cysteine mutagenesis
Post on 14-Feb-2017
239 Views
Preview:
TRANSCRIPT
1
J. Biol. Chem. (3/11/02)
Evaluation of Critical Structural Elements of UDP-sugar Substrates and Certain Cysteine
Residues of a Vertebrate Hyaluronan Synthase
Philip E. Pummill and Paul L. DeAngelisψ
Dept. of Biochemistry and Molecular Biology
Oklahoma Center for Medical Glycobiology
Univ. of Oklahoma Health Sciences Center
940 Stanton L. Young Blvd., Oklahoma City, OK 73104
ψ To whom correspondence should be addressed. Phone: (405) 271-2227 Fax: (405) 271-3092;
email: paul-deangelis@ouhsc.edu
Running Title: Elements of UDP-Sugar Precursors and Cysteines of HAS
Key Terms: hyaluronic acid, hyaluronate, or hyaluronan; synthase; polysaccharide;
glycosyltransferase; sugar-nucleotide; active site
* This work was supported by a National Institutes of Health grant (GM56497) to P.L.D.
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on April 9, 2002 as Manuscript M202456200 by guest on A
pril 12, 2018http://w
ww
.jbc.org/D
ownloaded from
2
ABSTRACT
The hyaluronan [HA] synthases catalyze the addition of two different monosaccharides from
UDP-sugar substrates to the linear heteropolysaccharide chain. In order to accomplish this task,
the HA synthases must be able to bind and to transfer from both UDP-sugar substrates. Until
now, it has been impossible to distinguish between these two abilities. We have created a mutant
of xlHAS1, a HA synthase from Xenopus laevis, that allows for the examination of the enzyme’s
ability to bind substrate only. The ability of different compounds to protect the xlHAS1(C337S)
mutant enzyme from loss of activity due to treatment with N-ethylmaleimide, a cysteine
modifying reagent, yields information on the relative affinity of a variety of nucleotides and
nucleotide-sugars. We have observed that the substrate-binding selectivity is more relaxed than
the specificity of catalytic transfer. The only attribute that appears to be absolutely required for
binding is a nucleotide containing two phosphates complexed with magnesium ion. The role of
certain cysteine residues in catalysis was also evaluated. C307 of xlHAS1 may play a role in
catalysis or in maintaining structure. Mutation of C337 raises the UDP-GlcUA Michaelis
constant (Km), suggesting that this residue participates in UDP-GlcUA substrate binding or in
catalytic complex formation.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
3
INTRODUCTION
HA1 is a glycosaminoglycan composed of alternating repeats of the disaccharide (→4)-β-D-
GlcUA(1→3)-β-D-GlcNAc(1→). This polysaccharide is abundant in vertebrates where it plays
structural, recognition, and signaling roles (1). The enzymes that catalyze the formation of HA,
the HA synthases, are dual-action glycosyltransferases that catalyze the transfer of both GlcUA
and GlcNAc (2, 3). These membrane-associated enzymes utilize UDP-linked sugar precursors.
We have reported previously that xlHAS1 is highly specific for the authentic HA substrates,
UDP-GlcUA and UDP-GlcNAc; the C4 epimers or UDP-glucose will not support HA
biosynthesis (4).
The vertebrate, the streptococcal, and the viral enzymes are comparable in size and have
regions or short sequence elements with considerable similarity (2). A few of these putative
elements, for example the DXD-containing motif, are similar to other glycosyltransferases that
produce various α- or β-linked polysaccharides from UDP-sugars (5-7). However, the exact role
of these motifs in the structure and/or the function of the polypeptide are only recently being
investigated. In view of the close amino acid sequence similarities among many
glycosyltransferases, it is quite likely that these residues are involved in binding common
determinants of UDP-sugars (e.g. uridine ring, phosphate groups) and/or catalyzing the transfer
of sugars residues.
1 The abbreviations used are: HA, hyaluronan, hyaluronate, or hyaluronic acid; HAS, HA synthase; Glc, Glucose;
GlcUA, glucuronic acid; GlcNAc, N-acetylglucosamine; Gal, Galactose; GalUA, galacturonic acid; GalNAc, N-
acetylgalactosamine; Tris, tris(hydroxymethyl)aminomethane; DTT, dithiothreitol; EDTA,
ethylenediaminetetraacetic acid; NEM, N-ethylmaleimide; Km, Michaelis constant; and Ki, inhibition constant.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
4
X-ray crystal structures have been obtained for several different glycosyltransferases from
bacteria, a bacteriophage, and vertebrates that utilize UDP-sugars as well as for a bacterial UDP-
glucose dehydrogenase (8-18). All of these structures show extensive hydrophobic interactions
with the uracil ring and hydrogen bonding with the functional groups of the uracil as well as with
the ribose hydroxyls and phosphates (Table 1). Many of these transferases use the DXD motif to
coordinate the divalent metal cation and interact with the phosphate groups of UDP (5-7, 14-16).
In lieu of a three-dimensional structure or active-site labeling data, the direct measurement or
analysis of the binding of substrates to glycosyltransferases is often quite difficult or even
impossible due to the low relative affinity of nucleotide-sugars for the enzymes. For example,
the Km value of xlHAS1 for UDP-sugars ranges from ~100 µM to almost 1 mM, depending on
experimental conditions (ref. 4; Table 2). Direct binding assays would require any washing steps
removing unbound substrate to be completed in a few seconds time. Equilibrium dialysis
experiments would require long times (which can be problematic for labile UDP-sugars) and
yield relatively weak signals.
The situation is further complicated by the extreme difficulty in purifying the vertebrate
HASs in a native state; most binding assessments should be performed on enriched or purified
membrane preparations because many other nucleotide binding proteins exist. Therefore, we
employed an indirect method, utilizing protection from inactivation mediated by a chemical
modification agent, NEM, to assess the relative affinity of a vertebrate HAS for a wide range of
compounds. These molecules have some or most of the structural elements of the authentic
UDP-sugars for HA biosynthesis. Our assumption is that a compound interacting with the
substrate-binding pocket or cleft will block NEM’s access to the site and protect the enzyme
from chemical modification and subsequent inactivation. We found that some structural
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
5
elements of the UDP-sugar substrates, including the two phosphate groups, are critical for
binding to xlHAS1. However, we have also found that some compounds with variations in the
sugar, base, or ribose can bind to xlHAS1 at a putative substrate-binding site but do not support
HA biosynthesis. This observation indicates that the substrate-binding requirements of the
enzyme are more relaxed than the catalytic requirements. Protection experiments suggest that
one or more cysteines might be part of or close to a putative substrate-binding site. We also
found that several cysteines of xlHAS1 were dispensable, but C307 may play a direct or a
structural role.
EXPERIMENTAL PROCEDURES
Production of Recombinant xlHAS1 Wild Type and Cysteine Mutant Enzymes – All reagents
were from Sigma or Fisher unless noted otherwise. The construction and the use of the xlHAS1
expression plasmid for studies in yeast were previously described (4, 19). Basically, the xlHAS1
polypeptide was cloned into the pYES2 vector (Invitrogen) under control of the GAL1 promoter
to form pYES/DG+. Site-directed mutagenesis was performed on pYES/DG+ using the
QuikChange Kit (Stratagene). Seven cysteine codons were altered using pairs of synthetic
oligonucleotides containing either the partially degenerate codon (TYS, where Y = C or T; S = G
or C) or the serine codon (TCT) to obtain a variety of mutants. Plasmids derived from
independent transformants were sequenced to verify the presence of mutations at the various
cysteine codons. The entire open reading frame of each mutant was also verified by sequencing.
The following mutants were generated: C117F, C117L, C117S, C210S, C239S, C298F, C298L,
C298S, C304S, C307S, C337S, C239S/C337S, C304S/C337S, and C307S/C337S. The plasmids
were transformed into Saccharomyces cerevisiae BJ5461 yeast (a pleiotrophic protease deficient
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
6
strain; Yeast Genetic Stock Center, Berkeley) by the lithium acetate/poly(ethyleneglycol)
method (20).
Yeast with recombinant plasmids were routinely grown to a suitable biomass in uracil-
deficient synthetic media with 0.1% glucose and 5% glycerol until OD600 was 0.3. Upon
induction with galactose (1 % final), xlHAS1 wild type or mutant enzyme accumulated in the
plasma membrane fraction. Crude membranes were prepared by disruption with silica/zirconia
beads (0.5 mm) in a MiniBead-Beater-8 (Biospec) and harvested by ultracentrifugation. The
membrane pellet was suspended in 50 mM Tris, pH 7.5, 0.1 mM EDTA, 1 µM E-64, 1 mM
benzamidine, 0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, and 5 µg/ml pepstatin.
Protein was quantitated by the Coomassie dye-binding assay (Pierce) using a bovine serum
albumin standard (21).
Polysaccharide Synthase Assays and Analyses – The incorporation of sugars into high
molecular-weight HA polysaccharide was monitored using UDP-[14C]GlcUA (~290 mCi/mmol;
NEN Life Sciences Products Inc.) and/or UDP-[3H]GlcNAc (29.2 Ci/mmol; NEN Life Sciences
Products Inc.) precursors as described previously (4, 19). Briefly, crude membranes were
incubated at 30°C in Tris buffer, pH 7.5, with MgCl2 and the UDP-sugar precursors.
Unincorporated, labeled UDP-sugars were separated from the HA product using paper
chromatography. HA at the origin of the paper strip was detected by liquid scintillation counting.
Assays were set so that <5% of the radiolabeled substrate was consumed and the enzyme
concentration was in the linear range. All HAS assays throughout this work were performed in
duplicate and the values were averaged.
The apparent Km values for the substrates were obtained by holding one radiolabeled UDP-
sugar at a constant and saturating concentration while titrating the other UDP-sugar. The
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
7
apparent Ki values (concentration of inhibitor required to reduce activity by 50%) of various
compounds were obtained by determining the HAS activity in the presence of varying
concentrations of the inhibitory compounds.
Chemical Modification and Enzyme Protection – For protection experiments, the enzyme
was first incubated with 200-1000 µM protecting compound on ice for 10 minutes. The enzyme
was then treated with 1 mM NEM at 15°C (5 µl reaction volume). After 15 minutes, the reaction
mixtures were diluted to 50 µl with DTT-containing buffer to quench any residual NEM and then
the residual HAS activity was determined.
For affinity experiments, the enzyme was treated with 1 mM NEM at 15°C in the absence or
presence of increasing concentrations of a protecting compound. The apparent affinity values of
various compounds for xlHAS1 were obtained indirectly by assessing their ability to protect the
enzyme from chemical inactivation. The apparent affinity values equal the concentration of
protecting compound required to yield 50% of the maximum protected activity. All kinetic data
was analyzed by graphing with rectangular hyperbola transformation in Sigma-Plot (Jandel
Scientific).
Immunochemical Detection of Polypeptides – The xlHAS1 and mutant proteins were
quantitated by Western blot analysis for assessment of the relative specific HAS activity. After
SDS-PAGE separation, the proteins in the gel were transferred to nitrocellulose by semi-dry
transfer. The blot was blocked with BSA and incubated with the primary reagent composed of
serum (1:1,000) from rabbits immunized with a fusion protein containing 1-166 residues of
xlHAS1 (gift of I. Dawid, ref. 22). Protein A-alkaline phosphatase detection with 5-bromo-4-
chloro-3-indolyl phosphate and nitroblue tetrazolium was used to visualize the immunoreactive
bands.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
8
RESULTS
Mutant Enzyme Expression and HAS Activity – The importance of some of the various
cysteines in xlHAS1 that are conserved among many Class I HASs (bacterial, viral, and
vertebrate) was assessed by site-directed mutagenesis. The membrane preparations containing
the various enzymes were tested for enzyme expression and HAS activity. The results are shown
in Table 2. The cysteine to serine mutation was typically found to be the least altering to protein
expression and activity in comparison to substitution with leucine or phenylalanine. The HAS
specific activity varied slightly among cysteine to serine mutants except for enzymes containing
the C307S mutation. xlHAS1(C307S) and xlHAS1(C307S/C337S) were expressed at levels
similar to that of wild type, but xlHAS1(C307S) retained less than 10% of wild type activity
while xlHAS1(C307S/C337S) had no detectable HAS activity. The Km values were similar for
all mutants except the series with the C337S mutation; UDP-GlcUA binds with lower affinity to
these mutants as assessed by higher Km values (Table 2).
Loss of HAS Activity Due to NEM Modification – In 1979, it was reported that a cysteine
modifying reagent, p-chloromercuribenzoate, inhibited the release of HA by streptococcal HAS
(23). We found that this reagent inactivated xlHAS1-catalyzed polymerization of HA (data not
shown). We tested the effect of NEM, a more selective cysteine modifying reagent, on the HAS
activity of the wild type and mutant enzymes. Membranes were incubated with varying
concentrations of NEM and the residual HAS activity was determined. xlHAS1 wild type and all
cysteine to serine mutants were inactivated by low levels of NEM, retaining <10% of their HAS
activity after treatment with 200 µM NEM (Fig. 1 and data not shown). xlHAS1 mutants
containing the C337S mutation were slightly more resistant to NEM-mediated loss of HAS
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
9
activity, with IC50 values (the concentrations of NEM that reduced the HAS activity by 50%) of
~75 µM NEM compared to ~35 µM NEM for all other mutants and wild-type (Fig. 1 and data
not shown). Results similar to wild type were obtained for xlHAS1(C307S) when more total
protein was used to accommodate the lower activity of this mutant (data not shown).
Protection from Loss of HAS Activity Due to NEM Modification – To determine if the
authentic HA substrate UDP-sugars, UDP-GlcUA and UDP-GlcNAc, could protect the wild-type
and mutant enzymes from loss of HAS activity due to NEM modification, membranes were
incubated with substrates before NEM treatment. If a substrate binds to the active site, then
NEM added later will be excluded from the site and the site’s modification rate will be
decreased. The HAS activity was then determined and compared to the activity of a parallel
aliquot of enzyme not treated with NEM. When preincubated with substrates, only
xlHAS1(C337S) could be protected from loss of HAS activity due to NEM. The modification of
C337 probably inactivates the enzyme by an indirect mechanism because the C337S mutant still
retains HAS activity. This mutant enzyme allows analysis of substrate binding characteristics
because an “irrelevant” (i.e. non-protectable) inactivation pathway has been eliminated. The
UDP-sugar protection effect also required Mg++ because no protection was observed when 10
mM EDTA chelator was added instead of Mg++ (data not shown).
Certain other structurally related compounds also protected xlHAS1(C337S) to various
extents from NEM-mediated loss of HAS activity (Table 3). The protecting compounds include
several UDP-sugars, thymine-containing nucleotides, and several other nucleotide triphosphates.
No nucleotide monophosphate protected the enzyme from NEM-mediated loss of HAS activity
(Table 3 and data not shown). The monosaccharides GlcUA and GlcNAc provided little
protection (Table 3).
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
10
The ability of some molecules to protect xlHAS1(C337S) from NEM-mediated loss of HAS
activity allowed for the investigation of the enzyme's apparent affinities for the various
compounds. Apparent affinity values were determined by titrating the protectant and measuring
the residual HAS activity after NEM treatment. UDP-GlcNAc yielded the highest maximum
protection, protecting ~60% of the HAS activity observed in the control not treated with NEM
(Fig. 2A). UDP-GalNAc, the C4 epimer, protected less than 20% of the control activity,
therefore, no apparent affinity value was obtained for this compound. The apparent affinity
values for all of the other compounds tested were about 10-4 M, except for UDP and UTP (Fig.
2B). Although UDP and UTP provided higher maximum protection than many of the other
compounds, xlHAS1(C337S) displayed approximately 10-fold lower apparent affinity (~10-3 M)
for UDP and UTP. When UDP or UTP were included together with UDP-GlcNAc, an apparent
affinity value similar to that of UDP-GlcNAc alone (~10-4 M) was obtained.
Inhibition of HAS Activity – The protection from NEM-mediated inactivation observed with
the added compounds is presumably due to the compounds binding to the enzyme and shielding
one or more cysteine residues from NEM modification. This cysteine(s) is hypothesized to be
either part of or near a substrate-binding site. To test this assumption of active site occupancy,
the compounds were tested for their ability to inhibit HAS activity (Table 4). As shown in
Figure 3, many of the compounds that protect xlHAS1(C337S) from NEM-mediated loss of HAS
activity (solid bars) also inhibit wild-type xlHAS1 (open bars). Similar inhibition was observed
with xlHAS1(C337S) (data not shown). Apparent Ki values were determined at two different
substrate concentrations and are listed in Table 4. The Ki values were higher when tested under
the higher authentic substrate concentration conditions. Km values of xlHAS1 were determined
for both substrates in the presence of UMP, UDP, and UDP-Glucose (Table 5). Although UMP
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
11
had little effect on the Km values, both UDP and UDP-Glucose raised the values considerably.
There was little effect on Vmax with any inhibitor. Overall, these alterations in kinetics are the
hallmark of competitive inhibition.
DISCUSSION
During the final preparation of this manuscript, it was reported that NEM inactivated HASs
from Group A and C Streptococcus (24, 25). These enzymes contain six and four cysteine
residues, respectively. The xlHAS1 polypeptide has 19 cysteines, therefore it is not surprising
that this enzyme is sensitive to treatment with NEM. When xlHAS1 is treated with biotin-
maleimide, the Western blot band shifts to ~5 kDa larger (data not shown), indicating that there
are ~10 free, readily available cysteines. xlHAS1 enzymes containing the C337S mutation were
slightly more resistant than wild type enzyme to loss of HAS activity due to NEM, indicating
that this residue is responsible in part for the NEM-mediated loss of activity. There are
obviously other cysteine residues that are modified by NEM since none of the cysteine to serine
mutants or double mutants tested thus far were completely resistant to NEM-mediated loss of
HAS activity.
The mutation of C337 to serine caused a large decrease in the enzyme's apparent affinity for
UDP-GlcUA (but not UDP-GlcNAc, Table 2), suggesting that C337 is somehow involved in the
binding of UDP-GlcUA or in GlcUA transfer. This result indicates that C337 is probably close
to the substrate-binding or catalytic site. The C337 residue, however, is probably not buried in a
substrate pocket or cleft because UDP-sugars could not protect the wild-type enzyme from
NEM-mediated inactivation.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
12
In a recent study on a rat glucosylceramide synthase, it was found that C207 was the primary
residue involved in the inactivation by NEM (26). It has also been recently found that the C226S
mutation in the HAS from equisimilis (24) and the C225S mutation in the HAS from pyogenes
(25) caused a reduction of about 90% and 50%, respectively, in the HAS activity. Interestingly,
based on sequence alignments, these residues roughly correspond to C307 in xlHAS1, which is
conserved in all known Class I HASs. As shown in Table 1, the C307S mutant lost almost all
HAS activity. A double mutant with C307S/C337S lost all measurable HAS activity. This
finding suggests that C307 plays a role in substrate binding, catalysis, or enzyme folding; the
latter explanation may not be as likely due to the mutant xlHAS1 enzyme's proper membrane
localization and good expression level.
Even though NEM inactivates the streptococcal HASs, it has been determined that no
cysteines are required for enzyme activity in these HASs (24, 25). Although it was speculated
that one or more of these cysteines are located in or near the active sites, the localization of the
cysteines to a substrate-binding site was not demonstrated by substrate protection from
inactivation. We show here that some of the 19 cysteines in xlHAS1 might be involved in
substrate binding or catalysis, based on kinetic and protection data. This involvement might not
be direct, but it is clear that loss or modification of some of these cysteines has significant effects
on the vertebrate enzyme’s ability to function as a HAS.
The fact that xlHAS1 is very specific for the substrates utilized in the HAS reaction could be
due to specificity at either the binding step or the catalytic step; our results in this report indicate
that the latter is most likely. Table 3 and Figure 3 show that many different compounds are able
to bind to the enzyme and protect it from NEM-mediated loss of HAS activity. The protection
observed with these non-substrate compounds was usually lower than that observed with the
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
13
authentic UDP-sugar substrates (Table 3). One of the most important structural elements appears
to be the pyrophosphate moiety because none of the nucleotide monophosphates tested were able
to protect the enzyme. It appears that xlHAS1 is able to bind purine-containing nucleotides (eg.
ATP, GDP) as well as many different pyrimidine-containing nucleotide-sugars (eg. UDP-
glucose). This finding suggests two potential hypotheses: (i) the enzyme can accommodate these
different shapes or (ii) the protection seen with these compounds is primarily due to interactions
with the phosphate groups. Neither simple phosphate ion nor pyrophosphate ion, however, were
able to protect xlHAS1(C337S) from NEM-mediated loss of HAS activity at a concentration of 1
mM (data not shown). This lack of effectiveness could be due to (a) the charge state differences
among these various phosphate ions, (b) the smaller size of these compounds (i.e. less sterically
hindered of access to pocket or cleft) allowing modification of cysteines that are normally
protected by other larger pyrophosphate-containing compounds, and/or (c) a requirement for
important interactions with the nucleotide base.
Mg++ was required for the protection phenomenon, thus it appears that the HAS enzyme
binds a UDP-sugar/metal complex. As mentioned earlier, the DXD-containing motif has been
implicated in coordination of the divalent metal cation and interaction with the phosphate groups.
All known Class I HASs contain a DXD-containing motif as described by Wiggins and Munro
(27) as well as an XDD motif similar to that seen in the putative UDP-sugar transferase SpsA of
Bacillus subtilis (7,8). When the first aspartate in DXD or the second aspartate in XDD was
mutated to glutamate in mouse HAS1, there was a 99% or greater loss of HAS activity (28).
These aspartate residues are probably involved in interactions with Mg++ and/or the phosphate
groups.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
14
Recently, the crystal structure for SpsA with dTDP has been obtained (8). This structure
shows that SpsA is able to accommodate the methyl group at position 5 of the pyrimidine ring.
Although the substrate specificity or transfer activity of SpsA has yet to be determined, it is
obvious that this protein has the ability to bind both dTDP and UDP (7,8). Our findings indicate
that xlHAS1 can not only accommodate a methyl group at position 5 of the pyrimidine ring, but
also a bromine or iodine atom at position 5, or a thiol at position 2. This suggests that there are
no intimate or essential interactions between xlHAS1 and positions 2 and 5 of the pyrimidine
ring. Interestingly, of the enzymes listed in Table 1, only half show an interaction with the
carbonyl at position 2 of the uridine in the crystal structure. xlHAS1 may interact with the
nucleotide base primarily by the hydrophobic effect due to its relatively promiscuous binding of
nucleotides, as assessed by the protection data.
When the two structures mentioned above for SpsA with different nucleotides are compared,
a shift in the contacts with the ribose ring can be seen to accommodate the loss of the 2' hydroxyl
(7,8). Since xlHAS1 can not only bind dTDP but also ddTTP, similar shifts are probably made
to accommodate the loss of either the 2' hydroxyl or both the 2' and 3' hydroxyls. The proteins
shown in Table 1 interact with the ribose hydroxyls but the requirements and importance in
catalysis or binding are not known. In the case of xlHAS1, contacts with the ribose hydroxyls
may not be essential.
Many of the compounds that provide substantial protection from NEM-mediated loss of HAS
activity also appear to be competitive inhibitors of xlHAS1. The Ki values of these inhibitors are
higher in the presence of increased substrate concentration (Table 4). UDP and UDP-Glucose
alter the Km for the two HAS substrates but do not significantly affect the Vmax (Table 5); these
results are indications of competitive inhibition.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
15
The apparent affinity obtained from Km studies is a measure of both the enzyme's ability to
bind the substrate as well as its ability to catalyze the addition of the sugar to the growing HA
chain. The apparent affinity values for various nucleotides (obtained from Figure 2) are
measurements of the ability of the enzyme to bind a substrate analog without concern for
catalytic ability. The protection from NEM observed with all compounds is probably due to
protection of one or more cysteines at a putative substrate-binding site. The UDP-GlcNAc
binding site is the most likely candidate because the most protection was observed with this
substrate. However, at this stage, it is impossible to determine which particular cysteines are
being protected and whether the cysteines participate in catalysis in the putative substrate-
binding site.
In Figure 3, dTDP, UDP, and thioUDP show higher inhibition of xlHAS1 in comparison to
their protection ability. xlHAS1(C337S) was found to have a much lower apparent affinity for
UDP and UTP than for the UDP-sugars (Fig. 2B). This result could be explained in several
ways, including (i) relative steric hindrance, (ii) multisite inhibition, and/or (iii) allosteric
regulation. As described below, we believe the first two effects may be responsible, in part, for
the observed disparity.
First, it is possible that the lack of sugar moieties on these nucleotides exposes one or more
cysteines in a substrate-binding pocket, which are normally protected by UDP-sugars, to
modification by NEM. The different levels of maximal protection observed in Figure 2 suggest
that there may be variations in the number of cysteines protected by the different compounds.
Specifically, it appears that at least one cysteine is protected by the GlcNAc portion of the UDP-
GlcNAc molecule. This cysteine is not efficiently protected by any of the other compounds,
leading to lower maximal protection values observed for all other compounds (Fig. 2).
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
16
Interestingly, UDP-GalNAc gave the lowest maximal protection, indicating that the position of
the C4 hydroxyl is probably critical for efficient or high affinity UDP-GlcNAc binding.
A second potential explanation for higher inhibition than protection is that nucleotides
without sugar moieties may be able to bind to both putative substrate-binding sites, thus
competing with both substrates simultaneously. This competition would explain the difference
in UDP-GlcNAc Km values observed with different concentration of UDP-GlcUA with xlHAS1
(Table 2), streptococcal HASs (29), and mouse HASs (30). The protection ability of the UDP-
like compounds, however, might be due to binding at only one of these sites (probably the UDP-
GlcNAc site), thus yielding a lower value in our NEM modification experiments.
A third possible explanation for the observation of greater inhibition compared to protection
is allosteric regulation; a site distinct from the catalytic sites would modulate polymerization
upon binding UDP. HA is extruded out of the cell once it is produced, therefore, it would be
difficult for the cell to determine the amount of HA produced directly. However, UDP, the
byproduct of the HAS reaction, may serve as a measure of synthesis rate or extent. Thus, the
local UDP concentration level near the synthase might serve as an internal indicator of the
amount of HA produced. However, the kinetics finding that UDP and UTP act as competitive
inhibitors does not support the solely allosteric control hypothesis. The sensitivity of the
vertebrate HASs to UDP may be an adequate potential negative feedback loop to control
synthesis levels by a competitive mechanism.
No three-dimensional structure for any HAS is available, thus the direct contacts between
substrate and enzyme are not known. Our work is the first assessment of the critical elements of
UDP-sugars required for binding to any HAS. A flexible xlHAS1 binding pocket probably
interacts with the hydrophobic nucleotide base and a metal-complexed pyrophosphate group.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
17
The identification of these critical elements of the substrate may allow for the future design of
HAS inhibitors to curtail HA polymer production in certain disease states. Based on sequence
similarities, it is probable that the streptococcal and vertebrate HASs interact with the same
elements. Also, we have made tentative assignments of the roles of two conserved cysteines
found in all vertebrate HASs. Further work on the details of the catalytic mechanism should
illuminate the nature of sugar transfer specificity.
Acknowledgements – We thank Dr. Ann Achyuthan for technical assistance in creating several
of the cysteine mutants and performing some chemical modification trials. We also thank Tasha
Arnett, Wei Jing, and Carissa White for aid in performing the many synthase activity assays and
for comments on the manuscript.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
18
REFERENCES
1. Laurent, T.C., and Fraser, J. R. E. (1992) FASEB J. 6, 2397-2404
2. Spicer, A. P., and McDonald, J. A. (1998) J. Biol. Chem. 273, 1923-1932
3. Weigel, P. H., Hascall, V. C., and Tammi M. (1997) J. Biol. Chem. 272, 13997-14000
4. Pummill, P. E., Achyuthan, A. M., and DeAngelis, P. L. (1998) J. Biol. Chem. 273, 4976-
4981
5. Breton, C. and Imberty, A. (1999) Curr. Opin. Struct. Biol. 9, 563-571
6. Ünligil, U. M. and Rini, J. M. (2000) Curr. Opin. Struct. Biol. 10, 510-517
7. Tarbouriech, N., Charnock, S. J., and Davies, G. J. (2001) J. Mol. Biol. 314, 655-661
8. Charnock, S. J. and Davies, G. J. (1999) Biochemistry 38, 6380-6385
9. Vrielink, A., Rüger, W., Driessen, H. P. C. and Freemont, P. S. (1994) EMBO J. 15, 3413-
3422
10. Moréra, S., Imberty, A., Aschke-Sannenborn, U., Rüger, W., and Freemont, P. S. (1999) J.
Mol. Biol. 292, 717-730
11. Moréra, S., Larivière, L., Kurzeck, J., Aschke-Sannenborn, U., Freemont, P. S., Janin, J., and
Rüger, W. (2001) J. Mol. Biol. 311, 569-577
12. Gastinel, L. N., Cambillau, C., and Bourne, Y. (1999) EMBO J. 18, 3546-3557
13. Ramakrishnan, B. and Qasba, P. (2001) J. Mol. Biol. 310, 205-218
14. Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H.
(2001) EMBO J. 20, 638-649
15. Persson, K., Ly, H. D., Dieckelmann, M., Wakarchuk, W. W., Withers, S. G., and Strynadka,
N. C. J. (2001) Nature Struct. Biol. 8, 166-175
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
19
16. Pedersen, L. C., Tsuchida, K., Kitagawa, H., Sugahara, K., Darden, T. A., and Negishi, M.
(2000) J. Biol. Chem. 275, 34580-34585
17. Ünligil, U. M., Zhou, S., Yuwaraj, S., Sarkar, M., Schachter, H., and Rini, J. M. (2000)
EMBO J. 19, 5269-5280
18. Campbell, R. E., Mosimann, S. C., van de Rijn, I., Tanner, M. E., and Strynadke, N. C. J.
(2000) Biochmistry 39, 7012-7023
19. DeAngelis, P. L., and Achyuthan, A. M. (1996) J. Biol. Chem. 271, 23657-23660
20. Gietz R. D., Schiestl R. H., Willems A. R., Woods R. A. (1995) Yeast 11, 355-360
21. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254
22. Rosa, F., Sargent, T. D., Rebbert, M. L., Michaels, G. S., Jamrich, M., Grunz, H., Jonas, E.,
Winkles, J. A., and Dawid, I. B. (1988) Dev. Biol. 129, 114-123
23. Sugahara, K., Schwartz, N. B., and Dorfman, A. (1979) J. Biol. Chem. 254, 6252-6261
24. Kumari, K., Tlapak-Simmons, V. L., Baggenstoss, B. A., and Weigel, P. H. (2001) J. Biol.
Chem. epub ahead of print, M110638200
25. Heldermon, C. D., Tlapak-Simmons, V. L., Baggenstoss, B. A., and Weigel, P. H. (2001)
Glycobiology 11, 1017-1024
26. Marks, D. L., Dominguez, M., Wu, K., and Pagano, R. E. (2001) J. Biol. Chem. 276, 26492-
26498
27. Wiggins, C. A. R., and Munro, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7945-7950
28. Yoshida, M., Itano, N., Yamada, Y., and Kimata, K. (2000) J. Biol. Chem. 275, 497-506
29. Tlapak-Simmons, V. L., Baggenstoss, B. A., Kumari, K., Heldermon, C., and Weigel P. H.
(1999) J. Biol. Chem. 274, 4246-4253
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
20
30. Itano, N., Sawai, T., Yoshida, M., Lenas, P., Yamada, Y., Imagawa, M., Shinomura, T.,
Hamaguchi, M., Yoshida, Y., Ohnuki, Y., Miyauchi, S., Spicer, A. P., McDonald, J. A., and
Kimata, K. (1999) J. Biol. Chem. 274, 25085-25092
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
21
Table 1
Polypeptide-Substrate Contacts in Enzymes Utilizing UDP-sugars
The residues reported to make distinct interactions with the nucleotide portion of the substrate
based on X-ray crystallography and the nature of the protein/substrate interaction are listed for
eight different enzymes that utilize UDP-sugars. The enzymes are: 1, Bacillus subtilis SpsA
(7,8); 2, bacteriophage T4 β-glucosyltransferase (9-11); 3, bovine β1,4-galactosyltransferase T1
(12,13); 4, bovine α1,3-galactosyltransferase (14); 5, Neisseria meningitidis LgtC
galactosyltransferase (15); 6, human β1,3-glucuronyltransferase I (16); 7, rabbit N-
acetylglucosaminyltransferase I (17); and 8, Streptococcus pyogenes UDP-glucose
dehydrogenase (18).
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
22
Enzyme #
Interaction 1 2 3 4 5 6 7 8
uridine
stacking
Y11 F213
G214
I238
V243
R191
F226
Y139
W195
I198
Y11 Y84 I187 V215
uridine O2 V136 D8 H190 S253
uridine N3 D39 I238 R189 V136 D8 D113 D144 N251
uridine O4 R71 I238 R189 N10 N251
ribose 2'
OH
D98 E272 P187 F134 A6 P82
D195
D212 D402
ribose 3'
OH
T9 E272 D252
V253
R202
V226
I104 D195 D212 G257
M319
α-PO4 K13 S189
R191
R269
Q187
K250
R156 R117
V321
Y249
K320
β-PO4 R191
R195
Y265
R269
K279
W314
H78
G247
R310 S322 E145
K320
metal-PO4 D99 E163 D254
M344
H347
D225
D227
D103
D105
H244
D194
D196
D213
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
23
Table 2
Enzyme Expression, HAS Activity, and Substrate Affinity
Membrane preparations were tested for enzyme expression, HAS activity, and substrate affinity
(apparent Km) as described in Experimental Procedures. Protein expression and HAS activity
were categorized as: >50% of wild type (+++), 10-50% of wild type (++), 0.1-10% of wild type
(+), or <0.1% of wild type (–). Constant precursor concentrations were 1.2 mM UDP-GlcUA for
the UDP-GlcNAc Km determination and 2.4 mM UDP-GlcNAc for the UDP-GlcUA Km
determination, unless noted otherwise by a footnote. All data points were obtained in duplicate.
Standard deviations are given for experiments performed at least three times.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
24
xlHAS1
Enzyme
Protein
Expression
HAS
Activity
UDP-GlcNAc
Km (µµµµM)
UDP-GlcUA
Km (µµµµM)
Wild Type +++ +++ 400 ± 100, 260a, 700b 190 ± 40, 110c
C117F – – n.d. n.d.
C117L +++ ++ n.d. n.d.
C117S +++ +++ 340 120
C210S +++ ++ 400 120
C239S +++ +++ 320 110
C298F + + n.d. n.d.
C298L + + n.d. n.d.
C298S +++ +++ 430 160
C304S +++ +++ 470 180
C307S +++ + n.d. n.d.
C337S +++ +++ 400 ± 100, 880b 700 ± 170
C239S/C337S ++ +++ 1000 930
C304S/C337S +++ ++ 1100 890
C307S/C337S ++ – n.d. n.d.
n.d. - not determined
Alternative concentrations of substrate were used to assess these particular Km values: a, 0.6 mM
UDP-GlcUA; b, 2.4 mM UDP-GlcUA; c, 1.2 mM UDP-GlcNAc.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
25
Table 3
Nucleotide Protection of xlHAS1 from NEM Inactivation
Four independent sets of protection experiments are shown. Membranes containing xlHAS1
(C337S) were incubated with 200 µM (Runs 1-3) or 1 mM (Run 4) of the indicated substrate or
analog compound for 10 minutes on ice followed by treatment with 1 mM NEM for 10 minutes
at 15°C. The membranes were then diluted 10-fold into assay buffer (containing 10 mM DTT to
quench the remaining NEM) and assayed as described in Experimental Procedures. The values
(averaged duplicates) indicate the percentage of HAS activity remaining compared to an
identical incubation performed without NEM (i.e. higher percentage means better protection).
Without protection under these conditions, ~80-90% of the enzyme is inactivated by the NEM.
The best protectant in all cases is UDP-GlcNAc. At least slight protection is observed for all
pyrophosphate-containing nucleotides.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
26
Protection (%)
Compound Run # 1 2 3 4
none 8 19 13 16
ATP 26 29 38
CTP 20 33 17 40
GTP 29 26 29
ITP 27 31 13 35
dTMP 13 12
dTDP 73 42 38
dTTP 69 40 39
ddTTP 65 46 23 51
UMP 12 11 15
UDP 47 43 25 41
UTP 60 44 25 46
5-BromoUTP 63 42 37
5-IodoUTP 57 40 34
2-ThioUDP 63 54 40
UDP-Mannose 59 22 57
UDP-Xylose 16 35
UDP-Galactose 30 52
UDP-GalUA 49 38 25 38
UDP-GalNAc 34 34 15 27
UDP-Glucose 69 41 29 53
UDP-GlcUA 55 42 23 39
UDP-GlcNAc 77* 68* 44* 69*
GlcUA 15 13
GlcNAc 19 14
*compound providing most protection in a given run.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
27
Table 4
Inhibition Constants for Uridine Nucleotides
Apparent Ki values were determined for each compound by titrating each compound (0-4.5 mM)
into the standard synthase assay; the Ki value equals the inhibitor concentration required to give
50% inhibition. The results from two independent experiments are shown. All compounds
completely inhibited HAS activity at elevated concentrations. Values were determined at low
substrate concentration (0.3 mM UDP-GlcUA and 0.6 mM UDP-GlcNAc) or at high substrate
concentration (1.2 mM UDP-GlcUA and 2.4 mM UDP-GlcNAc). Ki values increase with higher
substrate concentrations, indicating a competitive mode of inhibition.
low substrate high substrate
Ki (µM) Ki (µM)
Compound Exp. 1 Exp. 2 Exp. 1 Exp. 2
UDP 150 130 550 600
UDP-Glucose 290 230 1100 1200
UTP 600 450 1000 ≥1500
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
28
Table 5
Effect of Various Nucleotides on Apparent Substrate Affinity
Apparent Km and Vmax values were determined for xlHAS1 in the presence of 200 µM (Exp. 1) or
300 µM (Exp. 2) of the indicated compound. The constant UDP-sugar concentrations were 1.2
mM UDP-GlcUA for the UDP-GlcNAc Km determination or 2.4 mM UDP-GlcNAc for the
UDP-GlcUA Km determination. Vmax values are in units of pmoles of sugar transferred per
minute.
UDP-GlcNAc
Km (µµµµM)
UDP-GlcUA
Km (µµµµM)
UDP-GlcNAc
Vmax
UDP-GlcUA
Vmax
Compound Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2
none 250 270 130 170 160 180 110 90
UMP – 310 – 250 – 200 – 150
UDP 720 ≥1500 200 460 150 210 100 100
UDP-Glucose 480 630 300 690 130 180 110 130
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
29
FIGURE LEGENDS
Fig. 1. NEM Effect on HAS Activity. Membranes containing wild-type xlHAS1 or assorted
mutant enzymes (60 µg total protein) were incubated with varying concentrations of NEM for 15
minutes at 15°C. The membranes were then diluted 10-fold into assay buffer and assayed as
described in Experimental Procedures. Results for representative mutants are shown. Legend:
wild type (solid circle), C239S (solid diamond), C304S (solid triangle), C337S (solid square),
C239S/C337S (open diamond), C304S/C337S (open triangle). The mutants containing the
C337S mutation are more resistant to NEM-mediated inactivation relative to the enzymes with
C337.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
30
Fig. 2. Protecting Compound Affinities. Apparent affinity values were determined for each
compound by titrating in the compound up to 3 mM prior to 1 mM NEM treatment at 15°C for
10 minutes. The membranes were then diluted 10-fold into assay buffer (containing 10 mM
DTT to quench the remaining NEM) and assayed for HAS activity with 0.6 mM UDP-GlcUA
and 1.2 mM UDP-GlcNAc. Results are given as percentage form of [(activity of NEM-treated,
compound-protected enzyme) / (activity of untreated control with same amount of compound)] –
[(activity of NEM-treated, unprotected enzyme) / (activity of untreated control with no
compound)]. Averages of duplicate results from up to four independent experiments are shown
along with rectangular hyperbola curves. Panel A, UDP-Glucose (solid triangle, solid line),
UDP-Galactose (open triangle, dashed line), UDP-GlcUA (solid square, solid line), UDP-
GalUA (open square, dashed line), UDP-GlcNAc (solid diamond, solid line), and UDP-GalNAc
(open diamond, dashed line). Panel B, UDP (solid circle), UTP (solid square), UDP-GlcNAc
(solid diamond, solid line), equimolar UDP + UDP-GlcNAc (open circle, dashed line), and
equimolar UTP + UDP-GlcNAc (open square, dashed line). Although the rectangular hyperbola
curves for UDP + UTP gave a maximum protection of ~40% and ~70% of control, respectively,
the apparent affinities were about 10-fold lower than that obtained for the other compounds.
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
31
Fig. 3. Comparison of Protection from NEM Inactivation Versus Inhibition of HAS
Activity. For protection experiments, percentages of control activity for Run 4 from Table 3 are
represented graphically with solid bars. For inhibition experiments, membranes containing
xlHAS1 were assayed with or without 1 mM of the indicated compound in addition to 0.6 mM
UDP-GlcUA and 1.2 mM UDP-GlcNAc. Open bars indicate the percent inhibition of HAS
activity compared to the parallel reaction without added compound. UDP-GlcNAc is best
protectant, but nucleotides with at least two phosphates protect a moderate amount.
(*, authentic substrate is not applicable for inhibition study)
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
32
Fig. 1
0
0.2
0.4
0.6
0.8
1
1.2
0 50 100 150 200
[NEM] µµµµM
Fra
ctio
n o
f C
ontr
ol A
ctiv
ity
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
33
Fig. 2
A
B
[Protectant] mM
0.0 0.5 1.0 1.5 3.0
% P
rote
ctio
n
0
10
20
30
40
50
60
70
[Protectant] mM
0.0 0.5 1.0 1.5 3.0
% P
rote
ctio
n
0
10
20
30
40
50
60
70
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
34
Fig. 3
0
20
40
60
80
100
none
AT
P
CT
P
GT
P
ITP
dTM
P
dTD
P
dTT
P
ddT
TP
UM
P
UD
P
UT
P
5-B
rom
oUT
P
5-Io
doU
TP
2-T
hioU
DP
UD
P-M
anno
se
UD
P-X
ylos
e
UD
P-G
alac
tose
UD
P-G
alU
A
UD
P-G
alN
Ac
UD
P-G
luco
se
UD
P-G
lcU
A
UD
P-G
lcN
Ac
% C
ontr
ol A
ctiv
ity (
Pro
tect
ion)
% I
nhib
ition
of
HA
S A
ctiv
ity
* *
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Philip E. Pummill and Paul L. DeAngelisresidues of a vertebrate hyaluronan synthase
Evaluation of critical structural elements of UDP-sugar substrates and certain cysteine
published online April 9, 2002J. Biol. Chem.
10.1074/jbc.M202456200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on April 12, 2018
http://ww
w.jbc.org/
Dow
nloaded from
top related