UDP-galactose 4-epimerase from Kluyveromyces fragilis Evidence for independent mutarotation site Amrita Brahma and Debasish Bhattacharyya Division of Drug Development, Design and Molecular Modeling, Indian Institute of Chemical Biology, Calcutta, India UDP-galactose 4-epimerases from the yeast Kluyvero- myces fragilis and Escherichia coli are both homodimers but the molecular mass of the former (75 kDa/subunit) is nearly double that of the latter (39 kDa/subunit). Protein databank sequence homology revealed the possibility of mutarotase activity in the excess mass of the yeast enzyme. This was confirmed by three independent assay protocols. With the help of specific inhibitors and chem- ical modification reagents, the catalytic sites of epimerase and mutarotase were shown to be distinct and inde- pendent. Partial proteolysis with trypsin in the presence of specific inhibitors, 5¢-UMP for epimerase and galactose for mutarotase, protected the respective activities. Similar digestion with double inhibitors cleaved the molecule into two fragments of 45 and 30 kDa. After separation by size-exclusion HPLC, they manifested exclusively epi- merase and mutarotase activities, respectively. Epimerases from Kluyveromyces lactis var lactis, Pachysolen tanno- philus and Schizosaccharomyces pombi also showed asso- ciated mutarotase activity distinct from the constitutively formed mutarotase activity. Thus, the bifunctionality of homodimeric yeast epimerases of 65–75 kDa/subunit appears to be universal. In addition to the inducible bifunctional epimerase/mutarotase, K. fragilis contained a smaller constitutive monomeric mutarotase of % 35 kDa. Keywords: bifunctional enzyme; domain separation; muta- rotase; UDP-galactose 4-epimerase; yeast. UDP-galactose 4-epimerase (hereafter called epimerase), which reversibly converts UDP-galactose into UDP-glu- cose, is the first enzyme of the ÔLeloir pathwayÕ of galactose metabolism [1,2]. Clinically, this is related to the disease ÔgalactosemiaÕ [3]. It belongs to the rare class of enzymes that utilizes noncovalently bound NAD as cofactor through the transient formation of enzyme-bound NADH (class II oxidoreductase). This is unlike classical dehydrogenases where NAD acts as cosubstrate (class I oxidoreductase). Although epimerase is ubiquitously present from bacteria to mammals, its quaternary structure, size and NAD require- ment vary. The bacterial and yeast enzymes are homo- dimers with bound NAD, whereas, mammalian enzymes are monomeric and require extraneous NAD. The X-ray crystallographic structures of human and Escherichia coli epimerases have been determined at high resolution [4–6]. An intriguing fact of epimerase biochemistry is the significant difference in the size of protein isolated from different sources even though their mechanisms of action are the same. Whereas the molecular mass of the yeast enzyme varies between 65 and 75 kDa/subunit (homo- dimeric), those of E. coli and mammalian systems are 39 kDa/subunit (homodimeric) and 40 kDa (monomeric), respectively. With the availability of gene sequencing data and the development of the amino-acid sequence homology search facility of the Data Bank, it was possible to compare these enzymes with nonrelated proteins [7,8]. This revealed that all epimerases contain a conserved ÔRossman foldÕ sequence identified as the NAD-binding site at the extreme N-terminus; the E. coli enzyme has strong sequence homo- logy with the yeast enzyme constituting the N-terminal half, and the C-terminal part of the yeast enzymes bears homology with mutarotase (Scheme 1, where N and C represent the N-terminus and C-terminus) [9]. Mere sequence homology, however, does not predict manifesta- tion of enzyme activity. Mutarotase is another ubiquitous enzyme found in organisms from microbes to mammals and of molecular mass 34–38 kDa, with 10-fold variation in specific activity [10,11]. This enzyme is also known in yeast and is of comparable size [12,13]. Thus Ôepimerase associated muta- rotaseÕ, if that exists, should be a second one. A preliminary report on epimerase from Saccharomyces cerevisiae supports this hypothesis [14]. In continuation of our studies on the epimerase of Kluyveromyces fragilis [15–18], we examined its bifunctionality. The results are presented here. Further, we show that epimerases of comparable size from other yeast strains are also associated with mutarotase activity. Materials and methods Reagents 1,2-Cyclohexanedione, Gly-Gly, hydroxylamine-HCl, p-chloromercuribenzoate, UDP-Gal, UDP-Glu, 5¢-UMP, Correspondence to D. Bhattacharyya, Division of Drug Development, Design and Molecular Modeling, Indian Institute of Chemical Biology, 4, Raja S.C. Mallick Road, Jadavpur, Calcutta 700032, India. Fax: 91 33 2473 5197/0284, E-mail: debasish@iicb.res.in (Received 13 September 2003, revised 16 October 2003, accepted 28 October 2003) Eur. J. Biochem. 271, 58–68 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03902.x protein molecular mass markers (for SDS/PAGE and HPLC), all sugars and their derivatives were from Sigma. 5,5¢-dithiobis-(2-nitrobenzoic acid) was from Pierce, and diethylpyrocarbonate was from Aldrich. Glucose dehydro- genase, glucose oxidase, horseradish peroxidase and trypsin were from SRL, Mumbai, India. Hydroxyapatite was synthesized as described [19]. Yeast nitrogen base, peptone, yeast extract, malt yeast extract and agar were from HiMedia, Mumbai, India. Urea was recrystallized from hot ethanol to remove cyanate contamination, if any. Yeast strains Kluyveromyces fragilis (renamed Kluyveromyces marxianus var marxianus, ATCC strain no. 10022), Kluyveromyces lactis var lactis and Pachysolen tannophilus were purchased from Microbial Type Collection Center and Gene Bank, IMTECH, Chandigarh, India. Yeast Gal 10 mutant was from ATCC (Manassas, VA, USA; strain no. 204836). Schizosaccharomyces pombi was a gift from D. J. Chattyo- padhyay, University of Calcutta. Growth of cells Liquid media containing 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 3% galactose were used. Cells were grown for 16 h at 30 °C under aerobic conditions with shaking until the turbidity reached 0.4 at 650 nm after 10-fold dilution with water. To induce epimerase, cells were grown in the presence of galactose as the only carbon source. Glucose was used instead of galactose for the Gal 10 mutant, which was devoid of the epimerase gene, and also for K. fragilis where induction of epimerase was not sought. Purification of enzymes Epimerase from K. fragilis. Yeastcells(10gwetweight), suspended in 20 m M potassium phosphate, pH 7.0, were subjected to toluenization under shaking in two steps: with 15% (v/v) toluene at 30 °C for 90 min followed by dilution to 4% toluene at 4 °C for 16 h. All subsequent steps were carried out at 4 °C. Partially lysed cells were centrifuged at 16 500 g for 30 min to remove debris. Ammonium sulfate was added to the supernatant up to 55% saturation, and the precipitate after dialysis was slowly stirred with hydroxy- apatite (5 g wet weight) where epimerase remained unab- sorbed. After recovery of the enzyme, it was passed through a DEAE-cellulose column (6.0 · 0.5 cm) equilibrated with 20 m M potassium phosphate, pH 8.0. After a complete wash, the epimerase was eluted with the same buffer containing 0.5 M NaCl. The eluted enzyme in 500 lL aliquots was passed through Centricon Nylon Membrane filters (Millipore; cut off limit 100 kDa) under 200 g for 15 min to remove trace amounts of low molecular mass contaminants. It was stored in aliquots in 20 m M potassium phosphate, pH 8.0, containing 0.5 M NaCl at )20 °C. Partial purification of epimerase from other yeast strains. Yeast cells (5 g wet wt) were subjected to toluenization and centrifugation as described above. Ammonium sulfate was added up to 80% saturation to the supernatant. The precipitate, after dialysis against 20 m M potassium phos- phate, pH 7.4, was fractionated using a precalibrated Sephadex G-200 column (118.0 · 0.5 cm), equilibrated with the same buffer, at a flow rate of 15 mLÆh )1 .Fraction size was 1.5 mL. Elution was followed in terms of epimerase and mutarotase activities. Purification of mutarotase from capsicum (bell or green pepper, Capsicum frutescens). The enzyme was purified from fresh capsicums up to the dialysis step as described in [11]. Purification of UDP-glucose dehydrogenase. This was partially purified from bovine liver up to the heat denatur- ation step as described in [20] when the preparation was free of contaminating epimerase activity. Assay of enzymes Epimerase. Continuous conversion of UDP-Gal into UDP- Glu by epimerase was monitored spectrophotometrically at 340 nm in the presence of the coupling enzyme, UDP- glucose dehydrogenase, and NAD at 25 °C [21]. In brief, 22.5 m M UDP-Gal, 12 m M NAD and 20 m M UDP-glucose dehydrogenase together with 500 lL0.2 M Gly-Gly, pH 8.8, were made up to 1 mL with water. Epimerase was then added. One unit of enzyme was defined as the amount that converts 1 lmol UDP-Gal into UDP-Glu per min under standard assay conditions. Mutarotase. Conversion of a- D (+) to b- D (+) Glu by mutarotase was followed by three independent assay protocols [10,22]. In all presentations, the spontaneous rate of conversion served as control. One unit of mutarotase activity corresponds to the conversion of 1 lmol of a-Glu to b-Glu per min at 25 °C, pH 7.4. Polarographically. The enhanced rate of change of specific rotation for the said conversion by mutarotase was followed spectropolarimetrically. The sugar (28 m M )wasdissolvedin ice-cold 1 m M Tris/HCl, pH 7.4, immediately before addi- tion of the enzyme, and the reaction rate for 10 min was recorded. The first-order rate constant was obtained from theslopeofthestraight-lineplot: ln½ða 0 À a e Þ=ða t À a e Þ ¼ Kt ð1Þ where K is the rate constant, a 0 , a t and a e are the observed angular rotations at time zero, t, and equilibrium, respect- ively [10]. Scheme 1. Alignment of peptides indicating amino acid sequence homology. Ó FEBS 2003 Mutarotase activity of epimerase (Eur. J. Biochem. 271)59 With glucose oxidase. The same conversion was followed using glucose oxidase in 0.1 M potassium phosphate, pH 6.0, which specifically converted the b-anomer into glucuronic acid with quantitative liberation of hydrogen peroxide. The latter was spectrophotometrically estimated at 460 nm with horseradish peroxidase in the presence of 1% o-dianisidine [10]. With glucose dehydrogenase. The anomer conversion was also followed using the NAD-dependent glucose dehydro- genase reaction, which specifically converts the b-form into glucuronic acid [22]. The reaction was carried out in 5 m M Tris/HCl, pH 7.4, containing 20 m M NAD and 2.8 m M substrate. Formation of NADH was followed spectro- photometrically taking e M 340nm ¼ 6.3 · 10 )3 M )1 Æcm )1 .For convenience and reliability, mutarotase was assayed using glucose dehydrogenase, unless otherwise stated. Modification reactions Epimerase (0.5 mgÆmL )1 ) was incubated with 1.25 m M 5¢-UMP and 100 m ML -arabinose at 25 °C in the presence of 50 m M sodium phosphate, pH 7.5, to reduce NAD to NADH (ÔReductive inhibitionÕ) [23]. For dissociation of the dimeric structure, epimerase (0.05 mgÆmL )1 ) was dialyzed against low-salt buffer (1 m M potassium phosphate, pH 7.5) at 4 °C for 16 h [24]. Cysteine residues were modified by allowing epimerase (0.5 mgÆmL )1 ) to react with 20 m M p-chloromercuribenzoate in the presence of 100 m M Tris/HCl, pH 7.4, at 25 °C for 2 h [17,25]. Alternatively, the enzyme (0.25 mgÆmL )1 )wastreatedwith0.2m M 5,5¢-dithiobis-(2-nitrobenzoic acid) in the presence of 20 m M sodium phosphate, pH 7.0 [26]. Histidine residues were modified by treating the enzyme (0.5 mgÆmL )1 )with 0.25 m M diethylpyrocarbonate in 20 m M sodium phos- phate, pH 7.5, at 20 °C. Modification of histidine was reversed by treatment with hydroxylamine/HCl [27]. Argi- nine residues were modified after reaction of epimerase (0.5 mgÆmL )1 ) with 2.0 m M 1,2-cyclohexanedione in 0.2 M sodium borate, pH 9.0, for 3 h at 37 °C [28]. Except for the p-chloromercuribenzoate reaction, all modifications were monitored spectrophotometrically. Reversible folding Epimerase (1.0 mgÆmL )1 ) was denatured with 8 M urea in 20 m M sodium phosphate, pH 7.5, containing 2 m M 2-mercaptoethanol at 25 °C for 10 min. Under these conditions, the molecule is known to be denatured and inactivated, with dissociation of the constituent molecules. Refolding/re-activation was initiated by 20-fold dilution of the denaturant with the same buffer in the presence or absence of 1 m M extraneous NAD at 25 °C [15,16]. Partial proteolysis Epimerase (1.0 mgÆmL )1 )wastreatedwithtrypsin(50:1; w/w) in 20 m M potassium phosphate, pH 8.0, in the presence of 2.5 m M 5¢-UMP or 6.7 m MD (+)-galactose or both at 4 °C for 4 h. The reaction was followed in terms of epimerase and mutarotase activities, and the digest was analyzed by SDS/PAGE (15% gel) and HPLC. HPLC The purity of epimerase was determined by using a Waters Protein Pak300 size-exclusion HPLC column (fractionation range 20–300 kDa). The column was equilibrated with 20 m M sodium phosphate, pH 7.0, containing 0.2 M NaCl or the same buffer containing 8 M urea at a flow rate of 0.5 mLÆmin )1 . It was then calibrated with the marker proteins: alcohol dehydrogenase (150 kDa), BSA (66 kDa), ovalbumin (43 kDa), lysozyme (14 kDa) and cytochrome c (14.3 kDa). To separate fragments of epimerase after partial trypsi- nization, a Waters Protein Pak125 size-exclusion HPLC column (fractionation range 5–80 kDa) was used. The column was equilibrated with 20 m M sodium phosphate, pH 7.0, and elution was followed at 280 nm at a flow rate of 0.5 mLÆmin )1 . It was calibrated with the marker proteins: BSA (66 kDa), ovalbumin (43 kDa), trypsin (21 kDa), myoglobin (19 kDa) and cytochrome c (14.3 kDa). In both cases of HPLC, linear dependence of log (molecular mass) vs. elution volume was observed. Other methods PAGE was performed by following standard procedures, and gels were stained with Coomassie Brilliant Blue RC-250 (Sigma) or silver nitrate. The following markers were used in SDS/PAGE; phosphorylase B (92 kDa), BSA (66 kDa), ovalbumin (43 kDa) and RNase A (10.5 kDa). Optical absorbance was recorded with a SICO 200 UV-VIS (India) or Analytik Jena Specord 200 (Germany) spectrophoto- meter. A Jasco P 1020 spectropolarimeter was used to measure specific rotation of sugars. Protein concentration was determined with Bio-Rad Protein Assay Reagent (catalog no. 10044) with BSA as reference. Results Purification of epimerase Epimerase from K. fragilis has so far been purified using salt fractionations [21]. The degree of purity achieved was variable, and occasional contamination of proteases was suspected. To remove these, a protocol has been developed using ammonium sulfate fractionation, hydroxyapatite treatment, DEAE-cellulose chromatography, and ÔfiltrationÕ by Centricon. A 40-fold purification gave a homogeneous preparation (Table 1). Epimerase after hydroxyapatite treatment was found to be free from proteases, as the SDS/PAGE profile of the fraction remained unchanged after incubation at 37 °C for 6 h or at 4 °Cfor96hto account for thermolabile proteases [29]. Hydroxyapatite is known to effectively remove proteases from yeast cell extracts [30]. The specific activity recovered was 72–75 U per mg protein compared with 70 UÆper mg protein reported previously [15,17]. Demonstration of purity This was verified by production of a single band on SDS/ PAGE and PAGE (10% gels) at pH 8.8, even after overloading of the samples, after staining with Coomassie 60 A. Brahma and D. Bhattacharyya (Eur. J. Biochem. 271) Ó FEBS 2003 Blue or silver nitrate (Fig. 1, upper panel). On SDS/PAGE, the molecular mass was found to be % 75 kDa with respect to the markers, consistent with previous observations [15,16]. The elution profile of the purified enzyme from Protein Pak300 size-exclusion HPLC showed a single sharp and symmetrical peak with retention time, R t ¼ 12.5 ± 0.1 min under conditions stated above. The profiles were indistinguishable when followed at 280 or 220 nm, indicating the absence of contaminating proteins or peptides of different sizes and probably any bio-organic compounds. To ensure that no protein was coeluted, epimerase was pretreated with 8 M urea to dissociate adhering proteins. The denatured protein emerged as a single peak of R t ¼ 11.25 ± 0.1 min (Fig. 1, lower panel, A, B and C). The lower retention time of the unfolded protein was due to expansion of the molecule in spite of dissociation of the subunits [31]. The Waters 745B data module system used in HPLC, can identify peaks of area abundance 0.01%. No peaks of such low intensity appeared in the chromatograms even after overloading of the samples. As the possibility of the coexistence and copurification of two proteins of identical molecular mass, charge and subunit composition at such an advanced stage of purity was insignificant, homogeneity of the preparation was confirmed. Assay for mutarotase Strong mutarotase activity associated with the purified epimerase was demonstrated by three independent proce- dures. In all cases, the mutarotase from capsicum served as control. Polarographically. The enhanced rate of change in the specific rotation of a- D (+)-Glu over the spontaneous rate of conversion was found to be dependent on epimerase concentration (Fig. 2A). A linear dependence of log (specific rotation) with time (up to 10 min) indicated first-order dependence of the reaction (Fig. 2A, inset). Further, the slopes of the straight lines were found to be linearly dependent on epimerase concentration. When the same data were plotted according to Eqn (1), linear dependence was again observed for the initial 10 min. The derived specific activity was 600–700 U per mg protein depending on the batch. Whereas the specific activity of mammalian mutarotase was 425–1500 U per mg protein [10], that of Fig. 1. Demonstration of purity of epimerase. (A) SDS/PAGE (10% gel) of 20 lg phosphorylase B (97 kDa) (lane 1) and 7 lg epimerase (lane 2). (B)PAGE(10%gel)atpH8.8of10lg BSA (lane 1) and 10 lg epimerase (lane 2). (C) Elution profiles of epimerase from Protein Pak300 size-exclusion HPLC column: (a) 5 lg, monitored at 280 nm, (b) 0.25 lg, monitored at 220 nm and (c) 5 lg after equilibration of the column and the sample with 8 M urea in the presence of the buffer. Table 1. Purification of epimerase from K. fragilis. Fraction Total activity (units) Total protein (mg) Specific activity (units/mg) Fold purity Crude 2110 1125 1.87 – 55% (NH 4 ) 2 SO 4 precipitation 627 163 3.86 2.1 Hydroxyapatite treatment 550 12.5 44.0 23.5 DEAE-cellulose chromatography 500 8.3 60.2 32.2 Centricon ÔfiltrationÕ 387 5.3 73.0 39.0 Ó FEBS 2003 Mutarotase activity of epimerase (Eur. J. Biochem. 271)61 capsicum was 150 U per mg protein [11]. Thus the low activity of yeast mutarotase was comparable to its phylogenic position. With glucose oxidase. The enhanced rate of conversion of a- D (+)-Glu by epimerase was observed using the coupling enzyme glucose oxidase. The reactions followed linear kinetics up to 2 min (Fig. 2B). The initial rates were found to be related to epimerase concentration in a linear manner, thereafter reaching a plateau (Fig. 2B, inset). The derived specific activity was 450–550 U per mg protein. With glucose dehydrogenase. Mutarotation was demon- strated most conveniently with the coupling enzyme glucose dehydrogenase. Under the assay conditions, the reaction followed linear kinetics for at least 5 min with epimerase. Also the rates were linearly dependent on enzyme concen- tration (Fig. 3). The specific activity observed was 550– 650 U per mg protein. When the rates of conversion (turnover) were compared with that of capsicum mutarotase on a weight by weight basis, the dependencies were very similar (Fig. 3, inset A). As the molecular mass of yeast mutarotase is about fourfold higher than that of the capsicum enzyme, its catalytic efficiency appears to be higher by the same factor. A similar result was obtained with the polarographic assay. Yeast mutarotase showed a typical Michaelis–Menten relation with the substrate a- D (+)-Glu, yielding a linear Lineweaver–Burk plot (Fig.3,insetB,C).TheK m derived was 22.2 m M ,compared with 19 m M for the capsicum mutarotase. The specific activity and purity data for the yeast enzyme indicated that it was bifunctional. Stability of mutarotase activity The pH optima of mutarotase from both kidney cortex and capsicum are broad, with maxima at % 7.4, and activity at pH 4.0 and 8.0 was 70–72%. For yeast mutarotase, the pH optimum was 7.5, with 60–70% of activity under those conditions. When incubated at 30, 40, 50, and 60 °Cfor3 h, residual activities of capsicum mutarotase were 100%, 100%, 90%, and 11%, respectively; for epimerase residual activities were 85%, 30%, 10%, and 0.5% and for yeast mutarotase they were 89%, 45%, 30%, and 0.8%, respect- ively. Thus the yeast mutarotase was less stable than its capsicum counterpart. Substrate specificity Mutarotases from different sources show significant sub- strate specificity, with 60-fold variation of turnover [10,11]. D (+)-Glu and D (+)-Gal are effective substrates for yeast mutarotase, as are also standard substrates for most of the mutarotases. Catalytic activity was lower with D (+)-fucose and D (–)-fructose, which are poor substrates, if at all, for capsicum or pig kidney mutarotase. The substitution or removal of the equatorial OH on C2 is known to abolish all substrate interactions. 2-Deoxy- D (+)-glucose and 2-deoxy- D (+)-galactose, which act as inhibitors for the kidney enzyme, appeared to be poor substrates for yeast mutarotase. Thus the substrate specificity of yeast mutarotase appeared to be comparable to that of other mutarotases (Table 2). Substrate specificity was determined polarographically in all cases because of restricted use of coupling enzymes. Interactions with inhibitors The addition of a large number of sugars to the mutarotase assay using D (+)-Glu as substrate often markedly reduces Fig. 2. Demonstration of mutarotase activity by epimerase observed using polarography and the coupling enzyme galactose oxidase. (A) Time kinetics of specific rotation of a- D (+)-glucose (28 m M ) in the presence of epimerase: (d) nil (spontaneous hydrolysis); (s)33n M ;(n)66n M ; (h)99n M . As the initial rotation of 111 ° could not be maintained exactly because of manual mixing of the reagents, the results were normalized. Inset: First-order rate dependency of these reactions for the initial 10 min. Symbols were the same as stated. Derived rates were 0.043, 0.095, and 0.152 min )1 . (B) Time kinetics of the same mutaro- tase assay using the coupling enzyme galactose oxidase. The initial rate of conversion for 2 min are presented with 33 n M (h), 57 n M (n), and 75 n M (s) epimerase. Inset: Dependence of reaction rate on enzyme concentration. The plateau was apparently due to limitation of the coupling enzyme in the assay. An enzyme concentration of 1 lgÆmL )1 corresponds to 6.66 n M . 62 A. Brahma and D. Bhattacharyya (Eur. J. Biochem. 271) Ó FEBS 2003 enzyme-catalyzed mutarotation in a competitive fashion, although some of these sugars themselves are substrates [10,11]. Of the five inhibitors tested, D (+)-Gal, L -arabinose and D -fructose were found to be effective in inhibiting yeast mutarotase to varying degrees, while L -fucose was ineffective. L -Deoxyglucose could not be tested because of its interfer- ence with the coupling enzyme glucose dehydrogenase. This inhibition pattern was similar to that of capsicum muta- rotase. None of these sugars was inhibitory to epimerase. Inhibitory effects of different nucleotides on epimerase but not on mutarotase was also observed (Table 3). Modification reactions The architecture of the functional site of epimerase has been mapped in detail, and involvement of several amino acids, e.g. cysteines [17,26,32], histidine [33] and arginine [34], has been suggested. Apart from these, Ôreductive inhibitionÕ was performed to reduce the surface NAD to NADH with inactivation [23]. Also dialysis of epimerase against low-salt buffer led to spontaneous dissociation to monomer with irreversible inactivation [24]. In the case of mutarotase, only histidine residues are known to be involved in catalysis [35]. These experiments were repeated to verify the effects on yeast mutarotase. During Ôreductive inhibitionÕ, epimerase was inactivated by 4 h (residual activity 1 ± 1%; k ¼ 0.0114 min )1 ). Low- salt incubation also led to elimination of residual epimerase activity. In all chemical modification reactions, epimerase was inactivated to 0–5% of residual activity by 30–60 min in a time-dependent manner. The k derived for the p-chloro- mercuribenzoate, 5,5¢-dithiobis-(2-nitrobenzoic acid) and diethylpyrocarbonate reactions were 0.035, 0.025 and 0.022 min )1 , respectively. Upon hydroxylamine/HCl treat- ment after diethylpyrocarbonate modification, 92% of activity was recovered. Under identical conditions, yeast and capsicum mutarotase resisted inactivation to the extent of 85–100%, except in the case of diethylpyrocarbonate where complete inactivation occurred. The extent of reversible re-activation by hydroxylamine/HCl treatment Fig. 3. Demonstration of mutarotase activity by epimerase observed using the coupling enzyme glucose dehydrogenase. Enhanced rates of mutarotation of a- D (+)-glucose in the presence of 33 n M (n), 66 n M (h), and 99 n M (s) epimerase for the initial 4.5 min are shown. The relative rates were 0.9, 1.7 and 2.74, respectively. Inset A: Rate of the same reaction expressed with an equal amount (wt/ wt) of yeast epimerase (s) and capsicum mutarotase (d). Inset B: Dependence of the reaction rate on substrate concentration at an epimerase concentration of 200 n M .InsetC: Lineweaver–Burk plot of the same reaction. Table 2. Substrate specificity of mutarotase. The concentrations of the sugars and deoxy-sugars were 28 and 30 m M , respectively. The con- centration of capsicum mutarotase was 100 n M . Reactions were fol- lowed polarimetrically, and the initial rates, where first-order kinetics were observed, were used. Substrate Activity (U per mg enzyme) Mutarotase (associated with epimerase) Capsicum mutarotase D (+)-Glucose 600 150 D (+)-Galactose 300 260 D (+)-Fucose 50 Not a substrate D ())-Fucose 30 Not a substrate 2-Deoxy- D (+)-galactose 6 Not a substrate 2-Deoxy- D (+)-glucose 7 Not a substrate Table 3. Inhibitory effects of different sugars and nucleotides on muta- rotase and epimerase activities. Concentrations of the substrate D (+)-glucose, sugar inhibitors and nucleotide inhibitors were 5.6, 5.5, and 5.0 m M , respectively. ND, not determined because of interference with coupling enzyme. Capsicum mutarotase assay was performed with glucose dehydrogenase as coupling enzyme. Inhibitor % Inhibition Mutarotase (associated with epimerase) Capsicum mutarotase a Epimerase D -Galactose 33 40 n.d. D -Fructose 22 25 0 L -Fucose 1.5 2 1 L -Arabinose 90 61 0 L -Deoxyglucose n.d. 0 0 UMP 0 0 44 UDP 0 0 25 UTP 0 0 13 a From ref [11]. Ó FEBS 2003 Mutarotase activity of epimerase (Eur. J. Biochem. 271)63 was 90%. Similar results were observed for other mutaro- tases [35]. Thus cysteine and arginine residues could not be involved in the catalytic site of yeast mutarotase. As treatments with low-salt buffer and p-chloromercuribenzo- ate led to dissociation of the molecule without affecting mutarotase activity, it was certain that mutarotase func- tionality did not require a dimeric structure. These results have been summarized in Table 4. Reversible refolding The refolding and re-activation pattern of epimerase after denaturation by 8 M urea is known in detail [15,16,18], although similar studies on mutarotase are still scarce. To optimize the re-activation yield, refolding was performed at 200 l M epimerase with capsicum mutarotase serving as control. An initial linear dependence of re-activation rate was observed in all cases. Relative re-activation rates and maximum recoveries were as follows: 1.1 and 82% for yeast mutarotase, 3.2 and 88% for epimerase, and 6.8 and 91% for capsicum mutarotase. In another set, refolding was initiated in the absence of extraneous NAD. In this case, no recovery for epimerase was observed without affecting the recovery of mutarotase activity. This indicated that matur- ation of the mutarotase site has no relation to formation of the epimerase site (results not shown). Partial proteolysis with trypsin Epimerase was sensitive to trypsin as it was degraded to small peptides without accumulation of a stable intermedi- ate [36]. However, when trypsinized in the presence of 2.5 m M 5¢-UMP, its mutarotase activity was lost in a time- dependent manner without affecting the epimerase activity (Fig. 4A). SDS/PAGE (15% gel) of the digest showed the disappearance of the original 75-kDa band with appearance of a single 45-kDa fragment (result not shown). Proteolysis under identical conditions in the presence of 6.7 m M galactose or fructose led to reversal of this protection; epimerase activity was completely lost whereas mutarotase activity was 90% protected (Fig. 4B). In this case, the molecular mass of the reduced fragment was 30 kDa. Thus it was apparent that the inhibitors were capable of protecting the respective functional domains but not the rest of the molecule. Finally, when epimerase was trypsi- nized in the presence of the two inhibitors together, Table 4. Inhibition of mutarotase and epimerase activities of yeast enzyme after chemical modifications. Reaction conditions have been described in the text. 1,2-CHD, 1,2-Cyclohexanedione; p-CMB, p-chloromercuribenzoate; DTNB, 5,5¢-dithiobis-(2-nitrobenzoic acid); DEPC, diethylpyrocarbonate. Reactions Residual activities (%) Epimerase Mutarotase (associated with epimerase) Capsicum mutarotase ÔReductive inhibitionÕ 582 93 Incubation with low salt buffer 0 90 100 p-CMB 0 95 95 DTNB 10 96 90 DEPC (re-activation by hydroxylamine/HCl) 10 (92) 0 (91) 0 (90) 1,2-CHD 0 97 90 Fig. 4. Retention of epimerase and mutarotase activities after partial proteolysis with trypsin in the presence of inhibitors. Time-dependence of residual activities of yeast enzyme: epimerase (d) and mutarotase (s) activities after partial proteolysis with trypsin in presence of (A) 2.5 m M 5¢-UMP, (B) 6.7 m M galactose, or (C) the inhibitorstogether.(D)SDS/PAGE (15% gel)profileofthedouble inhibitor digestfor4(lane1), 8 (lane 2) and12h(lane3). 64 A. Brahma and D. Bhattacharyya (Eur. J. Biochem. 271) Ó FEBS 2003 protection of either of the activities was observed (Fig. 4C). SDS/PAGE of the digest demonstrated the disappearance of the parent molecule, with generation of the two fragments of 45 and 30 kDa (Fig. 4D). As the catalytic efficiencies of the 45-kDa and 30-kDa fragments (in terms of efficiency/ mol of catalytic site) remained within ± 5% with respect to epimerase and mutarotase, respectively, an allosteric rela- tion between the two sites did not appear to exist. Separation of catalytic domains The result shown in Fig. 4C suggest the existence of two functionally independent domains in epimerase. Based on the different molecular masses, their elution was followed using a precalibrated Protein Pak125 size-exclusion HPLC column. Whereas the native epimerase was eluted at the void volume (R t ¼ 5.88 ± 0.05 min), the 5¢-UMP-protec- ted and galactose-protected fragments were eluted at R t ¼ 9.40 ± 0.05 and 10.42 ± 0.07 min, which corres- pond to molecular masses of 43 and 28 kDa, respectively, with respect to the molecular mass markers. The absence of any detectable fraction at the void volume indicated complete digestion of the parent molecule. HPLC of the digest generated in presence of the two inhibitors led to separation of two peaks of identical retention times as stated above. The fractions eluted first and second expressed exclusively epimerase and mutarotase activities, respectively (Fig. 5A–D). Recovery of the epimerase and mutarotase domains was 80 ± 5 and 90 ± 5%, respectively, in terms of activity. Existence of two yeast mutarotases During induction of epimerase in K. fragilis, mutarotase activity corresponding to 150 kDa but not 38 kDa was expected to be increased. Thus K. fragilis was grown in galactose (to induce epimerase) or glucose (to retain epimerase at basal level) medium. Further, three other strains, K. lactis var. lactis, P. tannophilus and S. pombi, were grown in galactose medium. A Sephadex G-200 column was used to separate the two mutarotase activities from the crude cell lysates. To confirm the absence of mutarotase activity corresponding to epimerase but the presence of its constitutively formed mutarotase, the Gal 10 mutant strain served as a control (Fig. 6). In the case of the four wild-type yeast strains, the chromatograms clearly separated two mutarotase activities corresponding to 150 and 35 kDa. It further showed that with induction of epimerase, parallel induction of mutaro- tase corresponding to 150 kDa occurred (Fig. 6A,D,E). As expected, growth of K. fragilis and Gal 10 mutant in glucose medium did not show induction or manifestation of mutarotase activities corresponding to a molecular mass of 150 kDa (Fig. 6B,C). To reduce the possibility that the lower molecular mass mutarotase was not derived from its higher counterpart, a cocktail of protease inhibitors was added at the time of lysis of the cells. The chromatographic patterns thereby remained unaltered. Also, to rule out salt- induced multimerization of the smaller mutarotase [24], one set of extraction and gel filtration chromatography was performed in 5 m M sodium phosphate, pH 7.5, with K. fragilis strain. No difference was observed. Discussion Among epimerases, yeast and E. coli enzymes are well studied. However, it was an enigma that, although an identical mechanistic pathway and many enzymatic prop- erties are shared, the size of the yeast enzyme is almost double that of the E. coli. enzyme. Being extracellular and devoid of cysteine bridges, synthesis of bigger molecules is not warranted for stability unless specific requirements are attributed. Mehta [36] reported that, when trypsinized in the presence of 5¢-UMP under specified conditions, the epi- merase from K. fragilis was reduced to half its size (homodimer, % 38 kDa/subunit) with retention of activity. This indicated that nearly half of the molecule had no role in epimerization. A plausible explanation subsequently came from the sequence homology profiles where the C-terminal part was suspected to be associated with mutarotase activity (Scheme 1). This hypothesis has been verified by applying three independent mutarotase assays to this enzyme (Figs 2 and 3) after assessing its physical homogeneity (Fig. 1). The Fig. 5. Size-exclusion HPLC of epimerase after partial proteolysis with trypsin. The digest was fractionated isocratically on a Waters Protein- Pak125 column and was followed at 280 nm. (A) Elution profile of catalase (240 kDa) served to determine the void volume (V o ). (B–D) Elution profiles of epimerase treated with trypsin in the presence of 5¢-UMP, galactose, or both, respectively. Proteolytic conditions are described in the text. Inset: Calibration line of log (molecular mass) vs. retention time for standard proteins as described in the text. Down- ward and upward arrows indicate the void volume and the positions of fragmented epimerase, respectively. ÔRÕ stands for regression coeffi- cient. Ó FEBS 2003 Mutarotase activity of epimerase (Eur. J. Biochem. 271)65 substrate and inhibitor specificity of this newly reported function is in general agreement with those of other mutarotases (Tables 2 and 3). It is well known that large proteins consist of multiple domains [37,38], and multifunctional proteins are Ôgenerated by folding of contiguous stretches of chains to yield autonomous domainsÕ [39]. Also the mutarotases from most of the sources are monomers of 37–38 kDa [10–12]. As about half of the K. fragilis enzyme was not involved in epimerization, it was pertinent to ask whether the two functions of K. fragilis epimerase operated from different sites. Interaction with specific inhibitors and chemical modification reactions suggested two independent catalytic sites (Tables 2 and 4). Differences in the kinetics of refolding of this bifunctional enzyme with reappearance of activity also indicated this. As 5¢-UMP protects epimerase against trypsinization both in yeast [36] and E. coli [40], we investigated whether galactose (or fructose), a competitive inhibitor for mutarotase, could induce similar stabilization. The results were indeed positive (Fig. 4). When partial proteolysis was carried out in the presence of the inhibitors together, both activities were retained. SDS/ PAGE of the digest showed complete disappearance of Fig. 6. Sephadex G-200 gel filtration profiles of different yeast extracts with respect to epimerase and mutarotase activities. In all sets, open and filled circles represent epimerase and mutarotase activities, respectively. (A) K. fra- gilis grown in galactose medium. V o was measured using blue dextran and (1) catalase (240 kDa), (2) alcohol dehydrogenase (150 kDa), (3) BSA (66 kDa), (4) haemoglo- bin (64 kDa), (5) ovalbumin (43 kDa), and (6) myoglobin (19 kDa). Inset: log (molecular mass) vs. V e calibration curve for standard proteins. (B) K. fragilis growninglucose medium. (C) Gal 10 mutant grown in glucose medium. (D) K. lactis var lactis grown in galactose medium. (E) P. tannophilus also grown in galactose medium. A result identical with those in (D) and (E) was obtained with S. pombi grown in galactose medium. The amount of sample loaded in these sets was similar but not identical. 66 A. Brahma and D. Bhattacharyya (Eur. J. Biochem. 271) Ó FEBS 2003 the parent molecule, with generation of two fragments of molecular mass 45 and 30 kDa (Fig. 4). This was consistent with the notion that multidomain proteins are connected by a proteolytically sensitive hinge region [41]. Finally, the functional domains were separated by size- exclusion HPLC (Fig. 5). This proved conclusively the bifunctional character of yeast epimerase operating from two independent regions. As enhancement or inhibition of any one of the activities was observed in none of the partial proteolysis experiments, allosteric regulation between the sites was unlikely. We also wanted to know whether 130–150-kDa epi- merases from other yeast strains were also associated with mutarotase activities and that too in addition to the constitutively formed mutarotase of 38 kDa. For this, cell lysates of K. fragilis (with or without induction of epi- merase), K. lactis var lactis [42], P. tannophilus [43] and S. pombi [12] were fractionated using a Sephadex G-200 column. In all cases, mutarotase activity was found associated with the epimerase which was distinct from the second one appearing in the 35–39-kDa region (Fig. 6). Thus the bifunctional character of yeast epimerase appeared to be universal. The biological significance of epimerase and mutarotase gene fusion in yeast may be questioned in the light of its constitutively expressed mutarotase gene. It is evident that the mutarotase gene is co-induced when cells are grown in galactose medium. In the metabolic pathway, galactose is first phosphorylated by galactokinase before entering the Leloir pathway. Galactokinase has an absolute specificity for the a-anomer and thus rapid conversion of b fi a anomer is essential to utilize the b-form [44]. When cells grow in the exponential phase, constitutively formed mutarotase may not be adequate, leading to the necessity of induced mutarotase. 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