Molecular Basis for Severe Epimerase Deficiency Galactosemia

Galactosemia is an inherited disorder characterized by an inability to metabolize galactose. Although classical galactosemia results from impairment of the second enzyme of the Leloir pathway, namely galactose-1-phosphate uridylyltransferase, alternate forms of the disorder can occur due to either galactokinase or UDP-galactose 4-epimerase deficiencies. One of the more severe cases of epimerase deficiency galactosemia arises from an amino acid substitution at position 94. It has been previously demonstrated that the V94M protein is impaired relative to the wild-type enzyme predominantly at the level of Vmax rather thanKm. To address the molecular consequences the mutation imparts on the three-dimensional architecture of the enzyme, we have solved the structures of the V94M-substituted human epimerase complexed with NADH and UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-GalNAc. In the wild-type enzyme, the hydrophobic side chain of Val94 packs near the aromatic group of the catalytic Tyr157 and serves as a molecular "fence" to limit the rotation of the glycosyl portions of the UDP-sugar substrates within the active site. The net effect of the V94M substitution is an opening up of the Ala93 to Glu96 surface loop, which allows free rotation of the sugars into nonproductive binding modes.1I3M1I3N1I3K1I3L Galactosemia is an inherited disorder characterized by an inability to metabolize galactose. Although classical galactosemia results from impairment of the second enzyme of the Leloir pathway, namely galactose-1-phosphate uridylyltransferase, alternate forms of the disorder can occur due to either galactokinase or UDP-galactose 4-epimerase deficiencies. One of the more severe cases of epimerase deficiency galactosemia arises from an amino acid substitution at position 94. It has been previously demonstrated that the V94M protein is impaired relative to the wild-type enzyme predominantly at the level of Vmax rather thanKm. To address the molecular consequences the mutation imparts on the three-dimensional architecture of the enzyme, we have solved the structures of the V94M-substituted human epimerase complexed with NADH and UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-GalNAc. In the wild-type enzyme, the hydrophobic side chain of Val94 packs near the aromatic group of the catalytic Tyr157 and serves as a molecular "fence" to limit the rotation of the glycosyl portions of the UDP-sugar substrates within the active site. The net effect of the V94M substitution is an opening up of the Ala93 to Glu96 surface loop, which allows free rotation of the sugars into nonproductive binding modes.1I3M1I3N1I3K1I3L 2-(N-morpholino)ethanesulfonic acid Galactosemia is a rare, potentially lethal genetic disease that is inherited as an autosomal recessive trait and results in the inability of patients to properly metabolize galactose (1Holton J.B. Walter J.H. Tyfield L.A. Scriver C.R. Beaudet A.L. Sly S.W. Valle D. Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2000: 1553-1587Google Scholar). Clinical manifestations include intellectual retardation, liver dysfunction, and cataract formation, among others. Although deficiencies of any of the three enzymes participating in the Leloir pathway for galactose metabolism (Scheme FS1) can result in symptoms of galactosemia, the classical form of the disease arises from impairment of galactose-1-phosphate uridylyltransferase, the second enzyme in the pathway (1Holton J.B. Walter J.H. Tyfield L.A. Scriver C.R. Beaudet A.L. Sly S.W. Valle D. Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2000: 1553-1587Google Scholar). Of particular interest is the third enzyme in the pathway, namely UDP-galactose 4-epimerase, hereafter referred to as epimerase. This NAD+-dependent enzyme plays a key role in normal galactose metabolism by catalyzing the interconversion of UDP-galactose and UDP-glucose as indicated in Scheme FS1. Interestingly, the human form of epimerase has also been shown to interconvert UDP-GlcNAc and UDP-GalNAc (2Maley F. Maley G.F. Biochim. Biophys. Acta. 1959; 31: 577-578Crossref PubMed Scopus (49) Google Scholar, 3Piller F. Hanlon M.H. Hill R.L. J. Biol. Chem. 1983; 258: 10774-10778Abstract Full Text PDF PubMed Google Scholar, 4Kingsley D.M. Kozarsky K.F. Hobbie L. Krieger M. Cell. 1986; 44: 749-759Abstract Full Text PDF PubMed Scopus (242) Google Scholar). This type of activity has not been observed in the epimerase from Escherichia coli. Two types of human epimerase-based galactosemia have been identified thus far: peripheral and generalized. While the peripheral form can be quite common among some ethnic groups and is usually considered benign, the generalized form of the disease is clinically severe and extremely rare (5Gitzelmann R. Helv. Paediatr. Acta. 1972; 27: 125-130PubMed Google Scholar, 6Gitzelmann R. Steinmann B. Helv. Paediatr. Acta. 1973; 28: 497-510PubMed Google Scholar, 7Gitzelmann R. Steinmann B. Mitchell B. Haigis E. Helv. Paediatr. Acta. 1977; 31: 441-452PubMed Google Scholar, 8Alano A. Almashanu S. Maceratesi P. Reichardt J. Panny S. Cowan T.M. J. Invest. Med. 1997; 45 (abstr.): 191PubMed Google Scholar). The most severe form of epimerase deficiency galactosemia characterized to date arises from a homozygous mutation encoding the substitution of a methionine residue for a valine at position 94 (9). This substitution impairs enzyme activity to ∼5% of wild-type levels with respect to UDP-galactose and to ∼25% of wild-type levels with respect to UDP-GalNAc (10Wohlers T.M. Fridovich-Keil J.L. J. Inherit. Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar). The mutant protein is impaired relative to the wild-type enzyme predominantly at the level ofVmax rather than Km (10Wohlers T.M. Fridovich-Keil J.L. J. Inherit. Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar). Previous biochemical analyses on the epimerase from E. colihave suggested that its reaction mechanism proceeds through abstraction of the hydrogen from the 4′-hydroxyl group of the sugar by a catalytic base and transfer of a hydride from C-4 of the sugar to C-4 of the NAD+, leading to a 4′-ketopyranose intermediate and NADH (11Frey P.A. Dolphin D. Poulson R. Avramovic O. Pyridine Nucleotide Coenzymes: Chemical, Biochemical, and Medical Aspects. John Wiley & Sons, Inc., New York1987: 461Google Scholar). A limited but well-defined rotation of this intermediate is thought to occur in the active site, thereby allowing return of the hydride from NADH to the opposite side of the sugar. Recently, the three-dimensional structure of human epimerase complexed with NADH and UDP-glucose was solved by x-ray crystallographic analyses to 1.5-Å resolution (12Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. Biochemistry. 2000; 39: 5691-5701Crossref PubMed Scopus (142) Google Scholar). A ribbon representation of one subunit of the homodimeric protein is displayed in Fig. 1. As can be seen, the overall fold of the enzyme can be described in terms of two structural motifs: the N-terminal domain defined by Met1–Thr189and the C-terminal region formed by Gly190–Ala348. The N-terminal domain adopts the three-dimensional architecture referred to as a Rossmann fold. Strikingly, the NADH and UDP-glucose ligands are positioned within the active site such that C-4 of the sugar lies at ∼3.5 Å from C-4 of the dinucleotide (12Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. Biochemistry. 2000; 39: 5691-5701Crossref PubMed Scopus (142) Google Scholar). Additionally, Oη of Tyr157 and Oγ of Ser132 are located at 3.1 Å and 2.4 Å, respectively, from the 4′-hydroxyl group of the sugar moiety. It is believed that the low barrier hydrogen bond formed between the sugar and the side chain of Ser132facilitates the removal of the 4′-hydroxyl hydrogen by the phenolic acid chain of Tyr157 and the transfer of the hydride from C-4 of the sugar to C-4 of the nicotinamide ring (12Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. Biochemistry. 2000; 39: 5691-5701Crossref PubMed Scopus (142) Google Scholar). To address the molecular consequences of the V94M substitution in human epimerase, we have crystallized and solved the x-ray structures of the mutant protein complexed with UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-GalNAc, all to 1.5-Å resolution. These investigations have allowed for a more complete understanding of the three-dimensional consequences this mutation imparts on the active site geometry of the enzyme and provide a molecular explanation for the observed enzymatic impairment. The V94M form of human UDP-galactose 4-epimerase was constructed and overexpressed in the yeast Pichia pastoris, as described (9Wohlers T.M. Christacos N.C. Harreman M.T. Fridovich-Keil J.L. Am. J. Hum. Genet. 1999; 64: 462-470Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 12Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. Biochemistry. 2000; 39: 5691-5701Crossref PubMed Scopus (142) Google Scholar). Protein samples employed for crystallization trials were purified according to the protocol of Ref. 12Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. Biochemistry. 2000; 39: 5691-5701Crossref PubMed Scopus (142) Google Scholar. Ternary complexes of the protein were prepared by treating the epimerase samples (15 mg/ml in the final dialysis buffer) with 5 mm NADH and 20 mm UDP-sugars and allowing the solutions to equilibrate for 24 h at 4 °C. Large crystals of each of the complexes were grown from 100 mmMES1 (pH 6.0), 8–9% (w/v) poly(ethylene glycol) 3400, and 75 mm MgCl2 by macroseeding into batch experiments at 4 °C. Typically, the crystals grew to maximum dimensions of 0.3 × 0.2 × 0.7 mm in ∼1–2 weeks. For x-ray data collection, the crystals were transferred to cryoprotectant solutions in two steps. First they were slowly transferred to intermediate solutions containing 20% poly(ethylene glycol) 3400, 500 mm NaCl, and 20% (v/v) methanol. After equilibration in the methanol-containing solutions, the crystals were subsequently transferred into similar solutions that had been augmented with 4% (v/v) ethylene glycol. All of the crystals were suspended in loops of 20-μm surgical thread and immediately flash-frozen in a stream of nitrogen gas. The four V94M protein·NADH·UDP-sugar complexes crystallized in the space group P212121 with typical unit cell dimensions of a = 78.1 Å, b= 89.9 Å, and c = 96.9 Å. Each asymmetric unit contained one dimer, and only subunit I in the coordinate file will be discussed under "Results and Discussion." Native x-ray data sets to 1.5-Å resolution were collected at the Advanced Photon Source, Structural Biology Center beamline 19-BM. These data were processed with HKL2000 and scaled with SCALEPACK (13Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar). Relevant x-ray data collection statistics are presented in TableI. The V94M protein·NADH·UDP-glucose structure was solved via AMORE (14Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar), employing the previously determined wild-type protein·NADH·UDP-glucose structure as the search model (12Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. Biochemistry. 2000; 39: 5691-5701Crossref PubMed Scopus (142) Google Scholar). The other three structures were solved via difference Fourier techniques. Manual adjustments of the models using the program Turbo (15Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory. Silicon Graphics, Mountain View, CA1991Google Scholar) and subsequent least squares refinements with the package TNT (16Tronrud D.E. Ten Eyck L.F. Matthews B.W. Acta Crystallogr. Sect. A. 1987; 43: 489-501Crossref Scopus (874) Google Scholar) reduced the R-factors to 17.5, 18.1, 18.5, and 17.8%, respectively, for the ternary complexes with UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-GalNAc. Relevant refinement statistics can be found in Table II. Figs.1, 2, 3, and 5 were prepared with the software package, MOLSCRIPT while Fig. 4 was generated with the program BobScript (17Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar, 18Esnouf, R. (1996) BobScript, version 2.01, J. Appl. Cryst. (1991) 24, 946–950.Google Scholar).Table IX-ray data collection statisticsResolutionIndependent reflectionsCompletenessRedundancyAvg I/Avg ς(I)Rsym 1-aRsym = (Σ‖I − Ī‖/ΣI) × 100.Å%%Protein · NADH · UDP-glucose50.0–1.50108,99499.16.741.04.71.55–1.50 1-bStatistics for the highest resolution bin.10,33395.33.83.523.9Protein · NADH · UDP-galactose50.0–1.50107,56697.46.640.05.51.55–1.50940686.03.73.324.0Protein · NADH · UDP-GlcNAc50.0–1.50107,98598.46.434.45.81.55–1.50953588.03.62.527.6Protein · NADH · UDP-GalNAc50.0–1.50107,21497.37.037.95.31.55–1.50940086.24.23.027.91-a Rsym = (Σ‖I − Ī‖/ΣI) × 100.1-b Statistics for the highest resolution bin. Open table in a new tab Table IIRelevant least-squares refinement statisticsBound ligandUDP-glucoseUDP-galactoseUDP-GlcNAcUDP-GalNAcResolution limits (Å)30.0–1.5030.0–1.5030.0–1.5030.0–1.50R-factor 2-aR-factor = (Σ‖Fo −Fc‖/Σ‖Fo‖) × 100 where Fo is the observed structure-factor amplitude and Fc is the calculated structure-factor amplitude. (overall) %/reflections17.5 /108,66818.1 /107,19118.5 /107,69217.8 /107,109R-factor (working) %/reflections17.4 /97,80118.1 /96,47218.4 /96,92218.0 /96,398R-factor (free) %/reflections19.8 /10,68720.3 /10,71921.4 /10,77021.5 /10,711No. of Protein Atoms 2-bThese include multiple conformations for 1) UDP-Glu: Glu24, Glu63, and Ser81 in subunit I and Ser59, Glu61, Ser81, Met83, Lys120, Thr134, Thr177, Ile217, Asp231, Glu233, Ile252, and Ser342 in subunit II; 2) UDP-galactose: Glu24, Glu63, Ser81, Thr189, and Asn207 in subunit I and Glu24, Glu63, Gln114, Lys120, Thr134, Thr177, Thr189, Ile217, and Gln261 in subunit II; 3) UDP-GlcNAc: Glu24, Glu63, and Asn138 in subunit I and Glu61, Ile118, Thr134, Thr177, and Asn207 in subunit II: 4) UDP-GalNAc: Lys120 and Arg256 in subunit I and Glu3, Glu24, Gln114, Ile118, Thr134, Asn207, Asp231, and Gln282 in subunit II.5413541453885403No. of Hetero-atoms 2-cThese include for 1) UDP-glucose: 2 NADH, 2 UDP-glucose, 4 Cl−, 1 Mg2+, 3 ethylene glycols, and 977 waters; 2) UDP-galactose: 2 NADH, 2 UDP-galactose, 3 Cl−, 1 Mg2+, 2 ethylene glycols, and 935 waters; 3) UDP-GlcNAc: 2 NADH, 2 UDP-GlcNAc, 4 Cl−, 1 Mg2+, and 842 waters; 4) UDP-GalNAc: 2 NADH, 1 UDP-GlcNAc, 1 UDP-GlcNAc, 4 Cl−, 1 Mg2+, and 925 waters.1155110710141097Weighted root mean square deviations from idealityBond lengths (Å)0.0100.0100.0120.010Bond angles (degrees)2.102.212.142.17Trigonal planes (Å)0.0050.0050.0050.005General planes (Å)0.0090.0110.0110.012Torsional angles (degrees)15.615.815.615.72-a R-factor = (Σ‖Fo −Fc‖/Σ‖Fo‖) × 100 where Fo is the observed structure-factor amplitude and Fc is the calculated structure-factor amplitude.2-b These include multiple conformations for 1) UDP-Glu: Glu24, Glu63, and Ser81 in subunit I and Ser59, Glu61, Ser81, Met83, Lys120, Thr134, Thr177, Ile217, Asp231, Glu233, Ile252, and Ser342 in subunit II; 2) UDP-galactose: Glu24, Glu63, Ser81, Thr189, and Asn207 in subunit I and Glu24, Glu63, Gln114, Lys120, Thr134, Thr177, Thr189, Ile217, and Gln261 in subunit II; 3) UDP-GlcNAc: Glu24, Glu63, and Asn138 in subunit I and Glu61, Ile118, Thr134, Thr177, and Asn207 in subunit II: 4) UDP-GalNAc: Lys120 and Arg256 in subunit I and Glu3, Glu24, Gln114, Ile118, Thr134, Asn207, Asp231, and Gln282 in subunit II.2-c These include for 1) UDP-glucose: 2 NADH, 2 UDP-glucose, 4 Cl−, 1 Mg2+, 3 ethylene glycols, and 977 waters; 2) UDP-galactose: 2 NADH, 2 UDP-galactose, 3 Cl−, 1 Mg2+, 2 ethylene glycols, and 935 waters; 3) UDP-GlcNAc: 2 NADH, 2 UDP-GlcNAc, 4 Cl−, 1 Mg2+, and 842 waters; 4) UDP-GalNAc: 2 NADH, 1 UDP-GlcNAc, 1 UDP-GlcNAc, 4 Cl−, 1 Mg2+, and 925 waters. Open table in a new tab Figure 3Comparison of the hydrogen bonding patterns around the UDP when bound to either the wild-type enzyme or the V94M protein. Possible electrostatic interactions between the native enzyme and the UDP-glucose ligand are indicated by thedashed lines in a. The UDP-glucose is highlighted in yellow bonds for clarity. Thedashed lines indicate distances equal to or less than 3.2 Å. Interactions between the V94M protein and the UDP (in the V94M·NADH·UDP-glucose model) are indicated (b). The sugar moiety is not shown due to its free rotation in the active site of the V94M-substituted epimerase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Superposition of the regions near the active sites for the abortive complexes of wild-type enzyme and the V94M protein with bound UDP-GlcNAc. The wild-type and V94M proteins are depicted in red and black, respectively. Note the significant rotation of the N-acetylglucosamine moiety out of the active site pocket in the V94M protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Electron density in the vicinity of the UDP-GlcNAc binding pocket. The map was contoured at 2ς and calculated with coefficients of the form (Fo −Fc), where Fo was the native structure factor amplitude and Fc was the calculated structure factor amplitude. The UDP-GlcNAc ligand, the NADH, and Tyr157 were omitted from the x-ray coordinate file for the electron density map calculation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Recent biochemical studies on the human V94M-substituted UDP-galactose 4-epimerase revealed that the enzyme was kinetically impaired relative to the wild-type protein predominantly at the level of Vmax rather than Km (10Wohlers T.M. Fridovich-Keil J.L. J. Inherit. Metab. Dis. 2000; 23: 713-729Crossref PubMed Scopus (47) Google Scholar). Specifically, the Km values for the wild-type and mutant forms of the protein, when assayed with UDP-galactose, were 0.27 ± 0.01 and 0.15 ± 0.02 mm, respectively. The Vmax values, however, were significantly different for the wild-type and V94M proteins at 1.22 versus0.036 mmol of UDP-galactose/mg/min, respectively. Additionally, the apparent Km values for the wild-type and V94M enzymes, with UDP-GalNAc as the substrate, corresponded to 0.287 ± 0.05 mm and 0.445 ± 0.01 mm, respectively. For the x-ray investigation presented here, four different crystal forms of the V94M enzyme were prepared, namely the complexes of protein with NADH and the following ligands: UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-galNAc. All of the structures were solved to 1.5-Å resolution and refined to R-factors equal to or less than 18.5%. The relative location of the galactosemic mutation with respect to the active site of the native enzyme can be seen in Fig.2 a. As expected, the main structural perturbation imposed by the V94M substitution occurs in the helical loop defined by Ala93 to Glu96, which connects the fourth β-strand to the fourth major α-helix of the Rossmann fold. This change in loop structure is similar in all of the V94M protein models described here and is independent of the identity of the sugar ligand occupying the active site. In the wild-type enzyme, the side chain of Val94 points toward the active site and is located at ∼3.5 Å from the catalytic Tyr157. Additionally, the carbonyl oxygen of Val94 forms a hydrogen bond with the side chain hydroxyl group of Ser97, which further serves to tighten down that portion of the polypeptide chain backbone abutting the UDP-sugar binding pocket. This loop in the wild-type enzyme is well ordered with an average temperature factor of 22.0 Å2 for all of the atoms lying between Ala93 and Glu96. The corresponding temperature factors for the V94M protein·NADH·UDP-sugar complexes are significantly higher, however, at ∼77 Å2. Indeed, residual electron density in maps calculated with (Fo − Fc) coefficients suggests that alternate conformations of this loop are present in the crystalline lattice but at lower occupancies. It was not possible to build these alternate conformations into the electron density with any certainty, however, and hence they were not included in the protein models. A superposition of the polypeptide chains near residue 94 for the wild-type enzyme and the V94M protein complexed with UDP-galactose is displayed in Fig. 2 b. The mutation at position 94 results in a significant change in the backbone dihedral angles of the preceding alanine residue. Specifically, in the wild-type enzyme, Ala93 adopts φ and ψ angles of approximately −82 and 106°, respectively, while in the V94M protein, the corresponding angles are −124° and 148°. As a result of these changes in torsional angles, the side chain of Met94 in the mutant protein extends out toward the solvent, and the hydrogen bond between the carbonyl oxygen of residue 94 and Oγ of Ser97 is no longer present. It should be noted that if the loop between Ala93 and Glu96 were to adopt the wild-type conformation in the V94M protein, the larger side chain of Met94 could not be accommodated in the active site without significant steric clashes, and this, presumably, explains in part the dramatic changes in backbone conformation starting at position 93. The net effect of this three-dimensional perturbation is an opening of the active site, thereby allowing free rotation of the sugar moiety. Indeed, from electron density maps calculated with (Fo − Fc) coefficients, it is clear that the sugars in the V94M structures complexed with either UDP-glucose or UDP-galactose adopt multiple conformations that are not well defined. Because of this, it is not possible to describe in detail the carbohydrate/protein interactions in these two particular structures. It is possible, however, to compare the interactions between the protein and the UDP moieties in the wild-type and the V94M proteins with either bound UDP-glucose or UDP-galactose. Potential hydrogen-bonding interactions observed between the wild-type enzyme and the substrate are depicted in a schematic representation in Fig.3 a, while those for the V94M·NADH·UDP-glucose complex are shown in Fig. 3 b. As indicated, the side chains forming hydrogen bonds to the UDP-glucose in the wild-type protein include Ser132, Tyr157, Asn187, Asn207, Arg239, Asp303, and Arg300. Only Ser132 and Tyr157 interact solely with the carbohydrate portion. All of the other side chains are primarily involved in UDP binding. As can be seen in Fig. 3 a, the uracil ring of the UDP-glucose is anchored to the native enzyme via the backbone carbonyl group of Asn224 and the peptidic NH group of Phe226. Two additional water molecules serve to bridge the C-4 carbonyl group of the base to the protein. These interactions are also observed in the various V94M mutant protein models (Fig. 3 b). In the wild-type protein, the 2′- and 3′-hydroxyl groups of the uridine ribose are hydrogen-bonded to the carboxylate group of Asp303 and a water molecule, respectively. The guanidinium group of Arg300 interacts with both α- and β-phosphoryl oxygens of UDP when bound to wild-type enzyme, while the side chain of Arg239 forms an electrostatic interaction with a β-phosphoryl oxygen. Similar interactions are, indeed, observed in the V94M enzymes as indicated in Fig. 3 b. The only significant differences between the wild-type enzyme and the V94M protein occur at the glucose moiety. In the wild-type protein, the glucose moiety is firmly anchored in place by interactions with the side chains of Ser132 and Tyr157 and the carbonyl oxygen of Lys92, which is located in the loop containing the V94M mutation. These interactions are missing in the mutant proteins with bound UDP-glucose or UDP-galactose due to the free rotation of the glycosyl groups in the active site. The limited change in Km observed between the wild-type enzyme and the V94M protein is a function of the fact that the nucleotide portion of the UDP-sugar substrate provides most of the binding interactions. Unlike that observed for the V94M protein·NADH·UDP-glucose or the V94M protein·NADH·UDP-galactose complexes, the sugar moieties in the V94M proteins with either bound UDP-GlcNAc or UDP-GalNAc are visible in the electron density maps as shown in Fig.4 for the UDP-GlcNAc species. Note that the 6′-hydroxyl group of the N-acetylglucosamine adopts two conformations and that the sugar is rotated away from the nicotinamide ring of the NADH. Residual electron density in maps calculated with (Fo − Fc) coefficients suggests that each of these sugars adopt alternate conformations at lower occupancies. Because of the quality of the residual electron density, however, it was not possible to unambiguously model these alternate conformations into the electron density; hence, they were not included in the coordinate files. Interestingly, the electron density map calculated for the V94M enzyme crystallized in the presence of UDP-GalNAc clearly demonstrated that the ligand had been converted to UDP-GlcNAc. This result is reminiscent of that observed with the epimerase from E. coli. All attempts to prepare an abortive complex of the bacterial enzyme with UDP-galactose failed (19Thoden J.B. Frey P.A. Holden H.M. Biochemistry. 1996; 35: 5137-5144Crossref PubMed Scopus (153) Google Scholar). These experiments included reduction of the enzyme with dimethylamine/borane in the presence of UDP-galactose, UDP, UMP, or TMP and subsequent exchange of these nucleotides with UDP-galactose. In every case, the electron density maps always indicated the presence of UDP-glucose in the active site. Obviously, UDP-glucose binds more tightly to epimerase in the abortive complex, and although the enzyme had been reduced with dimethylamine/borane, enough residual activity remained to convert UDP-galactose to UDP-glucose. Most likely, the same phenomenon is occurring in the case of the human V94M-substituted epimerase with bound UDP-GalNAc. Within the last year, the three-dimensional structure of human wild-type epimerase with bound NADH and UDP-GlcNAc was solved to 1.5-Å resolution, and it was demonstrated that to accommodate the additionalN-acetyl group at the C-2 position of the sugar, the side chain of Asn207 rotates toward the interior of the protein and interacts with Glu199 (20Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. J. Biol. Chem. 2001; 276: 15131-15136Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Shown in Fig.5 is a superposition of the active site regions for the wild-type and V94M proteins with bound UDP-GlcNAc. As can be seen, in the V94M-substituted form, the sugar group of the ligand rotates out of the pocket and toward position 94. This type of rotation is blocked in the native enzyme due to the side chain of Val94. In the wild-type enzyme the distance between C-4 of the UDP-GlcNAc ligand and C-4 of the nicotinamide ring of the NADH is 3.0 Å. This distance in the V94M protein model is 9.4 Å. The 4′-hydroxyl group of the sugar in the wild-type enzyme is located at 2.8 Å from Oγ of Ser132 and 3.0 Å from Oη of Tyr157. Due to the drastic rotation of the sugar moiety in the active site pocket, these distances in the V94M protein complex are 9.5 and 10.2 Å, respectively. Key hydrogen bonds between the N-acetylglucosamine group of the ligand and the wild-type enzyme occur between the side chains of Asn187and the 6′-OH of the sugar, between both Ser132 and Tyr157 and the 4′-OH of the sugar, and finally, between the carbonyl group of Lys92 and the 3′-OH of the carbohydrate. These interactions are completely missing in the V94M enzyme with bound UDP-GlcNAc, where the hydroxyl groups simply form hydrogen bonds with solvent molecules. In summary, the x-ray studies described here provide a three-dimensional understanding of one example of severe epimerase deficiency galactosemia. In the normal enzyme, the hydrophobic side chain of Val94 provides a "molecular fence" to prevent sugar rotation out of the active site pocket, thereby preventing nonproductive binding. Upon substitution of Val94 by a methionine, the loop region connecting the fourth β-strand to the fourth α-helix of the Rossmann fold becomes disordered, adopts multiple conformations, and effectively opens up the sugar binding pocket to allow for free rotation of the sugar moiety in the active site and/or nonproductive substrate binding. In light of the structural results presented here, it is not surprising that the V94M mutation effects Vmax significantly more thanKm. Most of the binding interactions for the UDP-sugar substrates occur between the protein and the nucleotide, and these are not disrupted by the mutation. What the V94M substitution does, however, is allow the carbohydrate portions of the UDP-sugars to rotate freely, thereby limiting the time the ligand is bound in a productive mode near Oγ of Ser132 and Oη of Tyr157. As such,Vmax is severely affected. Interestingly, in previous work, it has been shown that the V94M epimerase is impaired to a 5-fold lesser extent with regard to UDP-GalNAc than to UDP-galactose (9Wohlers T.M. Christacos N.C. Harreman M.T. Fridovich-Keil J.L. Am. J. Hum. Genet. 1999; 64: 462-470Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The reason is, presumably, that the bulkier sugar moieties of UDP-GlcNAc and UDP-GalNAc can adopt fewer nonproductive binding modes. We are grateful to Drs. Dale Edmondson and Paige Newton-Vinson for generously allowing and helping us to use the fermenter and to Dr. W. W. Cleland for helpful discussions.