceptor substrates. A logical tool to utilize in the study of glycosyltransferase-substrate interactions is that of circular dichroism.
We have recently isolated a cDNA encoding N-acetylglucosaminyltransferase V (GlcNAc-T V,1 EC 2.4.1.155) and engineered it such that when transfected into Chinese hamster ovary cells, a soluble form of the enzyme is secreted into the culture medium (11, 12). Quantities sufficient for CD studies of this form of the enzyme can, therefore, be produced and purified to homogeneity. This glycosyltransferase transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to an oligosaccharide acceptor substrate by the following reaction:
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In the present study, the CD spectra of recombinant, soluble
GlcNAc-T V was studied in the absence and presence of its donor substrate, UDP-GlcNAc, an inhibitory substrate analog of its
oligosaccharide acceptor substrate (20), and the competitive inhibitor
UDP. The CD spectra of both enzyme-UDP-GlcNAc and enzyme-UDP mixtures could not be accounted for by additivity of the individual spectra, thus showing that CD is useful for studying GlcNAc-T V-UDP-GlcNAc complexes. Since, in the absence of oligosaccharide acceptor, UDP-GlcNAc and UDP bind to enzyme in a reversible bimolecular interaction, the CD data were analyzed with the binding model, E + S ES, and yielded calculated average
Kd values of 4.4 and 3.8 mM,
respectively. Interestingly, the CD spectrum of a mixture of the enzyme
and a trisaccharide acceptor substrate analog, which functions as a
competitive inhibitor, could be fully accounted for by the simple sum
of the individual spectra; moreover, the presence of the inhibitor had
no effect on the Kd of UDP-GlcNAc binding to
GlcNAc-T V.
UDP-GlcNAc and UDP were
products of Sigma, and the inhibitory trisaccharide acceptor analog,
n-octyl
6-O-[2-O-(2-acetamido-2-deoxy--D-glycopyranosyl)-6-deoxy-
-D-mannopyranosyl]-
-D-glucopyranoside, was obtained from Dr. Ole Hindsgaul (University of Alberta, Canada (21)). Recombinant, soluble GlcNAc-T V was purified from concentrated Chinese hamster ovary cell medium (12). After extensive dialysis against the buffer used for CD spectrascopy, 10 mM
Tris-HCl, pH 6.5, containing 0.1 mM EDTA, the enzyme was
concentrated at 3000 × g using Centricon-30 columns
(Amicon).
Solutions of either
3.2 or 3.6 µM GlcNAc-T V were scanned in a Jasco J-700
spectropolarimeter at ambient temperature using a cell of 1 mm
pathlength. Replicate scans were obtained at 0.2 nm resolution, 0.2 nm
bandwidth, and a scan speed of 50 nm/min, followed by spectral
averaging and smoothing. Conditions were such that the signal at 220 nm
was about 20 millidegrees with enzyme alone; the variation from
replicate scans was at most ±1 millidegree. CD spectra of GlcNAc-T V
plus UDP-GlcNAc or plus UDP were determined by titrating a concentrated
solution of the sugar nucleotide or nucleotide into the enzyme
solution, and control spectra were obtained on UDP-GlcNAc or UDP
solutions at the same concentrations. The CD spectrum of GlcNAc-T V
was also measured in the presence of the trisaccharide inhibitor, which
too was scanned in the absence of enzyme. Lastly, CD spectra were
determined on mixtures of GlcNAc-T V (3.6 µM),
trisaccharide inhibitor (1 mM), and various concentrations
of UDP-GlcNAc (1 µM-10 mM). The signal-to-noise ratio of all spectra was satisfactory above 205 nm;
data were not analyzed below 200 nm due to low signal-to-noise ratios.
The radiochemical assays were performed as described using the standard synthetic trisaccharide acceptor (20). The acceptor concentration was varied from 5 µM to 0.2 mM, and the UDP-GlcNAc donor concentration varied from 0.1 to 4 mM.
The CD spectrum of GlcNAc-T V between 200 and 260 nm is shown in
Fig. 1. The negative extrema located at 208 and 222 nm
are indicative of -helicity. When analyzed by the Convex Constraint Analysis program (22), the spectrum is consistent with the presence of
58% helicity, 5%
-structure, and 37% aperiodic conformation. The
secondary structure estimates from CD analysis were compared with those
predicted by a neuralnet system using the amino acid sequence of the
truncated enzyme (23). The latter predicted 45% helicity, 11%
-structure, and 44% aperiodic conformation, in reasonable agreement
with the experimental findings.
Since UDP-GlcNAc is but one of two required substrates of the enzyme,
it is possible to form stable enzyme-substrate complexes without
catalysis occurring. First, various concentrations of UDP-GlcNAc
(1-200 µM) were scanned in the 1-mm cell, but no
detectable ellipticity was observed between 200 and 300 nm (data not
shown). At higher concentrations, however, negative ellipticity was
measured between 200 and 250 nm, while positive ellipticity was noted
between 250 and 300 nm (Fig. 2). A
concentration-dependent change in the CD spectrum was noted
above 0.5 mM. For example, at 1 mM UDP-GlcNAc the CD spectrum is characterized by negative and positive extrema associated with broad bands at about 214 and 270 nm, respectively; at
10 mM distinct and well resolved negative and positive
bands appear at about 231 and 285 nm, respectively. In addition, at the
higher concentrations of UDP-GlcNAc there are negative CD bands between
200 and 225 nm and only minimal positive ellipticity between 250 and
280 nm. Similar spectral results were observed with UDP and may result
from dimerization or stacking of the nucleotides at these higher
concentrations.
The CD spectrum of a mixture of GlcNAc-T V (3.6 µM) and
UDP-GlcNAc (4 mM) is shown in Fig. 3, along
with reference spectra of enzyme and substrate alone. The sum of the
spectra of GlcNAc-T V and UDP-GlcNAc was found to be quite different
from that of the mixture (Fig. 3), establishing that this system does
not obey simple additivity rules. Comparable studies were then
performed at various concentrations of UDP-GlcNAc and a fixed enzyme
concentration. The CD spectrum difference, i.e. the spectrum
of the mixture minus the sum of the spectra of enzyme and substrate,
changed with increasing concentrations of UDP-GlcNAc between 0.2 and 2 mM; at 3 and 4 mM UDP-GlcNAc, the CD spectral
differences were essentially identical (Fig. 4). At the
lower concentrations of substrate (0.2-2 mM), the CD
difference extremum was negative and centered at about 210 nm; at about
250 nm the difference ellipticity was slightly positive. The apparent
limiting CD difference spectrum, e.g. occurring at 3, 4, and
10 mM UDP-GlcNAc, is characterized by negative extrema at
about 210 and 216-218 nm, with a weak positive extrema at about 276 nm. Measurements were then made to determine if the non-additivity noted above could be observed using UDP alone, since this nucleotide is
a competitive inhibitor of the enzyme (24). The results (Fig. 5) demonstrated that mixtures of UDP and enzyme also
yield spectra that fail to follow simple spectral additivity, similar
to the findings obtained with mixtures of UDP-GlcNAc and enzyme.
An inhibitory substrate analog of the enzyme has been synthesized in
which the 6-OH of the
(1,2)-linked mannose of the trisaccharide acceptor, to which GlcNAc is transferred by the enzyme, has been replaced by hydrogen (20). This analog displays a Ki of 60 µM, compared to the Km of 213 µM for the trisaccharide acceptor, determined using crude
enzyme from baby hamster kidney cells. This inhibitory acceptor analog
was chosen because it could be mixed simultaneously with the enzyme and
UDP-GlcNAc without a catalytic reaction. Interestingly, mixtures of the
enzyme with the trisaccharide inhibitor, characterized by a negative
extremum at 210 nm, could be fully accounted for by the individual
spectra (Fig. 6). The CD spectrum of the mixture of
enzyme (3.6 µM), UDP-GlcNAc (1 mM), and
inhibitor (1 mM) is, within experimental error, identical to the additive spectra of enzyme plus UDP-GlcNAc and inhibitor (Fig.
7), indicating that the inhibitor does not alter the
interaction of GlcNAc-T V and UDP-GlcNAc.
Since CD spectroscopy can be used to follow enzyme-UDP-GlcNAc
interactions, the CD difference spectra were analyzed to obtain information on the Kd of the reaction, E + S ES. We have defined the ellipticity at any
wavelength of enzyme, substrate, and enzyme-substrate mixture as
GlcNAc-T V,
UDP-GlcNAc, and
mixture, respectively, and the difference ellipticity as
, where
=
mixture
GlcNAc-T V
UDP-GlcNAc. For a
reversible bimolecular reaction, E + S
ES,
the Kd is given by [E]
[S]/[ES], where brackets denote concentrations of the
indicated species. Since [Et] = [E] + [ES] and [St] = [S] + [ES], where
[Et] and [St] refer to total enzyme and
substrate concentrations, respectively, the Kd can
be written as follows:
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(Eq. 1) |
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(Eq. 2) |
To characterize further the kinetic reaction mechanism of GlcNAc-T V,
activity was measured at various concentrations of UDP-GlcNAc and
trisaccharide acceptor. The enzymatic activity assay data were then
plotted as the reciprocal of the activities versus the reciprocal of the substrate concentrations (25). In Fig.
9A, for each concentration of UDP-GlcNAc, the
reciprocal of activities at different concentrations of trisaccharide
acceptor were plotted against the reciprocal of the concentrations of
acceptor, which resulted in a linear relationship. The slope of each
line decreased when the UDP-GlcNAc concentration increased. In Fig.
9B, for each concentration of trisaccharide acceptor, the
reciprocal of activities at different concentrations of UDP-GlcNAc were
plotted against the reciprocal of the concentrations of UDP-GlcNAc,
which also resulted in a linear relationship. The slope of the lines in
Fig. 9B decreased when the acceptor concentration
increased. These data demonstrate that the two-substrate reaction
follows a sequential Bi-Bi mechanism rather than a Ping-Pong
mechanism.
The CD spectra of mixtures of GlcNAc-T V and various
concentrations of its substrate, UDP-GlcNAc, could not be accounted for by simple additivity of the spectrum of each component alone. Since
both the enzyme and the substrate are optically active and exhibit
overlapping bands between 200 and 250 nm, it is difficult to attribute
with confidence the spectral differences to just one of the species.
The problem is exacerbated in the case of UDP-GlcNAc, which shows a
dramatic concentration-dependent change in its CD spectrum,
even in the absence of enzyme. Nevertheless, despite these caveats, it
was possible to analyze the spectral data and obtain an average
Kd of 4.4 mM for the equilibrium binding
of UDP-GlcNAc to GlcNAc-T V. The nucleotide moiety, UDP, also showed
this anomalous concentration-dependent change in its CD
spectrum and caused non-additive spectral changes, similar to those
observed with UDP-GlcNAc and enzyme. Calculation of an average
Kd for UDP (3.8 mM) yielded a value
similar to that for UDP-GlcNAc binding to the enzyme. The CD spectra of
UDP and enzyme mixtures appear to be very similar to those observed with mixtures of UDP-GlcNAc and enzyme, suggesting that the active site
binding environments of UDP and UDP-GlcNAc are similar. A comparison of
the Km of UDP-GlcNAc, 6 mM, and the
Ki for UDP, 2 mM, however, reveals a
difference in these kinetic parameters, which may reflect subtle
differences in the binding of these molecules in the active site. An
earlier study used CD spectroscopy to investigate bovine milk
galactosyltransferase and determined that only low amounts (10%) of
-helix were present (26). A decrease in the amount of
-helix was
observed when UDP-galactose was added, but no change was observed,
however, in the presence of UDP alone in contrast to our data obtained when UDP was added to GlcNAc-T V.
The CD spectra of mixtures of the enzyme and inhibitory substrate analog, which is also optically active, were fully accounted for by simple additivity of the individual spectra. Moreover, the substrate analog had no effect on the CD difference spectra of enzyme-substrate mixtures and the Kd derived from analysis of the spectral changes. These findings are consistent with an ordered mechanism in which the substrate UDP-GlcNAc first binds to GlcNAc-T V, and this complex then binds inhibitor. We cannot unequivocally rule out other models, particularly if the inhibitor binds to the enzyme and neither spectrum is altered by the interaction; however, we believe that this possibility is unlikely. It is clear, though, that by CD analysis the interactions of the acceptor substrate and sugar nucleotide substrate with the active site appear to be dissimilar.
The results from the GlcNAc-T V kinetic assays demonstrate that the
release of the catalytic products follows a sequential Bi-Bi mechanism.
Therefore, the total reaction for the enzyme appears to be an ordered
sequential Bi-Bi mechanism, which is similar to that proposed for
GlcNAc-T II (28) but is distinct from that proposed for the
(1,4)-galactosyltransferase, which appears to function by a random
mechanism (27). Kinetic data suggested that UDP-GlcNAc binds first to
GlcNAc-T II and induces a conformational change to which the
appropriate oligosaccharide acceptor could then bind (27); our results
on UDP-GlcNAc binding to GlcNAc-T V are supportive of this model.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U86699[GenBank].
We thank Sally Boardman for technical assistance with the CD spectral analysis and Drs. Suzanne Crawley, Monica Palcic, and John Wampler for helpful discussions. The trisaccharide inhibitor was graciously provided by Dr. Ole Hindsgaul.