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J. Biol. Chem., Vol. 277, Issue 24, 21610-21616, June 14, 2002

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Evaluation of Critical Structural Elements of UDP-Sugar Substrates and Certain Cysteine Residues of a Vertebrate Hyaluronan Synthase^*

Philip E. Pummill and Paul L. DeAngelis

From the Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

Received for publication, March 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The hyaluronan (HA) synthases catalyze the addition of two different monosaccharides from UDP-sugar substrates to the linear heteropolysaccharide chain. 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. Cys³⁰⁷ of xlHAS1 may play a role in catalysis or in maintaining structure. Mutation of Cys³³⁷ raises the UDP-GlcUA Michaelis constant (K_m), suggesting that this residue participates in UDP-GlcUA substrate binding or in catalytic complex formation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HA¹ is a glycosaminoglycan composed of alternating repeats of the disaccharide (4)--D-GlcUA(3)--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 (e.g. the DXD-containing motif) are similar to other glycosyltransferases that produce various - or -linked polysaccharides from UDP-sugars (5-7). However, the exact roles of these motifs in the structure and/or the function of the polypeptide have only recently been 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 sugar residues.

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 I). 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 K_m value of xlHAS1 for UDP-sugars ranges from ~100 µM to almost 1 mM, depending on experimental conditions (Ref. 4; Table II). 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.

View this table: [in this window] [in a new window]   Table I 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 numbered as follows: 1, B. subtilis SpsA (7,8); 2, bacteriophage T4 -glucosyltransferase (9-11); 3, bovine 1,4-galactosyltransferase T1 (12,13); 4, bovine 1,3-galactosyltransferase (14); 5, Neisseria meningitidis LgtC galactosyltransferase (15); 6, human 1,3-glucuronyltransferase I (16); 7, rabbit N-acetylglucosaminyltransferase I (17); and 8, Streptococcus pyogenes UDP-glucose dehydrogenase (18).

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 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 Cys³⁰⁷ may play a direct or a structural role.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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^TM kit (Stratagene). Seven cysteine codons were altered using pairs of synthetic oligonucleotides containing either the partially degenerate codon (TYS, where Y represents C or T and S represents 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 strain; Yeast Genetic Stock Center, Berkeley, CA) by the lithium acetate/poly(ethylene glycol) 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 A₆₀₀ was 0.3. Upon induction with galactose (1% final concentration), 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; PerkinElmer Life Sciences) and/or UDP-[³H]GlcNAc (29.2 Ci/mmol; PerkinElmer Life Sciences) 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 K_m 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 apparent K_i 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 min. The enzyme was then treated with 1 mM NEM at 15 °C (5-µl reaction volume). After 15 min, the reaction mixtures were diluted to 50 µl with dithiothreitol-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 semidry transfer. The blot was blocked with bovine serum albumin 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 (22)). Protein A-alkaline phosphatase detection with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium was used to visualize the immunoreactive bands.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 II. The cysteine to serine mutation was typically found to be the least altering to protein expression and activity in comparison with 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, whereas xlHAS1(C307S/C337S) had no detectable HAS activity. The K_m 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 K_m values (Table II).

View this table: [in this window] [in a new window]   Table II Enzyme expression, HAS activity, and substrate affinity Membrane preparations were tested for enzyme expression, HAS activity, and substrate affinity (apparent Km) as described under "Experimental Procedures." Protein expression and HAS activity were categorized as follows: >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. S.D. values are given for experiments performed at least three times.

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 activity, with IC₅₀ values (the concentrations of NEM that reduced the HAS activity by 50%) of ~75 µM NEM compared with ~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).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   NEM effect on HAS activity. Membranes containing wild-type xlHAS1 or assorted mutant enzymes (60 µg of total protein) were incubated with varying concentrations of NEM for 15 min at 15 °C. The membranes were then diluted 10-fold into assay buffer and assayed as described under "Experimental Procedures." Results for representative mutants are shown. Solid circle, wild type; solid diamond, C239S; solid triangle, C304S; solid square, C337S; open diamond, C239S/C337S; open triangle, C304S/C337S. The mutants containing the C337S mutation are more resistant to NEM-mediated inactivation relative to the enzymes with Cys337.

Protection from Loss of HAS Activity Due to NEM Modification-- To determine whether 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 with 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 Cys³³⁷ 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. nonprotectable) inactivation pathway has been eliminated. The UDP-sugar protection effect also required Mg²⁺, because no protection was observed when 10 mM EDTA chelator was added instead of Mg²⁺ (data not shown).

Certain other structurally related compounds also protected xlHAS1(C337S) to various extents from NEM-mediated loss of HAS activity (Table III). The protecting compounds include several UDP-sugars, thymine-containing nucleotides, and other nucleotide triphosphates. No nucleotide monophosphate protected the enzyme from NEM-mediated loss of HAS activity (Table III and data not shown). The monosaccharides GlcUA and GlcNAc provided little protection (Table III).

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 10⁴ 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 ~10-fold lower apparent affinity (~10³ 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 (~10⁴ M) was obtained.

View larger version (20K): [in this window] [in a new window]   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 min. The membranes were then diluted 10-fold into assay buffer (containing 10 mM dithiothreitol 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 the 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. 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). 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 or UTP gave a maximum protection of ~40 or ~70% of control, respectively, the apparent affinities were about 10-fold lower than that obtained for the other compounds.

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 IV). As shown in Fig. 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 K_i values were determined at two different substrate concentrations and are listed in Table IV. The K_i values were higher when tested under the higher authentic substrate concentration conditions. K_m values of xlHAS1 were determined for both substrates in the presence of UMP, UDP, and UDP-glucose (Table V). Although UMP had little effect on the K_m values, both UDP and UDP-glucose raised the values considerably. There was little effect on V_max with any inhibitor. Overall, these alterations in kinetics are the hallmark of competitive inhibition.

View larger version (19K): [in this window] [in a new window]   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 III are represented graphically with solid bars. For inhibition experiments, membranes containing xlHAS1 were assayed with or without a 1 mM concentration of the indicated compound in addition to 0.6 mM UDP-GlcUA and 1.2 mM UDP-GlcNAc. Open bars indicate the percentage inhibition of HAS activity compared with the parallel reaction without added compound. UDP-GlcNAc is the best protectant, but nucleotides with at least two phosphates provide a moderate amount of protection. *, authentic substrate is not applicable for inhibition study.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 Cys³³⁷ to serine caused a large decrease in the enzyme's apparent affinity for UDP-GlcUA (but not UDP-GlcNAc; Table II), suggesting that Cys³³⁷ is somehow involved in the binding of UDP-GlcUA or in GlcUA transfer. This result indicates that Cys³³⁷ is probably close to the substrate-binding or catalytic site. The Cys³³⁷ 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.

In a recent study on a rat glucosylceramide synthase, it was found that Cys²⁰⁷ 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 Cys³⁰⁷ in xlHAS1, which is conserved in all known class I HASs. As shown in Table I, the C307S mutant lost almost all HAS activity. A double mutant with C307S/C337S lost all measurable HAS activity. This finding suggests that Cys³⁰⁷ 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.

Although 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 III and Fig. 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 nonsubstrate compounds was usually lower than that observed with the authentic UDP-sugar substrates (Table III). 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 (e.g. ATP, GDP) as well as many different pyrimidine-containing nucleotide-sugars (e.g. 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 (i) the charge state differences among these various phosphate ions, (ii) the smaller size of these compounds (i.e. less sterically hindered access to pocket or cleft) allowing modification of cysteines that are normally protected by other larger pyrophosphate-containing compounds, and/or (iii) a requirement for important interactions with the nucleotide base.

Mg²⁺ 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 Mg²⁺ and/or the phosphate groups.

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 cannot 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 I, 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 cannot 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 I 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 K_i values of these inhibitors are higher in the presence of increased substrate concentration (Table IV). UDP and UDP-glucose alter the K_m for the two HAS substrates but do not significantly affect the V_max (Table V); these results are indications of competitive inhibition.

The apparent affinity obtained from K_m 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 Fig. 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 Fig. 3, dTDP, UDP, and thio-UDP show higher inhibition of xlHAS1 in comparison with 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 Fig. 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). 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 K_m values observed with different concentrations of UDP-GlcUA with xlHAS1 (Table II), 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 with 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 by-product 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. 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.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM56497 (to P. L. D.).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 Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104. Tel.: 405-271-2227; Fax: 405-271-3092; E-mail: paul-deangelis@ouhsc.edu.

Published, JBC Papers in Press, April 9, 2002, DOI 10.1074/jbc.M202456200

	ABBREVIATIONS

The abbreviations used are: HA, hyaluronan, hyaluronate, or hyaluronic acid; HAS, HA synthase; NEM, N-ethylmaleimide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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