Catalytic properties and crystal structure of UDP-galactose 4-epimerase-like l-threonine 3-dehydrogenase from Phytophthora infestans.

We report, for the first time, the three-dimensional structure and biochemical properties of a UDP-galactose 4-epimerase-like l-threonine 3-dehydrogenase (GalE-like L-ThrDH) from Phytophthora infestans, a plant disease-causing fungus. We identified GalE-like L-ThrDH using Kyoto Encyclopedia of Genes and Genomes (KEGG) database as a candidate target for the development of a new fungicide. The GalE-like L-ThrDH gene was expressed in Escherichia coli, and its product was purified and characterized. N-Acetylglycine was found to act as a competitive inhibitor of the enzyme (Ki =0.18 mM). The crystal structure of the unique hexameric GalE-like L-ThrDH was determined using the molecular replacement method at a resolution of 2.3 Å, in the presence of NAD+ and citrate, an analogue of the substrate. Based on the molecular docking simulation, N-acetylglycine molecule was modeled into the active site and the binding mode and inhibition mechanism of N-acetylglycine were elucidated.

The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima.

UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80 °C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism.

UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors.

Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 µM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

Piperine inhibits IL-1ß-induced IL-6 expression by suppressing p38 MAPK and STAT3 activation in gastric cancer cells.

Piperine, a kind of natural alkaloid found in peppers, has been reported to exhibit anti-oxidative and anti-tumor activities, both in vitro and in vivo. Interleukin-6 (IL-6) is an important cytokine that activates the signal transduction, promotes tumor cell metastasis, and induces malignancy, including in gastric cancer. However, the effects of piperine on IL-6 expression in gastric cancer cells have not yet been well defined. In this study, we investigated the effects of piperine on the IL-6 expression, and examined the underlying signaling pathways via RT-PCR, promoter studies and Western blotting in human gastric cancer TMK-1 cells. Our results showed that piperine inhibited interleukin-1ß (IL-1ß)-induced IL-6 expression in a dose-dependent manner. In addition, piperine also inhibited IL-6 promoter activity. Experiments with mitogen-activated protein kinase (MAPK) inhibitors and dominant negative mutant p38 MAPK indicated that p38 MAPK was essential for IL-6 expression in the TMK-1 cells. Additionally, signal transducer and activator of transcription 3 (STAT3) was also involved in the IL-1ß-induced IL-6 expression in gastric cancer cells. Piperine inhibited IL-1ß-induced p38 MAPK and STAT3 activation and, in turn, blocked the IL-1ß-induced IL-6 expression. Furthermore, gastric cancer cells pretreated with IL-1ß showed markedly enhanced invasiveness, which was partially abrogated by treatment with IL-6 siRNA, piperine, and inhibitors of p38 MAPK and STAT3. These results suggest that piperine may exert at least part of its anti-cancer effect by controlling IL-6 expression through the suppression of p38 MAPK and STAT3.

Computer-aided identification of Trypanosoma brucei uridine diphosphate galactose 4'-epimerase inhibitors: toward the development of novel therapies for African sleeping sickness.

Trypanosoma brucei, the causative agent of human African trypanosomiasis, affects tens of thousands of sub-Saharan Africans. As current therapeutics are inadequate due to toxic side effects, drug resistance, and limited effectiveness, novel therapies are urgently needed. UDP-galactose 4'-epimerase (TbGalE), an enzyme of the Leloir pathway of galactose metabolism, is one promising T. brucei drug target. We here use the relaxed complex scheme, an advanced computer-docking methodology that accounts for full protein flexibility, to identify inhibitors of TbGalE. An initial hit rate of 62% was obtained at 100 microM, ultimately leading to the identification of 14 low-micromolar inhibitors. Thirteen of these inhibitors belong to a distinct series with a conserved binding motif that may prove useful in future drug design and optimization.

Identification of novel inhibitors of UDP-Glc 4'-epimerase, a validated drug target for african sleeping sickness.

Novel inhibitors of Trypanosoma brucei and mammalian UDP-Glc 4'-epimerase were identified by screening a small library of natural products and commercially available drug-like molecules. The inhibitors possess low micromolar potency against the T. brucei and human enzymes in vitro, display a degree of selectivity between the two enzymes, and are cytotoxic to cultured T. brucei and mammalian cells.

UDP-galactose 4-epimerase from Kluyveromyces fragilis: analysis of its hysteretic behavior during catalysis.

UDP-galactose 4-epimerase serves as a prototype model of class II oxidoreductases that use bound NAD as a cofactor. This enzyme from Kluyveromyces fragilis is a homodimer with a molecular mass of 75 kDa/subunit. Continuous monitoring of the conversion of UDP-galactose (UDP-gal) to UDP-glucose (UDP-glu) by the epimerase in the presence of the coupling enzyme UDP-glucose dehydrogenase and NAD shows a kinetic lag of up to 80 s before a steady state is reached. The disappearance of the lag follows first-order kinetics (k = 3.22 x 10(-2) s(-1)) at 25 degrees C at enzyme and substrate concentrations of 1.0 nM and 1 mM, respectively. The observed lag is not due to factors such as insufficient activity of the coupling enzyme, association or dissociation or incomplete recruitment of NAD by epimerase, product activation, etc., but was a true expression of the activity of the prepared enzyme. Dissociation of the bound ligand(s) by heat followed by analysis with reverse-phase HPLC, TLC, UV-absorption spectrometry, mass spectrometry, and NMR showed that in addition to 1.78 mol of NAD/dimer, the epimerase also contains 0.77 mol of 5'-UMP/dimer. The latter is a strong competitive inhibitor. Preincubation of the epimerase with the substrate UDP-gal or UDP-glu replaces the inhibitor and also abolishes the lag, which reappeared after the enzyme was treated with 5'-UMP. The lag was not observed as long as the cells were in the growing phase and galactose in the growth medium was limiting, suggesting that association with 5'-UMP is a late log-phase phenomenon. The stoichiometry and conserved amino acid sequence around the NAD binding site of multimeric class I (classical dehydrogenases) and class II oxidoreductases, as reported in the literature, have been compared. It shows that each subunit is independently capable of being associated with one molecule of NAD, suggestive of two NAD binding sites of epimerase per dimer.

UDPgalactose 4-epimerase from Saccharomyces cerevisiae. A bifunctional enzyme with aldose 1-epimerase activity.

UDPgalactose 4-epimerase (epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme of the galactose metabolic pathway. The Saccharomyces cerevisiae epimerase encoded by the GAL10 gene is about twice the size of either the bacterial or human protein. Sequence analysis indicates that the yeast epimerase has an N-terminal domain (residues 1-377) that shows significant similarity with Escherichia coli and human UDPgalactose 4-epimerase, and a C-terminal domain (residues 378-699), which shows extensive identity to either the bacterial or human aldose 1-epimerase (mutarotase). The S. cerevisiae epimerase was purified to > 95% homogeneity by sequential chromatography on DEAE-Sephacel and Resource-Q columns. Purified epimerase preparations showed mutarotase activity and could convert either alpha-d-glucose or alpha-d-galactose to their beta-anomers. Induction of cells with galactose led to simultaneous enhancement of both epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property of the yeast epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5'-UMP and l-arabinose, epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4-epimerase is a bifunctional enzyme with aldose 1-epimerase activity. The active sites for these two enzymatic activities are located in different regions of the epimerase holoenzyme.

Prenatal exposure to high galactose adversely affects initial gonadal pool of germ cells in rats.

BACKGROUND: In rats, prenatal exposure to high concentrations of galactose may contribute to a condition that is equivalent to the premature ovarian failure (POF) component of human galactosaemia. We investigated if development of POF under experimental galactosaemia-like conditions was attributed to impaired germ cell migration. METHODS: Pregnant rats were fed pellets supplemented with, or without, 35% galactose from day 3 of conception continuing through parturition. Between days 12-15, embryos from one uterine horn were dissected out. Primordial germ cells (PGC) were histochemically localized and counted on the basis of binding of Dolichos biflorus agglutinin, a lectin specific for terminal N-acetylgalactosamine (GalNAc), to the surface glycoconjugate of the germ cells. The embryos from the other uterine horn were maintained until parturition. Liver activity of uridine diphosphate galactose 4-epimerase, the enzyme involved at multiple steps in the process of synthesis of GalNAc, was assayed in 1-2 day old female pups. RESULTS: The numbers of PGC at the day-specific sites on all days of examination were significantly lower (P

UDP-galactose 4-epimerase from Escherichia coli: equilibrium unfolding studies.

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with non-covalently bound NAD acting as cofactor. The enzyme can be reversibly reactivated after denaturation and dissociation using 8 M urea at pH 7.0. There is a strong affinity between the cofactor and the refolded molecule as no extraneous NAD is required for its reactivation. Results from equilibrium denaturation using parameters like catalytic activity, circular-dichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8-naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the native enzyme surface), elution profile from size-exclusion HPLC and light scattering have been compiled here. These show that inactivation, integrity of secondary, tertiary and quaternary structures have different transition mid-points suggestive of non-cooperative transition. The unfolding process may be broadly resolved into three parts: an active dimeric holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual secondary structure. Thermodynamical parameters associated with some transitions have been quantitated. The results have been discussed with the X-ray crystallographic structure of the enzyme.

Reversible folding of UDP-galactose 4-epimerase from Escherichia coli.

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39-kDa subunits having 1 or 2 molecules of NAD bound non-covalently/dimer. The enzyme can be dissociated and denatured by 8 M urea at pH 7.0 to a state having only 15% of residual secondary structure. Dilution of the denaturant by 20 mM potassium phosphate, pH 8.5, leads to functional reconstitution of the enzyme. No addition of extraneous NAD is required for reactivation, indicating a strong affinity of the cofactor for refolded molecule. The reactivation follows a second-order kinetics (k = 1.2 +/- 0.07 X 10(3) M(-1) s(-1) at 25 degrees C) with an energy of activation of 23.79 +/- 0.33 kJ/mol. The native, denatured and renatured states of the enzyme were characterized by far-ultraviolet CD spectra for secondary structure: protein fluorescence, interaction with extrinsic fluorescence probe ANS (1-anilino 8-naphthalene sulfonic acid) and ultraviolet absorption spectra for tertiary structure and size-exclusion HPLC, gel-filtration chromatography and light-scattering for quaternary structure. The folding process could be broadly divided into two distinct steps: (a) regain of secondary structure and dimerization were fast and were complete within 2 min and 9 min, respectively, and (b) regain of catalytic activity was slow and was complete by 45 min. No active holoenzyme could be identified. It appears that generation of the NAD-binding site and subsequent assembly of NAD is the rate-limiting step expressing catalytic activity.

Properties and kinetic analysis of UDP-glucose dehydrogenase from group A streptococci. Irreversible inhibition by UDP-chloroacetol.

UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucuronate is released last. UDP-xylose was found to be a competitive inhibitor (Ki, 2.7 microM) of the enzyme. The enzyme is irreversibly inactivated by uridine 5'-diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (ki/Ki) was found to be 2 x 10(3) mM-1 min-1. Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5'-diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.

Two tryptophans at the active site of UDP-glucose 4-epimerase from Kluyveromyces fragilis.

Efficient fluorescence energy transfer from aromatic residues to the pyridine moiety of the bound coenzyme (NAD) of UDP-glucose 4-epimerase from Kluyveromyces fragilis had been reported earlier (Mukherji, S., and Bhaduri, A. (1992) J. Biol. Chem. 267, 11709-11713). We have employed N-bromosuccinimide (NBS) to identify tryptophan as the exclusive aromatic donor in the energy transfer. The characteristic UV absorption spectrum associated with Trp oxidation is observed during NBS modification of two of the four Trp residues of native epimerase along with concomitant inactivation of the enzyme. Excellent correlation between the observed inactivation and abolition of fluorescence energy transfer to coenzyme from Trp in epimerase upon treatment with NBS implicates the involvement of the same two tryptophans in both catalytic activity and fluorescence energy transfer. SDS-polyacrylamide gel electrophoresis and fluorescence data preclude gross structural/conformational changes in epimerase due to NBS oxidation. The susceptible tryptophans do not reside at the substrate binding site as substrates and UMP fail to protect against NBS modification. However, failure of sodium borohydride to reduce the bound NAD in the NBS-inactivated epimerase suggests that the reactive tryptophans are close to the coenzyme. Tryptophan fluorescence lifetime values of 1.9 and 3.9 ns for the native and 3.5 ns for the NBS-modified epimerase, complemented by a linear Stern-Volmer plot (effective Stern-Volmer constant = 2.85 M-1) of acrylamide quenching, suggest that the two key tryptophans are buried close to an intrinsic quencher, presumably NAD.

The importance of binding energy in catalysis of hydride transfer by UDP-galactose 4-epimerase: a 13C and 15N NMR and kinetic study.

UDP-galactose 4-epimerase contains NAD+ irreversibly but noncovalently bound to the active site. Uridine nucleotides bind to the substrate site and induce a protein conformational change that increases the chemical reactivity of NAD+ at the coenzyme site. Activation of NAD+ by uridine nucleotides perturbs the 15N and 13C NMR chemical shifts of selectively enriched NAD+ bound to the coenzyme site. The proton-decoupled 15N NMR signal for enzyme-bound [carboxamide-15N]NAD+ does not change upon addition of UDP, indicating that activation is not brought about by a change in the binding of the carboxamide group. The 15N NMR signal of enzyme-bound [nicotinamide-1-15N]NAD+ is shifted upfield 3.0 ppm and the 13C NMR signal for [nicotinamide-4-13C]NAD+ is shifted downfield 3.4 ppm downfield by the binding of UDP at the substrate site. These changes are consistent with the induction of a distortion into the nicotinamide ring, in which positive charge is transferred from N-1 to C-4. The kinetic and thermodynamic effects of these perturbations are significant, as indicated by the nonenzymatic chemical reactivities of a series of N-alkyl nicotinamides differing in the inductive electron withdrawing effects of the alkyl substituents. A downfield change of 3.4 ppm in the 4-13C chemical shifts brought about by electron withdrawal in the model compounds corresponds to a 3200-fold increase in the rate of reduction by NaBH3CN in water, a 15,000-fold increase in 86% ethanol, and a 152 mV more positive reduction potential in this series. The distortion of NAD+ by the binding of UDP is a long-range effect that is transmitted from the substrate binding site to the coenzyme through the protein conformational change. This apparently distorts the pi-electron distribution in the nicotinamide ring and reduces the activation energy for its reduction. Activation of enzyme-bound NAD+ toward reduction apparently arises from a destabilization in the nicotinamide ring structure rather than from a stabilization of the transition state through attractive interactions between the nicotinamide ring and the enzyme.

An essential histidine residue for the activity of UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was completely inactivated by diethylpyrocarbonate following pseudo-first order reaction kinetics. The pH profile of diethylpyrocarbonate inhibition and reversal of inhibition by hydroxylamine suggested specific modification of histidyl residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of 1 essential histidine residue to be responsible for loss in catalytic activity of yeast epimerase. No major structural change in the quarternary structure was observed in the modified enzyme as shown by the identical elution pattern on a calibrated Sephacryl 200 column and association of coenzyme NAD to the apoenzyme. Failure of the substrates to afford any protection against diethylpyrocarbonate inactivation indicated the absence of the essential histidyl residue at the substrate binding region of the active site. Unlike the case of native enzyme, sodium borohydride failed to reduce the pyridine moiety of the coenzyme in the diethylpyrocarbonate-modified enzyme. This indicated the presence of the essential histidyl residue in close proximity to the coenzyme binding region of the active site. The abolition of energy transfer phenomenon between the tryptophan and coenzyme fluorophore on complete inactivation by diethylpyrocarbonate without any loss of protein or coenzyme fluorescence are also added evidences in this direction.

Distinct functional roles of two active site thiols in UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was earlier shown to have two conformationally vicinal thiols at the active site. Upon treatment with diamide, these thiols form a disulfide linkage across the subunits that results in coordinated loss of catalytic activity and coenzyme fluorescence (Ray, M., and Bhaduri, A. (1980) J. Biol. Chem. 255, 10777-10786). Employing a number of thiol-specific reagents, we now suggest discriminatory and nonidentical roles for these two thiols. Kinetic and statistical analysis of 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide modification reaction of epimerase show that only one thiol is essential for activity. Consecutive modification experiments clearly show that the same active thiol is modified in both cases. However, significant differences are observed when the reactivity of these reagents is monitored in terms of coenzyme fluorescence. Treatment with N-ethylmaleimide leads to a form of inactive enzyme that fully retains its fluorescent properties whereas modification with 5,5'-dithiobis-(2-nitrobenzoic acid), on the other hand, results in the loss of both activity and fluorescence. The closely spaced nonessential second thiol, which is not modified by N-ethylmaleimide is therefore involved in generating and maintaining the coenzyme fluorescence. Modification studies with a series of spin-labeled maleimide shows that only 3-(maleimidomethyl)proxyl causes partial quenching of coenzyme fluorescence. This suggests that the active thiol is situated at a distance of 4.5 A approximately from the coenzyme fluorophore.

Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator.

UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5'-diphosphate chloroacetol (UDC) and uridine 5'-diphosphate bromoacetol (UDB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a KD of 0.110 mM and kinact of 0.84 min-1 at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD+. The inactivation of epimerase by uridine 5'-diphosphate [2H2]chloroacetol proceeds with a primary kinetic isotope effect (kH/kD) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD+ at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD+ is proposed to be the chromophore with lambda max at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.

UDP-galactose 4-epimerase. Phosphorus-31 nuclear magnetic resonance analysis of NAD+ and NADH bound at the active site.

The phosphorus atoms of NAD+ bound within the active site of UDP-galactose 4-epimerase from Escherichia coli exhibit two NMR signals, one at delta = -9.60 +/- 0.05 ppm and one at delta = -12.15 +/- 0.01 ppm (mean +/- standard deviation of four experiments) relative to 85% H3PO4 as an external standard. Titration of epimerase.NAD+ with UMP causes a UMP-dependent alteration in the chemical shifts of the resulting exchange-averaged spectra, which extrapolate to delta = -10.51 ppm and delta = -11.06 ppm, respectively, for the fully liganded enzyme, with an interconversion rate between epimerase.NAD+ and epimerase.NAD+.UMP of at least 490 s-1. Conversely, the binding of 8-anilinonaphthalene-1-sulfonate, which is competitive with UMP, causes a significant sharpening of the epimerase.NAD+ resonances but very little alteration in their chemical shifts, to delta = -9.38 ppm and delta = -12.16 ppm, respectively. UMP-dependent reductive inactivation by glucose results in the convergence of the two resonances into a single signal of delta = -10.57 ppm, with an off-rate constant for UMP dissociation from the epimerase.NADH.UMP complex estimated at 8 s-1. Reductive inactivation by borohydride under anaerobic conditions yields a single, broad resonance centered at about delta = -10.2 ppm. The data are consistent with, and may reflect, the activation of NAD+ via a protein conformational change, which is known from chemical studies to be driven by uridine nucleotide binding. Incubation of epimerase.NAD+ with UMP in the absence of additional reducing agents causes a very slow reductive inactivation of the enzyme with an apparent pseudo-first-order rate constant of 0.013 +/- 0.001 h-1, which appears to be associated with liberation of inorganic phosphate from UMP.

Mechanism of inactivation of UDP-glucose 4-epimerase from Saccharomyces cerevisiae by D-xylose and L-arabinose.

In a previous paper (Cármenes et al., 1984) we reported that UDP-glucose 4-epimerase from Saccharomyces was inactivated both in vivo and in vitro (crude extracts) by L-arabinose or D-xylose. In this paper, we report that pure epimerase requires the presence of UMP or UDP to be inactivated by sugars and that the inactivation is due to the reduction of the epimerase NAD+, which is essential for epimerase activity. The inactivation rate is directly proportional to epimerase and sugar concentrations and hyperbolically proportional to UMP concentration. In situ experiments made with permeabilized cells showed that epimerase is inactivated in the same way when it is inside the cell. In vivo studies showed that epimerase is inactivated to a smaller extent when 1% D-galactose is present in the culture medium than when 1% ethanol is the main carbon source.