From the Department of Biochemistry and Molecular
Biology, University of Miami School of Medicine, Miami, Florida 33101 and § Department of Chemistry, Wayne State University,
Detroit, Michigan 48202
Received for publication, July 21, 2000, and in revised form, December 19, 2000
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ABSTRACT |
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UDP-galactose: UDP-Gal:-galactosyl-
1,3-galactosyltransferase
(
3GT) catalyzes the synthesis of galactosyl-
-1,3-
-galactosyl
structures in mammalian glycoconjugates. In humans the gene for
3GT
is inactivated, and its product, the
-Gal epitope, is the target of
a large fraction of natural antibodies.
3GT is a member of a family
of metal-dependent-retaining glycosyltransferases that
includes the histo blood group A and B enzymes. Mn2+
activates the catalytic domain of
3GT (
3GTcd), but the affinity reported for this ion is very low relative to physiological levels. Enzyme activity over a wide range of metal ion concentrations indicates
a dependence on Mn2+ binding to two sites. At physiological
metal ion concentrations, Zn2+ gives higher levels of
activity and may be the natural cofactor. To determine the role of the
cation, metal activation was perturbed by substituting Co2+
and Zn2+ for Mn2+ and by mutagenesis of a
conserved D149VD151 sequence motif that is
considered to act in cation binding in many glycosyltransferases. The
aspartates of this motif were found to be essential for activity, and
the kinetic properties of a Val150 to Ala mutant with
reduced activity were determined. The results indicate that the
cofactor is involved in binding UDP-galactose and has a crucial
influence on catalytic efficiency for galactose transfer and for the
low endogenous UDP-galactose hydrolase activity. It may therefore
interact with one or more phosphates of UDP-galactose in the Michaelis
complex and in the transition state for cleavage of the UDP to
galactose bond. The DXD motif conserved in many glycosyltransferases appears to have a key role in metal-mediated donor
substrate binding and phosphate-sugar bond cleavage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosyl-
-1,3-galactosyltransferase
(
3GT),1 a Golgi
membrane-bound enzyme, catalyzes the synthesis of galactosyl
-1-3-galactosyl
-OR structures in glycoconjugates (Refs. 1 and
2; see Fig. 1).
3GTcd and the products
of its action are found in most mammals but not in humans and
their closest relatives, the old world monkeys and apes (3, 4). In
these species the gene for
3GT is mutationally inactivated (4, 5),
and the absence of active enzyme allows the production of antibodies against the product of
3GT action, the
-Gal epitope (Fig. 1). Primates lacking this enzyme have natural antibodies (1-3% of circulating IgG, designated anti-Gal) that bind
-Gal (5) and facilitate immune defenses against pathogens but also present a barrier
to the xenotransplantation of organs from mammalian species that
produce active
3GT (6).
View larger version (11K):
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Fig. 1.
The reaction catalyzed by 3GTcd.
Like other glycoprotein glycosyltransferases, 3GTs are type-2
membrane proteins with a short N-terminal cytosolic domain, a
transmembrane helix, a stem, and a C-terminal catalytic domain (7-10).
Only a few subgroups of glycosyltransferases that function in the
processing of glycoproteins and glycolipids show global homology in
primary structure. At present, a 2.4-Å structure of the catalytic
domain of
-1,4-galactosyltransferase I is the only reported
three-dimensional structure of an enzyme of this type (11).
1,3-GT
is homologous to histo blood group glycosyltransferases A and B and to
Forssman glycolipid synthase but is not significantly similar in
overall sequence to
-1,4-galactosyltransferase I (12-13). Nevertheless, these and other divalent cation-dependent
glycosyltransferases share a DXD sequence motif that is
thought to represent at least part of a cation binding site (14-16),
suggesting that they may share a metal binding domain or substructure.
Recombinant forms of bovine and other 3GTs have been previously
expressed (18, 19); the cytosolic and transmembrane domains together
with a 67-residue stem can be deleted to produce a fully active soluble
enzyme (18). We have described the use of a bacterially expressed
soluble form of the catalytic domain of bovine
3GT (residues
80-367;
3GTcd) for the enzymatic production of substantial amounts
of
-Gal-containing oligosaccharides (20). Here we have investigated
the role of the metal cofactor in the catalytic mechanism using the
recombinant enzyme. Like many other glycoprotein glycosyltransferases,
3GTcd requires a divalent cation for activity that has been thought to be Mn2+ (1, 2). However, the reported affinity of the
enzyme for this ion (Kd of 6 mM) exceeds
the physiological concentration of Mn2+ by about 3 orders
of magnitude, raising questions about the cation dependence in
vivo. Studies of metal activation over a wide concentration range
reveal a high affinity binding site for Mn2+ and other
metals, including Zn2+, which is present at higher levels
in biological systems.
Glycosyltransferases that, like 3GT, catalyze "retaining"
reactions are expected to utilize double displacement mechanisms in
which the UDP to galactose bond is cleaved, with formation of an
intermediate before transfer of galactose to an acceptor. The mechanism
of the reaction catalyzed by
3GTcd is sequential, UDP not being
released before the completion of catalysis. However, a low level of
UDP-galactose hydrolytic activity indicates that the UDP to galactose
bond can be cleaved in the absence of a carbohydrate acceptor, possibly
via formation of an oxycarbenium intermediate. The properties of
3GT
activated by different metal ions and a mutant with a substitution in a
metal binding sequence motif indicate a role for the metal ion in
UDP-galactose binding and catalysis.
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EXPERIMENTAL PROCEDURES |
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Construction of pET15b E80rGT--
The
expression plasmid was generated from a cDNA of bovine
1,3-GT in
pSV-SPORT vector provided by Dr. L. Inverardi, Diabetics Research, Cell
Transplant Center, University of Miami School of Medicine. This clone
had a deletion corresponding to Tyr-64 to Phe-95 that included a
15-residue sequence previously reported to be essential for enzyme
activity. The region required for activity was restored by polymerase
chain reaction using the primers designated "extension" and
"BGT-C." The product was gel-purified and used as a template in a
second amplification using primers BE80GT-N and BGT-C as follows.
Extension (coding), GAAAGCAAGCTTAAGCTATCGGACTGGTTCAACCCATTTAAACGC; BE80GT-N (coding), CGAATATCATATGGAAAGCAAGCTTAAGCTATCG; BGT-C
(complementary), CGCGGATCCCAAAGTCAGACATTA-TTTCTAACCAC.
The product from the second polymerase chain reaction was directly used
for TA cloning. Positive colonies were isolated and screened by
restriction mapping with NdeI and BamHI. A clone
with the appropriate insert was selected and digested with the same restriction enzymes. The insert was purified by agarose gel
electrophoresis and cloned into a preparation of pET15b vector that had
been previously cleaved with NdeI and BamHI. The
product was transformed into Escherichia coli DH5
competent cells, and plasmids were prepared and characterized by
restriction mapping and DNA sequencing.
Bacterial Expression and Purification--
Cultures of E. coli BL21(DE3) transformed with pET15b rGT were grown in LB
medium containing 100 µg/ml ampicillin with rapid shaking (250rpm) at
37 °C. When the A600 nm of the culture reached 0.8-1.0, isopropyl-1-thio-
-S-galactopyranoside was added to
a concentration of 0.4 mM to induce expression of T7 RNA
polymerase and recombinant
3GTcd. The cultures were then incubated
at 27 °C for 20 h with slow shaking (200 rpm) and harvested by
centrifugation at 3,000 rpm for 20 min. Bacteria were washed with
washing buffer (20% sucrose, 20 mM Tris-HCl, pH 8.0),
suspended in 50 mM Tris-HCl, pH 8.0, containing 1 mM EDTA, 0.1 M NaCl, and lysed using a French pressure cell. The soluble fraction was collected by centrifugation at
12,000 rpm for 30 min.
The supernatant was applied to a to Ni2+ column at 4 °C,
and the column was subsequently washed with 10 volumes of 20 mM Tris HCl buffer, pH 7.9, containing 5 mM
imidazole and 0.5 M NaCl followed by 6 volumes of 20 mM Tris-HCl buffer, pH 7.9, containing 60 mM imidazole and 0.5 M NaCl. The enzyme was finally eluted
with 6 volumes of 20 mM Tris-HCl buffer, pH 7.9, containing
500 mM imidazole and 0.5 M NaCl. Purified
recombinant 3GTcd was precipitated between 10 and 80% saturation
with ammonium sulfate (can be stored in 20 mM MES buffer,
pH 6.0, containing 50% glycerol at
20 °C for up to 6 months).
Mutagenesis-- Mutagenesis was carried out by the polymerase chain reaction megaprimer method (21) with modifications described previously (22, 23). Mutant coding sequences were generated by amplifications using a T7 promoter and mutagenic and T7 terminator primers. The final product of amplification was cleaved using NdeI and BamHI and cloned into a pET15b vector that had been previously treated with the same enzymes. Mutants were characterized by automated DNA sequence analysis of the entire coding sequence in the expression vector, and the proteins were expressed as described for the wild-type enzyme.
Activity Measurements--
3GT assays typically included 2-4
µg/ml enzyme, 50 mM MES buffer, pH 6.0, 10 mM
lactose, 0.3 mM UDP-[3H]galactose
(specific activity, 500 cpm/nmol), 0.1% bovine serum albumin, and
metal cation in a total volume of 100 µl and were incubated at
37 °C for 5-15 min. In steady-state kinetic studies, the
concentrations of metal ion, lactose, and UDP-galactose were varied
with other substrates and cofactors at fixed concentrations. Blanks
were reactions from which the acceptor substrate is omitted. Studies at
high enzyme concentrations indicated that the enzyme has a low level of
UDP-galactose hydrolase activity, which contributes to the backgrounds
measured in this way; however, because hydrolase activity is only
0.25% of the transferase activity, the effects of the hydrolase
activity on the calculated transferase activities is insignificant.
Assays were terminated by adding ice-cold 0.1 M EDTA (100 µl). The reaction mixture was then applied to a 2-ml AG1-X8 (Bio-Rad)
column, and the radioactive product was eluted with 0.5 ml followed by
1 ml of water. The eluate was collected in a plastic vial, mixed with
10 ml of EcoLume (ICN Biomedicals, Costa Mesa, CA), and counted
in a liquid scintillation counter (LKB). One unit of enzyme activity is
defined as the amount of enzyme that catalyzes the transfer of 1 µmol
of galactose from UDP-galactose to lactose/min at 37 °C. Conditions
were chosen so that <25% of the radioactivity from the
UDP-[3H]galactose was incorporated into product in most
experiments. Kinetic data were analyzed by fitting to appropriate rate
equations using the curvefitter program of SigmaPlotTM. For metal
activation studies, data were fitted to equations describing activation
through binding to a single metal binding site (Equation 1) or
activation to V1 by binding to site 1 with
affinity K1 and to V2 by
additional binding to site 2 with a Kd of
K2 (Equation 2).
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
CD Spectroscopy--
Near and far UV CD spectra of recombinant
proteins were determined with a JASCO J-710/720 spectropolarimeter.
Twenty spectra were scanned for each sample at a speed of 100 nm/min,
averaged, and smoothed. Near UV CD spectra (250-320 nm) were
determined using a cell with a path length of 1 cm, and far UV spectra
(200-250 nm) were determined using a cell with a path length of 0.1 cm. Proteins were dissolved in 10 mM MES, pH 6.0, containing 50% glycerol at concentrations between 0.15 and 0.5 mg/ml.
Far UV CD data were analyzed to estimate secondary structure
composition using the k2d neural network program (24).
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RESULTS |
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Preparation and Properties of Bovine Recombinant
3GTcd--
3GTcd (residues 80-367 of bovine
1,3GT) was expressed cytoplasmically in E. coli as a
soluble active enzyme and was purified, reproducibly, in yields of more
than 7 mg/liter bacterial culture. Active enzyme is also produced by
expression at 37 °C, but the yield is lower by about 20% than at
27 °C. The enzyme migrates as a single component on SDS gel
electrophoresis with an apparent molecular weight of 36,000 (data not
shown). After precipitation between 10 and 80% saturation with
ammonium sulfate,
3GTcd is soluble at concentrations of up to 7.5 mg/ml in 20 mM MES, pH 6.0, containing 50% (v/v) glycerol.
A form of
3GTcd with an additional N-terminal truncation of six
residues was also expressed. In this case, when the expression was at
27 °C, a similar yield was obtained, but at 37 °C, essentially no
active enzyme was produced. Steady state kinetic studies with the
purified smaller enzyme, in which the concentrations of UDP-galactose
or lactose were varied at a fixed concentration of the second substrate
at 10 mM Mn2+, showed that the apparent
Km and Vm values were closely similar to
those of the larger enzyme (data not shown).
Fig. 2 shows the near and far UV CD
spectra of 3GTcd. CD spectra have not been previously reported for
either natural or recombinant forms of this enzyme. The near UV
spectrum has a similar magnitude to those of other proteins and can be
expected to be that of the correctly folded catalytic domain. The far
UV CD spectrum was analyzed using the k2d neural network program to
give estimates of 28%
helix and 38%
sheet; the predicted
spectrum from the analysis is a reasonable fit to the experimental data
(Fig. 2). The secondary structure predicted from the sequence from a
consensus of the PREDATOR (25, 26), PHD (27), and Quadratic Logistic (28) methods is 27%
helix and 18%
sheet. Differences in the
sheet content obtained from these analyses may reflect the inaccuracy of
-structure predictions from far UV CD spectra
resulting from the relatively weak ellipticity of
sheets compared
with
helices as well as the inherent limitations of sequence-based secondary structure predictions. The predictions are similar to the
secondary structure composition of the
-1,4-GT catalytic domain:
25% helix, 20%
sheet (11). The form of
GTcd with the
additional six-residue truncation had CD spectra closely similar to
those of the larger enzyme.
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3GTcd Has High and Low Affinity Binding Sites for
Mn2+ and Other Metals--
As previously reported for
natural
3GT, the bacterially expressed enzyme is inactive in the
absence of metal ions and is strongly activated by Mn2+.
Activity measurements in the presence of other metal ions at concentrations of 10 mM and in the absence of
Mn2+ indicated that Co2+ and Fe2+
also activate the enzyme but Zn2+, Ca2+, and
Mg2 do not. Detailed studies with Fe2+ were not
performed because of its tendency to oxidize in the assay system, but
the kinetic properties of the Co2+-activated enzyme were
characterized in detail (see below and Table
I). Enzyme activities at Mn2+
concentrations from 10 µM to 15 mM at fixed
concentrations of lactose (10 mM) and UDP-galactose
(0.3 mM) did not fit well to a rate equation describing the
dependence of activity on metal binding to a single site, giving
residuals that vary systematically with log[Mn2+].
However, the rate equation for a two-site model of metal activation gave a better fit (Fig. 3B).
The results of this analysis indicate that
3GTcd is activated by
binding of Mn2+ to a high affinity site with an apparent
Kd value of 80 ± 20 µM;
additional binding to a second site with an apparent Kd of 2.3 ± 0.5 mM gives a 6-fold
higher maximum activity at the substrate concentrations used in these
assays.
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This result led us to re-investigate the effects of other metal ions over a broader range of concentrations (10-10,000 µM). Although no activity was observed with Ca2+ or Mg2+ at any concentration, Zn2+ was found to activate up to a concentration of 500 µM and to progressively inhibit at higher concentrations. In the lower concentration range, Zn2+ produces a similar level of activity to Mn2+ and is more effective than Co2+ (Fig. 3). The data obtained with Zn2+ do not fit well to an equation describing activation by binding to a high affinity site and inhibition resulting from binding to a second lower affinity site, and the line through these data in Fig. 3A was generated by a nonlinear spline method. Inhibition by Zn2+ may result from multisite binding and/or denaturation at higher concentrations. The activity profile with Co2+ fits well to the equation for single-site activation with a Kd of 4.8 ± 0.5 mM and a lower maximum activity than for Mn2+. It is possible that this cation also binds to two sites, but that the enzyme form with a single metal ion bound to the high affinity site is inactive or has extremely low activity. The apparent binding constants for activation by different cations derived from these studies with fixed substrate concentrations are summarized in Table II.
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To determine whether 3GT, when partially activated by
Mn2+ binding to the high affinity site, can be further
activated by a different metal binding to a second lower affinity
second site, different metal ions (Ca2+, Mg2+,
Zn2+, Ni2+, Cu2+, Te3+,
and Eu3+) were added at concentrations of 10 mM
in the presence of 80 µM Mn2+. None enhanced
the activity. In the enzyme activated by 10 mM Mn2+, Cu2+ or Eu3+ were found to
strongly inhibit activity at micromolar concentrations, indicating that
they bind to a high affinity cation binding site(s). Because
Eu3+ is a very potent inhibitor, it was further
characterized and found to be a mixed inhibitor with respect to
Mn2+ (0.5-6 mM) with a small intercept effect
and large slope effect in a double reciprocal plot (Fig.
4). In contrast, Eu3+ is a
competitive inhibitor against Co2+ (2.2 to 15 mM; data not shown). These results are consistent with the
presence of two metal binding sites for Mn2+ and a single
site for Eu3+; it appears also that a binding of
Co2+ to a single site is required for catalysis. An
apparent Ki of 0.5 µM was calculated
from the inhibitory activity against Co2+ and
Kii and Kis values for inhibition
against Mn2+ were 11.3 ± 1.7 and 3.3 ± 0.7 µM, respectively.
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Steady State Kinetic Studies with Different -Galactoside
Acceptors Show That
3GTcd Catalyzes Galactose Transfer by a
Sequential Mechanism--
Bisubstrate reactions can utilize ping-pong
(double displacement) or sequential mechanisms; these can be
distinguished by steady state kinetic experiments in which acceptor and
donor substrate concentrations are varied. Galactose transfer to
lactose, lactosamine, and
-azido lactose was measured at
UDP-galactose concentrations from 0.03 to 0.6 mM and
different acceptor concentrations at a fixed 10 mM
concentration of MnCl2. The steady state kinetic parameters are summarized in Table III. In each
case, the data fitted best to Equation 3, and in double-reciprocal
plots for velocity and concentration of either substrate at a series of
fixed concentrations of the second substrate, gave a family of
intersecting lines (Fig. 5). In a
double-displacement mechanism, Kia is zero (Equation 4), and double-reciprocal plots produce families of parallel lines. These results indicate that the catalytic mechanism is sequential so
that both substrates bind to the enzyme before any product is released
(29). In a sequential mechanism, substrate binding can be random or
ordered, and if ordered, acceptor binding can precede UDP-galactose
binding or vice versa.
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3GTcd Catalyzes a Low Rate of UDP-galactose Hydrolysis,
Indicating That It Does Not Bind Acceptor before Donor Substrate in an
Obligatory Order--
UDP-galactose hydrolase activity was initially
noticed in the form of high backgrounds (3H release from
UDP-galactose in the absence of acceptor into a product that does not
bind to the anion exchange resin) at higher enzyme concentrations. The
activity increases linearly with enzyme concentration and time, up to
15 min. Hydrolase activity was characterized using a 10-20-fold higher
concentration of enzyme than in standard assays; it is
Mn2+-dependent (data not shown) and also
displays saturation kinetics with varying concentrations of
UDP-galactose (Fig. 6). The
kcat for hydrolysis is 0.25% of the
corresponding transferase activity with lactose as acceptor.
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The hydrolytic activity of 3GTcd indicates that the enzyme binds the
donor substrate and catalyzes cleavage of the bond between UDP and
galactose in the absence of a carbohydrate acceptor. Although the
enzyme complexes for the hydrolysis reaction may differ structurally from those for the transfer reaction, the hydrolase activity is inconsistent with a sequential mechanism with obligatory ordered binding of acceptor before donor substrate. The remaining alternative mechanisms are random sequential or ordered sequential binding of donor
and acceptor.
The kinetic parameters listed in Table III were calculated by fitting to Equation 3 designating UDP-galactose as substrate A and lactose as substrate B. The same rate equation applies to an ordered sequential mechanism and random equilibrium mechanism; the values for Kib = Kia × Kb/Ka given in Tables I and III represent the Kd of the enzyme-acceptor complex in the case of a random equilibrium mechanism.
Metal Cofactor Substitution Affects UDP-galactose Binding and, More
Strongly, Catalytic Efficiency--
Because Mn2+,
Co2+, and Zn2+ are active as cofactors for
3GTcd, the role of the metal ion can be investigated by determining
the effects of substituting different metals on different kinetic parameters. The three metal ions were used at concentrations of 10 mM, 10 mM, and 300 µM,
respectively, levels that gave optimal activities in assays conducted
at fixed substrate concentrations. Table I shows that the metal
substitution has a relatively small but significant effect on
kcat and on the Km for
acceptor (lactose), but Kia, the
Kd of UDP-galactose from the enzyme-UDP-galactose
complex in an ordered or random equilibrium sequential mechanism, was
increased 10-fold, and kcat/ Kia × Kb, a parameter reflecting
catalytic efficiency in a bisubstrate reaction (30), was reduced
11-38-fold.
These three metal ions are also effective cofactors for the low
hydrolase activity of 3GTcd. Hydrolase activity was measured at a
range of UDP-galactose concentrations with the same fixed concentrations of these metals (Fig. 6). As also shown in Table I,
metal substitution has similar effects on this activity, perturbing kcat and the Km for
UDP-galactose, which results in a larger reduction in catalytic
efficiency (kcat/Km), as observed with the transferase activity.
The Effects of Mutations in the Conserved DVD Sequence Indicates a
Crucial Role in Functional Properties Associated with Metal
Binding--
A sequence motif, DXD, with some less specific
conserved features in the adjacent sequence, is found in many
metal-dependent glycosyltransferases (14-16). The
aspartates have been shown to be crucial for activity in enzymes that
are highly divergent in function and sequence: yeast MNN1
-1,3-mannosyltransferase and large clostridial cytotoxins (15, 16).
A similar sequence, DXH, is essential in
UDP-GalNAc:polypeptide GalNAc-transferase (17). This region,
which appears to represent part of the binding site for a divalent
cation cofactor (15), is present in
3GTcd as
D149VD151. The role of this region in
3GTcd
was investigated by expressing mutants with substitutions for each of
the aspartates: D149N and D151N. Both enzymes
were expressed in soluble form and isolated in pure form in reasonably
good yields (about 4 mg/liter bacterial culture), but no catalytic
activity was detected in either protein. Near and far UV CD spectra of
the D149N mutant are closely similar to those of the
wild-type enzyme (Fig. 7), indicating
that the loss of activity is a direct effect of the mutation, but the
D151N mutant shows large changes around 265 nm in the near
UV range and 230 nm in the far UV range. Difference spectra for this
mutant are characteristic of an effect on tertiary, rather than
secondary, structure that affects the environment of a buried
tryptophan (see "Discussion"). These results indicate that
Asp149 has an essential role in catalysis, whereas
Asp151 may be required to form the correct structure in
this region for activity. Val150 is less conserved and has
a side chain that is unlikely to have a direct role in catalysis. To
perturb the structure associated with the essential aspartates but
retain catalytic activity so that quantitative changes in kinetic
parameters can be measured, a mutant of
3GTcd was constructed and
expressed containing a Val150 to Ala substitution. The
V150A mutant was also expressed in a soluble form in good
yield and purified. It is less active than the wild-type enzyme under
standard assay conditions, yet its CD spectrum was closely similar to
that of the wild-type protein (Fig. 7). Tables I and II summarize its
metal activation and kinetic properties. The activities with varying
[Mn2+] at fixed standard concentrations of the two
substrates were found to fit best to the equation for metal binding to
a single site. The apparent affinities of the enzyme for
Mn2+, Zn2+, and Co2+ were reduced
2-4-fold, and the extrapolated activity at saturating [Co2+] was very low, in keeping with an effect on cation
activation. The values of kinetic parameters determined at 10 mM Mn2+ indicate that the mutation produces a
2-fold reduction in kcat, a 6-fold increase in
the Km and Ki for UDP-galactose, and a more than 20-fold decrease in catalytic efficiency
(kcat/Kia × Kb).
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DISCUSSION |
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Expression of 3GTcd at 27 °C produces a stable enzyme in
high yield that is sufficiently soluble (up to 7.5 mg/ml) and stable to
be a suitable subject for structural analysis. The effects of
temperature on the expression of the fully active variant with six
additional residues deleted from the N terminus indicates that residues
80-85 enhance stability but not structure or activity.
The activity of 3GTcd is modulated by high and low affinity binding
sites for metal cations (Ka of >103 and
105-106
M
1) as previously observed with
-1,4GT (31). Inhibition by Eu3+ and Cu2+ is
associated with KI values in the low micromolar
range. The involvement of two metal ions in catalysis has been observed in several metalloenzymes that catalyze phosphoryl transfer reactions including 3'-5' DNA exonuclease (32), alkaline and acid phosphatases (33, 34), purple acid phosphatase (35, 36), and phosphoprotein Ser/Thr
phosphatase, PP-1 (37). In these enzymes, the metal ions form a bimetal
center that in purple acid phosphatase and PP-1 has been found to act
as a ligand for the phosphate, facilitating its stabilization and
correct orientation in the active site and also in generating a
hydroxide nucleophile involved in catalysis (35-37). An analogous role
for the metal as a ligand for phosphate(s) in the donor substrate in
3GT is supported by the present results.
In vivo only the high affinity site may be relevant since
the cellular concentrations of the ions that can activate 3GTcd are
in the micromolar range. In this concentration range, Zn2+
is as effective as Mn2+ (Fig. 3A). The
concentrations of different metals in the trans-Golgi lumen, the
cellular site of action of
3GT, are unknown. However, body fluids
that originate in part from this compartment such as milk have
Zn2+ concentrations (50-200 µM) that exceed
those of Mn2+ by about 2 orders of magnitude (38).
Co2+ is irrelevant as an activator in vivo
because of its low abundance relative to the level required for
activation. It is therefore possible that, as with methionine
aminopeptidase I (39), the biologically active cofactor has been
misidentified, and Zn2+ is the relevant cofactor for
3GT. However, previous studies with
-1,4GT-1 show that the enzyme
in Golgi vesicles is optimally activated by lower concentrations of
Mn2+ and that an endogenous high molecular weight molecule
participates in the activation of this glycosyltransferase in
vivo (40). Although the present studies indicate that
Zn2+ is the likely natural cofactor(s) of
3GT, the
possibility of the involvement of an endogenous macromolecule cannot be discounted.
There is limited information presently available on the mechanisms of
glycoprotein glycosyltransferases that catalyze retaining reactions.
The steady state kinetic properties of 3GTcd indicate, as for
-1,4GT (41), a sequential mechanism in which metal cofactor, donor,
and acceptor bind enzyme before catalysis, with no release of UDP
before the transfer of galactose to an acceptor. Thus, a double
displacement mechanism in which UDP is released with formation of an
intermediate covalently bound
-galactosyl derivative can be
eliminated. Present data do not distinguish whether substrate binding
is random or ordered. Although the low level of UDP-galactose hydrolase
activity makes obligatory binding of acceptor before donor substrate
seem unlikely, it cannot be eliminated since we have not established
that hydrolase activity is a catalytically competent step in the
overall reaction. Previous studies with bovine
3GTcd also show that
the enzyme binds more strongly to UDP-Sepharose than
1,4GT-1 but
does not bind to lactose- or N-acetyllactosamine-Sepharose (2); however, this does not prove that there is obligatory binding of
donor before acceptor substrate and may simply reflect the much weaker
affinity of the enzyme for acceptor as compared with donor substrate.
Ordered sequential binding of donor and acceptor substrates has been
reported for
1,4GT-1 and
-3 fucosyltransferase (41, 42), but
further studies are required to determine whether substrate binding to
3GT is ordered or random.
The effects of different metal cofactors on the steady state kinetic
parameters show that the metal strongly affects the affinity for
UDP-galactose (Kia) but has a lesser effect on
affinity for acceptor substrate (Kb and
Kib); this is borne out by the kinetic parameters
for UDP-galactose hydrolysis with different cofactors (Table I).
Although there are large standard errors in the values for the
catalytic efficiency for galactose transfer,
kcat/Kia × Kb, since these are compounded from the errors in
three experimentally determined parameters, it is clear that metal
substitution has its largest effect on this parameter. For
UDP-galactose hydrolysis, the substitution of Co2+ or
Zn2+ produces increases in the Km
similar to those for Kia for the transferase
reaction and greater reductions in catalytic efficiency
(kcat/Km); the
Km for UDP-galactose will closely approximate the
Kd of the E·S complex for hydrolysis because of
the low kcat. As discussed above, the transition state for the 3GTcd-catalyzed reaction may be similar to those in
other retaining glycosyltransferase such as glycogen phosphorylase and
its close relative, maltodextrin phosphorylase. In these enzymes, where
the reaction is analogous to the reverse of the
3GTcd-catalyzed reaction, the monosaccharide in the enzyme-bound oligosaccharide substrate that is transferred is rotated relative to its preferred structure in solution, and C1-O bond cleavage results in formation of
an oxycarbenium intermediate (43). A similar process may occur with the
UDP-galactose substrate of
3GT; the formation of such an
intermediate independent of acceptor substrate binding is consistent
with the low level of hydrolytic activity. Data supporting cleavage of
the nucleoside diphosphate-monosaccharide bond before transfer have
been previously reported for two inverting transferases,
-1,3-fucosyltransferase V (43, 44) and
-1,4GT-1 (45, 46),
suggesting that this may be a common intermediate in retaining and
inverting transferases. A plausible role for the metal would involve an
interaction with the phosphates of the UDP-sugar and of the UDP formed
by cleavage of the UDP to galactose bond.
The aspartates of the D149VD151 motif of
3GTcd are essential for catalysis as found for analogous residues in
yeast MNN1
-1,3-mannosyltransferase (15) and clostridial cytotoxins
(16) and for the aspartate and histidine of an analogous DXH
sequence in polypeptide GalNAc transferase (17). The Asp151
to Asn mutation changes the near and far UV CD spectra, indicating that
this substitution perturbs the local structure. Difference spectra for
the mutant show a peak in the far UV CD spectrum around 230 nm and a
broad peak in the near UV CD spectrum centered around 265 nm (Fig. 7);
both of these peaks are consistent with an effect of the mutation on
the environment of a buried tryptophan side chain (47). Since residues
of this sequence motif are essential for function in highly divergent
cation-dependent glycoprotein glycosyltransferases, which
are otherwise not significantly similar in sequence, it appears that
these enzymes may be distantly related or share a homologous domain
(14). Sequence comparisons suggest that the catalytic domains of
glycoprotein-processing glycosyltransferases have modular structures
and are composed of domains of different origins (17). The elucidation
of high resolution crystallographic structures for multiple
representatives is needed to clarify this issue. At present, the only
known structure is that of the truncated catalytic domain of
-1,4GT-1 (11); the structure of
3GTcd is presently unknown, but a
model for a putative sugar nucleotide binding domain of
3GTcd has
been described based on the use of a T4 phage DNA-modifying
glucosyltransferase (48) as a template. However, the phage enzyme
appears to be structurally unrelated to
-1,4 GT (11), whereas
secondary structure topology predicted for
3GTcd from multiple
alignment methods and the secondary structure content indicated by the
far UV CD spectrum are consistent with an
/
fold similar to that
found in
1,4GT-1. Also, the D252VD254
sequence in
1,4GT-1 has a role in binding the UDP-galactose substrate (11), in agreement with the role for
D149VD151 in
3GTcd indicated by the present
study which shows that the Val150 to Ala substitution
reduces the affinity for UDP-galactose and more strongly lowers the
catalytic efficiency for transferase and hydrolase activities. Although
there is insignificant global similarity between the sequences of the
catalytic domains of
3GTcd and
1,4GT family, this apparent
similarity between the two enzymes in structure-function relationships
suggests that part of their structures may have a common origin.
Unfortunately, the level of resolution of the structure of
1,4GT-1
does not provide any information about bound metal ions. The properties
of the Val150 to Ala mutant of
3GTcd suggest that the
mutation disrupts properties of the enzyme that are associated with
cation activation, supporting the view that this region has a role in
binding the metal ion and UDP-galactose.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM 58773.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biomedical Sciences, Florida Atlantic University, 777 Glades Rd., Boca Raton, FL 33431. Tel.: 561-297-0407; Fax: 561-297-2221; E-mail: kbrew@fav.edu.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M006530200
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ABBREVIATIONS |
---|
The abbreviations used are:
3GT, UDP-D-galactose:
-D-galactosyl-
-1,3-galactosyltransferase;
-1, 4GT-I,
UDP-D-galactose:
-D-N-acetylglucosaminyl
-1,4-galactosyltransferase;
cd, catalytic domain;
MES, 4-morpholineethanesulfonic acid.
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