UDP-galactose 4-epimerase from Kluyveromyces fragilis – catalytic sites of the homodimeric enzyme are functional and regulated Amrita Brahma*, Nupur Banerjee* and Debasish Bhattacharyya Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology (CSIR), Jadavpur, Kolkata, India Introduction UDP-galactose 4-epimerase, hereafter called epimerase, is an essential and ubiquitous enzyme that reversibly converts UDP-Gal to UDP-Glc. The epimerase from the yeast Kluyveromyces fragilis is a homodimer of nearly 75 kDa per subunit, and contains bound NAD + acting as cofactor [1–3]. Epimerases from Esc- herichia coli [4–6], Saccharomyces cerevisiae [7] and human sources [8] have been cloned and sequenced, and their X-ray crystallographic structures are known. The bacterial enzyme has two NAD + -binding sites Keywords catalytic sites; inhibitor; multimeric enzyme; regulation; UDP-galactose 4-epimerase Correspondence D. Bhattacharyya, Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology (CSIR), 4, Raja S. C. Mallick Road, Jadavpur, Kolkata 700 032, India Fax: +91 33 2473 5197 ⁄ 0284 Tel: +91 33 2499 5764 E-mail: debasish@iicb.res.in *These authors contributed equally to this work (Received 20 July 2009, revised 20 August 2009, accepted 16 September 2009) doi:10.1111/j.1742-4658.2009.07386.x UDP-galactose 4-epimerase from Kluyveromyces fragilis is a homodimer containing one catalytic site and one NAD + as cofactor per subunit. One 5¢-UMP, a competitive inhibitor, binds per dimer of epimerase as isolated and causes inactivation. Addition of 0.2 mm inhibitor to the enzyme in vitro leads to three sequential steps: first, the inhibitor binds to the unoccupied site; second, the inhibitor bound ex vivo is displaced allosterically; and finally, both sites are occupied by the inhibitor. These reactions have been monitored by kinetic lag in substrate conversion, coenzyme fluorescence, protection against trypsin digestion, and reductive inhibition. The transi- tion profiles indicate the existence of a stable intermediate with one inhibi- tor-binding site remaining unoccupied. Reductive inhibition of this intermediate reduced the activity to 58% ± 2%, with modification of one catalytic site. A change of conformation of the epimerase upon binding with substrate or inhibitor was evident from fluorescence emission spectra. The epimerase demonstrated a biphasic Michaelis–Menten dependency. The epimerase devoid of 5¢-UMP showed a Michaelis–Menten dependency that can be explained by assuming simultaneous operation of two catalytic sites. A monomeric form of the epimerase was devoid of such regulation. The inhibitory profile of 5¢-UMP also suggested negative cooperativity. Incubation of the epimerase with combinations of substrate analogs ren- dered one of the sites inactive, supporting the presence of two functional and regulated catalytic sites. Dissimilar kinetic patterns of the reconstituted enzyme after treatment with p-chloromercuribenzoate indicated stability of the dimeric enzyme against fast association–dissociation, which could otherwise generate multiple forms of the enzyme with functional heterogeneity. Abbreviations CHD, 1,2-cyclohexanedione; GG, glycylglycine; pCMB, p-chloromercuribenzoate; STI, soybean trypsin inhibitor. FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6725 away from the subunit contact region, and its mono- mers are functional [9]. The molecular mass of the yeast enzyme is almost double that of the bacterial and human epimerases. A blast search of the yeast epimerase revealed two fea- tures: its N-terminal half showed strong homology with the E. coli epimerase, and the C-terminal half showed homology with mutarotase [10]. The predicted mutarotase activity in K. fragilis epimerase was later demonstrated [11,12]. Furthermore, the enzyme can be cleaved by trypsin into two parts in the presence of epimerase and mutarotase inhibitors. They can func- tion independently as an epimerase and a mutarotase [11]. Interestingly, when trypsin digestion is performed in the presence of only the epimerase inhibitor, the mutarotase domain is fragmented, yielding a 45 kDa monomeric epimerase [11,13]. The yeast enzyme exhibits a stoichiometry of two NAD + molecules per dimer, similarly to E. coli and human epimerase, raising the possibility of the exis- tence of two catalytic sites. Binding of one molecule of nondialyzable 5¢-UMP, a competitive inhibitor, renders the enzyme inactive. This led to the assumption that there is regulation between the catalytic sites. Func- tionality could be restored after replacement of the inhibitor by a substrate [14]. Therefore, either the unbound site is nonfunctional or the bound inhibitor regulates its functionality. The second possibility is favored, as there is evidence that the activities of many metabolic enzymes are controlled in vivo. In the pres- ence of excess 5¢-UMP in vitro, the epimerase is com- pletely inactivated, signifying that the inhibitor blocks its two catalytic sites [15]. The background literature on epimerase shows that the nature of binding of 5¢-UMP in vivo and in vitro is different. First, the inhibitor bound as isolated, but not the one bound in vitro, shows a kinetic lag in catalysis [14]. Second, for removal of a nondialyzable inhibitor by its own counterpart added extraneously, there must be another binding site of the inhibitor in the enzyme. This site is presumably the second cata- lytic site. This observation supports the idea that the stoichiometry of bound 5¢-UMP ex vivo is indeed £ 1 per dimer. As the externally added inhibitor interacts specifically at the unoccupied binding site, a long-range interaction between the occupied and unoccupied sites is predicted (Scheme 1). Third, there is an arginine at the catalytic site of epimerase that can be modified by Scheme 1. Proposed model of conversion of E 1 to E 4 .E 1 , epimerase containing one 5¢ -UMP per dimer bound as isolated (native epimer- ase); [E 2 ], an intermediate of the conversion where the unoccupied 5¢-UMP-binding site of E 1 is occupied by the added 5¢-UMP (the bracket indicates its transient character); E 3 , stable intermediate where the 5¢-UMP bound ex vivo to E 1 is replaced allosterically by the added 5¢-UMP; E 4 , epimerase where both the 5¢-UMP binding sites are occupied by added 5¢-UMP; [E 2A ], product of reductive inhibition of [E 2 ] with L(+)-arabinose (the bracket indicates uncertainty about its existence); E 3A , product of reductive inhibition of [E 3 ] with L(+)-arabinose. The two lobes in all the structures indicate homodimeric epimerase; the flange at the middle of each lobe separates the epimerase (upper) and mutarotase (lower) domains of a monomer; the rectangular denting of the upper domains of each lobe indicates the binding site of 5¢-UMP ex vivo; the shaded rectangle indicates 5¢-UMP bound as isolated; the open rectangle indicates added 5¢-UMP; • , NAD + , , NADH, s, arginine at the active site; s over the 5¢-UMP binding site indicates protection against trypsinization; circumference of the lobe next to the site indicates susceptibility to the protease; +UMP and –UMP indicate its association and dissociation; the arrow on the top of the scheme indicates the direction of 5¢-UMP-dependent conversion of epimerase. The symmetrical pattern of the dimeric enzyme as shown is a working model only. Regulation of catalytic sites of yeast epimerase A. Brahma et al. 6726 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 1,2-cyclohexanedione (CHD), leading to inactivation [15]. Whereas 5¢-UMP added in vitro could completely protect the arginine from modification, the inhibitor bound ex vivo is incapable of doing so [16]. Fourth, degradation of epimerase by trypsin is initiated from the arginine of the catalytic site, leading to further unraveling of the molecule [11]. Trypsin digestion of epimerase could be completely prevented if the said arginine were protected by 5¢-UMP in vitro. However, the inhibitor bound ex vivo is incapable of preventing trypsin digestion [15]. Fifth, 5¢-UMP bound ex vivo does not participate in reductive inhibition (‘reductive inhibition’ is a specific reaction whereby epimerase bound NAD + , acting as cofactor, is reduced to NADH in the presence of 5¢-UMP, a competitive inhibitor, and l(+)-arabinose, a reducing sugar, lead- ing to complete inactivation of the enzyme [24] – NAD + as free nucleotide or bound to other enzymes is not sensitive to this reaction), whereas the inhibitor bound in vitro does [14]. Here, we provide a hypothesis for the pathway fol- lowed by epimerase as isolated (E 1 ) during its satura- tion with extraneously added 0.5 mm 5¢-UMP (E 4 ) (Scheme 1). An essential feature in this proposal is that the nature of the binding of 5¢-UMP in E 1 and that in E 4 are different, evidence for which has been men- tioned above. On the basis of the situation in Scheme 1, the minimum requirement for the conver- sion is the existence of two intermediates, E 2 and E 3 . In E 1 , one inhibitor-binding site is occupied and the other is vacant. In E 2 , the added 5¢-UMP binds to the unoccupied site of E 1 .InE 3 , the added inhibitor has removed the 5¢-UMP bound ex vivo by allostericity. A higher concentration of the inhibitor leads to the final product E 4 Thus, the model predicts that, in E 3 , the inhibitor occupies the high-affinity site, leaving the low-affinity site vacant. In summary, the conversion involves three steps: association of added inhibitor at low concentration; dissociation of the inhibitor bound ex vivo; and, finally, association of added inhibitor at the unoccupied site (Scheme 1). Theoretical considerations predict that E 2 should exist only as a transient intermediate. If E 2 is stable, it will prevent the spontaneous formation of E 3 .AsE 2 has no additional 5¢-UMP-binding site, its conversion to E 3 should be independent of added 5¢-UMP. A cor- ollary of the prediction is that half of the catalytic sites of E 1 and E 3 remain bound to 5¢-UMP, whereas both of the catalytic sites in E 2 and E 4 are occupied by the inhibitor. Thus, after incubation with trypsin, the residual activities of E 1 ,E 2 (if it exists), E 3 and E 4 are expected to be 0%, 50%, 50% and 100%, respectively, on the basis of selective protection by the added inhib- itor. Similarly, the formation of NADH and residual activity after reductive inhibition in the presence of only l(+)-arabinose for these enzyme–inhibitor com- plexes are expected to be 0, 1, 1 and 2 mol per dimer and 100%, 50%, 50% and 0%, respectively. To be more precise, as the catalytic sites are supposed to be regulated, inactivation of E 2 and E 3 after trypsiniza- tion or reductive inhibition leading to E 2A and E 3A (Scheme 1) may not be exactly 50%. While supporting Scheme 1, two physical properties of epimerase have been established. These are the stoi- chiometry of bound cofactor NAD + being two per dimer, and the stability of its dimeric structure with regard to fast dissociation–association. In the absence of this information, Scheme 1 is not valid. Subse- quently, we verified whether the Michaelis–Menten relationship is or is not maintained by the enzyme. When product inhibition and secondary reactions are not applicable for an enzyme, its deviation from the Michaelis–Menten relationship is a strong indication of allosteric regulation. Specific inactivation of one catalytic site of epimerase with substrate analogs has also been investigated to provide supportive evidence. Results Stoichiometry of bound NAD + Conversion of NAD + to NADH by reductive inhibi- tion of epimerase offered a sensitive method for deter- mination of stoichiometry of the bound cofactor. Reductive inhibition was applied to 33.3 nmol (5 mg) of epimerase. A control set of the enzyme under identi- cal conditions but in the absence of the reducing agents [5¢-UMP and l(+)-arabinose] retained 98% of the activity. Complete inactivation of the enzyme ensured quantitative reduction of NAD + to NADH. Dissociation of NADH from the enzyme was achieved with 8 m urea [17], and was quantified spectrofluori- metrically. Recovery of NADH was 63.0 ± 4.0 nmol, yielding a stoichiometry of 1.89 ± 0.12 per dimer, or close to 1.0 NADH per subunit (n = 4). Further improvement in quantification was restricted by the uncertainty of protein concentration determination and the incompleteness of conversion of NAD + to NADH. Therefore, with respect to the composition of cofactor, the catalytic sites of epimerase remain indistinguish- able. Stability of subunits According to Scheme 1, the subunits of epimerase (E 1 ) are asymmetric, of the type a:b, on the basis of bind- A. Brahma et al. Regulation of catalytic sites of yeast epimerase FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6727 ing of 5¢-UMP. Epimerase exists as a stable dimer [18], but this does not exclude fast association–dissociation of the dimer, leading to three types of functional entity (Eqn 1). Depending on the magnitude of the rate con- stants involved, there might be undetectable amounts of the monomers at equilibrium. Thus, noncompliance with the Michaelis–Menten relationship of epimerase, as described later, might originate from heterogeneity of the enzyme without regulatory behavior. 2a : b Ð 2a þ 2b Ð a : a þ b : b ð1Þ To check this, epimerase was treated with p-chloro- mercuribenzoate (pCMB), leading to inactivation and dissociation of the subunits without denaturation. Functionality was restored with reconstitution of the dimeric structure after reduction of the modified enzyme with dithiothreitol and NAD + [19]. The dura- tions of kinetic lag of catalysis for 0.004 units of native and reconstituted epimerase were found to be 83 s and 17 s, respectively, whereas the rates of substrate con- version in the steady state were very similar, being 2.52 lmol and 2.63 lmol of UDP-Gal per min, respec- tively (Fig. 1A). Furthermore, the Michaelis–Menten patterns of the native and reconstituted epimerase were constructed and were found to be entirely different (Fig. 1B). The kinetic features of the native enzyme remained unchanged when the enzyme was incubated in the assay mixture without the substrate for 30 min at 25 °C prior to activity measurements. These obser- vations collectively indicate that epimerase does not undergo rapid exchange of subunits during catalysis. Characterization of epimerase–inhibitor complexes The equilibrium intermediates formed during conver- sion of the inhibitor complex E 1 to E 4 in the presence of 0–0.6 mm 5¢-UMP were characterized by a kinetic lag in catalysis, ‘coenzyme fluorescence’ (described later), and inactivation by trypsin (Fig. 2A), and two other parameters of reductive inhibition, namely inacti- vation and formation of NADH (Fig. 2B). The last two parameters are sensitive enough to be measured with an accuracy of ± 0.25%. The dependency of the lag in catalysis followed a sharp decrease of 100% to 10% ± 2% in the presence of 0–0.2 mm 5¢-UMP. This indicated removal of the inhibitor bound as isolated in E 1 or modification of the inhibitor-binding site. A corresponding conforma- tional change at the cofactor-binding site was moni- tored from coenzyme fluorescence. There was an initial 25% rise in emission intensity in the presence of 0–0.2 mm 5¢-UMP. The intensity gradually returned to its original value in the presence of 0.5 mm inhibitor. The maximum emission was seen in the presence of 0.15 mm 5¢-UMP, which corresponded closely to the transition midpoint of abolition of the kinetic lag. In the case of inactivation by trypsin, the profile distinctly indicated the existence of a stable intermediate between 0.1 mm and 0.2 mm of the inhibitor. This intermediate retained 58% ± 2% of the residual activity, indicating that the added inhibitor could protect half of the catalytic sites, as expected for E 3 . All transitions were A B Fig. 1. Differences in catalytic properties of native and reconsti- tuted epimerase after dissociation with pCMB. (A) Pre-steady-state kinetics of 0.004 units of native (1) and reconstituted (2) epimerase. The horizontal dotted lines indicate initial absorbance of native (1) and reconstituted (2) epimerase. Durations of initial lag of catalysis are indicated by vertical dotted lines. The parallel nature of curves 1 and 2 indicate that, at steady state, the catalytic efficiencies of the two forms of epimerase are equal. (B) Michaelis–Menten plots of native (s—s) and reconstituted ( • — • ) epimerase. The bar indi- cates variation of results (n = 3). Results for the native epimerase are elaborated in Fig. 5. Inset: Lineweaver–Burk plot of the recon- stituted epimerase. Units of the ordinate and abscissa are lmolÆ- min )1 and mM )1 , respectively; values correspond to the original plot. Regulation of catalytic sites of yeast epimerase A. Brahma et al. 6728 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS completed with 0.5 mm 5¢-UMP, as higher concentra- tions of the inhibitor failed to cause additional change (Fig. 2A). Proteolysis of native epimerase by trypsin is initiated from an arginine residue located at the catalytic site [11]. The degradation can be prevented either by modi- fication of this residue by CHD or by incubation with 5mm 5¢-UMP [16]. 5¢-UMP can protect E 4 against trypsin, but not E 1 . Furthermore, as the arginine at the catalytic site of E 1 does not appear to be protected by 5¢-UMP, the amino acid could be modified by CHD, which in turn is expected to resist trypsin diges- tion. SDS ⁄ PAGE of CHD-modified E 1 showed genera- tion of a stable 45 kDa fragment after trypsin digestion, which was presumably the N-terminal domain of the epimerase (Fig. 2A, inset). Thus, suscep- tibilities of different epimerase–UMP complexes to trypsin may be used to follow interactions of the inhib- itor at the catalytic site. The protective effect of 5¢-UMP against modification of the arginine located at the catalytic site by CHD was similar to that observed for trypsin digestion. Native epimerase was modified by CHD in the pres- ence of 0, 0.1–0.2 and 0.5–1.0 mm 5¢-UMP, and the excess reagent was removed with a spin column. The results showed that, in these ranges, the enzymes were inactivated by 95% ± 5%, 48% ± 2%, and 8% ± 3%, respectively. This accords with the idea that externally added 5¢-UMP can protect the said arginine against modification but the inhibitor bound ex vivo is unable to do so, which is consistent with Scheme 1. The existence of an isolable intermediate in the con- version of E 1 to E 4 was also indicated by the profiles generated from reductive inhibition of E 1 in the pres- ence of 0–0.5 mm 5¢-UMP and 10 mml(+)-arabinose (Fig. 2B). These clearly indicate a three-state transition with a stable intermediate in the presence of 0.1– 0.2 mm 5¢-UMP. Formation of E 3A from E 3 represents the reductive inhibition of the stable intermediate. This intermediate possesses residual activity of 58% ± 2% as compared with E 1 , and NADH fluorescence of 63% ± 2% as compared with E 4 . The NADH fluores- cence was measured under denaturing conditions to remove interference from coenzyme fluorescence. This indicated that under the defined conditions of reduc- tive inhibition, one of the two NAD + molecules of the dimeric enzyme was converted to NADH. In control experiments, it was verified that the reagents carried over to the assay mixture did not cause inactivation of the coupled enzyme. Therefore, the results of Fig. 2A,B are in agreement with Scheme 1. Irreversible conversion If the conversion of Scheme 1 were reversible, the enzyme–inhibitor complexes would become unstable while the excess inhibitor was removed. The complexes E 3 and E 4 were dialyzed extensively against 50 mm A B Fig. 2. Dependence of physicochemical properties of native epim- erase (0.25–0.5 mgÆmL )1 ) in the presence of 0–6 mM added 5¢-UMP at pH 8.0. (A) The kinetic lag in converting UDP-Gal to UDP-Glc was followed. Coenzyme fluorescence was measured without dilution. Inactivation by trypsin was followed from residual epimerase activity after protease digestion for 4 h under the stipu- lated conditions. Results are expressed by taking activity of the native epimerase as 100%. Inset: SDS ⁄ PAGE of epimerase (20 lg) digested with trypsin (50 : 1, w ⁄ w) at pH 8.0 and at 4 °C. Lane 1: after 30 s of protease pulse. Lane 2: after 4 h of protease pulse. Lane 3: in the presence of 5 m M 5¢-UMP for 4 h. Lane 4: as lane 2, except that the epimerase used was modified by CHD. The upper and lower arrows indicate the positions of BSA (66 kDa) and ovalbumin (45 kDa) in the electrogram. (B) Reductive inhibition was performed with 5 m ML(+)-arabinose for 1 h at 25 °C, in the presence of varying concentrations of 5¢-UMP. Fluorescence was measured after treatment with 8 M urea (n = 2–3). In all measure- ments, baseline correction was performed. A. Brahma et al. Regulation of catalytic sites of yeast epimerase FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6729 sodium phosphate (pH 7.5) at 4 °C to remove unbound inhibitor. It was verified that the native epim- erase (E 1 ) could withstand inactivation due to dialysis under such conditions. The presence of 5¢-UMP in all of the dialyzed samples was confirmed by MS analysis (Fig. 3). The dialyzed enzyme complexes were sub- jected to reductive inhibition in the presence of 10 mm l(+)-arabinose. The residual activities of E 1 ,E 3 and E 4 were 80% ± 5%, 55% ± 5% and 8% ± 5%, respectively (n = 2). The kinetic lag in catalysis as observed with E 1 could not be reproduced with the dialyzed samples of E 3 and E 4. This indicated that the inhibitor-binding steps are irreversible. Change in tertiary structure A change in conformation of an enzyme is an obligatory requirement for regulation of its activity. In epimerase, one of four tryptophans per subunit is located at the catalytic site [20]. Thus, perturbation of the catalytic sites is likely to be reflected in the fluorescence spectra of its tryptophans (excitation at 295 nm). The change of conformation of epimerase during its interaction with 0–0.6 mm 5¢-UMP and 0–0.4 mm UDP-Gal for 5 min at 25 °C in two separate experimental sets was followed. The substrate served as a catalytic site-directed ligand. As compared with the native enzyme, the maximum emission of the treated samples remained unaltered, at 343.8 ± 0.5 nm, indicating retention of the micro- environment of the tryptophans. However, in the presence of maximum concentrations of 5¢-UMP and UDP-Gal as applied in this study, the emission intensity was reduced by 12% ± 2% and 17% ± 2%, respec- tively, indicating conformational change (Fig. 4A,B). Application of higher concentrations of the inhibitor or the substrate could not alter the extent of quenching. The conformers also attained equilibrium, as no further change in emission intensity was observed with increasing incubation period. Kinetic patterns To investigate whether the catalytic sites of epimerase are distinguishable on the basis of turnover, three forms of the enzyme, namely dimeric native epimerase (E 1 ), dimeric inhibitor free epimerase (E 0 ), and mono- meric epimerase (E M ), were used for kinetic analysis in the presence of 0–0.35 mm substrate (Figs 5–7). Fig. 3. MS analysis of the dissociated ligand of native epimerase. The observed peaks have been assigned as follows: 5¢-UMP, 2Na + , H + = 369.1 (obs. 368.99); 5¢-UMP, 2Na + ,2H + = 370.1 (obs. 369.87); 5¢-UMP, 3Na + = 391.1 (obs. 390.96); and 5¢-UMP, 3Na + ,H + = 392.1 (obs. 391.87). Commercially available 5¢-UMP-disodium salt, under similar experimental conditions, showed an identical mass pattern. The spectral zone of NAD + has not been included. Regulation of catalytic sites of yeast epimerase A. Brahma et al. 6730 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS Regulation of catalytic activity has been clearly dem- onstrated in the case of the native epimerase. The Michaelis–Menten relationship showed hyperbolic dependencies in the substrate concentration ranges 0–0.075 mm and 0.2–0.35 mm, whereas there was no significant variation of reaction rate between 0.075 mm and 0.2 mm (Fig. 5). Thus, the low-affinity site (the high-affinity and low-affinity sites of epimerase referred to in the text are related to the substrate UDP-Gal – A B Fig. 5. Michaelis–Menten plot of native epimerase with UDP-Gal as substrate. The enzyme concentration was 3.3 n M. (A, B) Linewe- aver–Burk plots with 0–0.05 m M and 0.2–0.35 mM substrate. Units of the ordinate and abscissa are lmolÆmin )1 and mM )1 , respec- tively; values correspond to the original plot. Derived values of K m and V max are presented in Table 1. Solid and hatched bars represent the presence of bound 5¢-UMP ex vivo and the gradual disappearance of the initial lag of catalysis, respectively. A B Fig. 4. Change of conformation of native epimerase in the pres- ence of (A) 0–0.6 m M 5¢-UMP and (B) 0–0.4 mM UDP-Gal. The emission intensity of native epimerase is considered to be 100% in either set. The ligands had no emission in this spectral zone. AB Fig. 6. Michaelis–Menten plot of inhibitor-free epimerase with UDP-Gal as substrate. The enzyme concentration was 1.65 n M. The open circles (s—s) indicate experimentally observed points. (A, B) Lineweaver–Burk plots with 0–0.05 m M and 0.05–0.35 mM sub- strate. Units of the ordinate and abscissa are lmolÆmin )1 and m M )1 , respectively; values correspond to the original plot. Derived values of K m and V max are presented in Table 1. The line ( • — • ) was constructed according to Eqn (2), using the parameters of Table 1. The other line is the best fit joining the experimentally observed points (s—s). Fig. 7. Michaelis–Menten plot of monomeric epimerase with UDP-Gal as substrate. Inset: Lineweaver–Burk plot with 0–0.35 m M substrate. Units of the ordinate and abscissa are lmolÆmin )1 and m M )1 , respectively; values correspond to the original plot. The enzyme concentration was 0.5 n M. Derived values of K m and V max are presented in Table 1. A. Brahma et al. Regulation of catalytic sites of yeast epimerase FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6731 high-affinity and low-affinity sites of 5¢-UMP have no relationship with the corresponding UDP-Gal-binding sites) became functional at a substrate concentration much higher than that required for saturation of the high-affinity site. Lineweaver–Burk plots were con- structed for the two dependencies (Fig. 5A,B). Derived K m and V max values were 0.01 mm and 2.88 lmolÆ min )1 Æmg )1 for the high-affinity site, and 1.0 mm and 5.56 lmolÆmin )1 Æmg )1 for the low-affinity site, respec- tively. The presence of 5¢-UMP bound to the enzyme as isolated was detected from the kinetic lag in cataly- sis and the characteristic MS pattern. When these anal- yses were performed, the enzyme was incubated with variable concentrations of the substrate for 1 min under the assay conditions, and was passed through a spin column to separate unbound ligands from the eluted enzyme. It was observed that, in the presence of up to 0.06 mm UDP-Gal, the inhibitor remained bound to the enzyme (Fig. 5, solid and hatched bars). Thus, the catalytic site of the native epimerase, which was free of the inhibitor, was nonfunctional at low substrate concentrations. In the case of inhibitor-free epimerase, the depen- dency of reaction velocity on substrate concentration cannot be represented by a single Michaelis–Menten relationship over the range of substrate concentrations used, because the corresponding Lineweaver–Burk plot had a poor correlation (R 2 = 0.8473, where R 2 is the regression coefficient). However, when two Linewe- aver–Burk plots were constructed for 0–0.02 mm and 0.05–0.2 mm substrate, a significant improvement in linear dependency was observed, R 2 being 0.960 and 0.999, respectively. These yielded K m and V max values of 0.011 mm and 2.08 lmolÆmin )1 mg )1 for the high- affinity site, and 0.178 mm and 1.76 lmolÆmin )1 Æmg )1 for the low-affinity site, respectively (Fig. 6A,B). It is noteworthy that the Lineweaver–Burk plot for the high-affinity site showed a downward curvature at a higher substrate concentration (Fig. 6A). This is an indication of the presence of a second operational site of low efficiency; otherwise, the plot would follow the linear trend [21]. It has been calculated that the low- affinity site contributed at most 10% towards the turn- over efficiency in the presence of 0.02 mm UDP-Gal. The Michaelis–Menten relationship of the mono- meric epimerase showed hyperbolic dependency and a linear Lineweaver–Burk plot between 0 mm and 0.35 mm UDP-Gal. Derived K m and V max values were 0.01 mm and 2.52 lmolÆmin )1 Æmg )1 , respectively, sug- gesting that the catalytic site was similar to the high- affinity site of the native and inhibitor-free epimerase (Fig. 7 and inset). All kinetic parameters are summa- rized in Table 1. Assessment of kinetic data When the epimerase reaction did not show a Michaelis– Menten relationship, it was assumed that the cata- lytic sites were operating simultaneously at unequal efficiencies. Under such conditions, the rate of an enzyme reaction (V) can be expressed as the sum of two Michaelis–Menten dependencies, as in Eqn (2) [21]. V ¼ V max ðHÞ ½S K m ðHÞ þ½S þ V max ðLÞ ½S K m ðLÞ þ½S ð2Þ where the subscripts H and L refer to high-affinity and low-affinity sites for the substrate. V max of the enzyme was obtained from the Lineweaver–Burk plot at infi- nite substrate concentration, because, under this condi- tion, both of the catalytic sites were operating at maximum efficiency; that is, V max = V max (H) + V max (L) . V max (H) was calculated after extrapolation of the linear portion of the Lineweaver–Burk plot using data points from the low substrate concentration. V max (L) was obtained by subtracting V max (H) from V max . From the values of K m and V max (Table 1), the dependency of V on [S] was calculated between 0 mm and 0.35 mm UDP-Gal and compared with the experi- mental data. For inhibitor-free epimerase, the correla- tion was quite satisfactory (R 2 = 0.982) (Fig. 6). In the case of native epimerase, Eqn (2) was not expected to be valid, as the catalytic sites were not operating simultaneously (Fig. 5). Analysis of Fig. 5 showed that contributions by the high-affinity and low-affinity sites to overall turnover were 34.2% and 65.8%, respec- tively, when maximum turnover by the enzyme was achieved. This is in agreement with the profiles of Fig. 2A,B, where inactivation of one catalytic site by trypsinization or reductive inhibition led to residual activities of 61.3% and 59.9%, respectively. Deviation from equal catalytic efficiency of the two functional Table 1. Kinetic properties of different forms of epimerase. The high-affinity and low-affinity sites refer to the substrate UDP-Gal. Results shown are within ± 5% error. Epimerase K m (mM UDP-Gal) V max (lmolÆmin )1 Æmg )1 ) Native epimerase High-affinity site 0.01 2.88 Low-affinity site 1.0 5.56 Inhibitor-free epimerase High-affinity site 0.011 2.08 Low-affinity site 0.178 1.76 Monomeric epimerase 0.01 2.52 Regulation of catalytic sites of yeast epimerase A. Brahma et al. 6732 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS sites is in agreement with Scheme 1 and Fig. 2, where inactivation of E 3 by trypsinization and reductive inhi- bition was 55% ± 5% and 58% ± 2%, respectively. Consistent deviation from inactivation by 50% in these reactions indicated that the catalytic sites of epimerase are nonidentical. Equation (2) was used further to assess the perfor- mance of the catalytic sites of the inhibitor-free epim- erase. It has been assumed that the efficiency of the catalytic sites at infinite substrate concentration reached 100%, although these values are different in absolute terms because of regulation. The analysis shows that raising the substrate concentration from 0.001 mm to 0.025 mm increased the activities of the high-affinity and low-affinity sites from 0.90% to 69.4% and from 0.06% to 12.26%, respectively. When the substrate concentration was further increased from 0.025 mm to 0.35 mm, the corresponding increases were 69.4–96.96% and 12.26–66.27%, respectively. Effects of inhibitor The range of inhibitor concentrations and the pattern of dependency of inhibition of regulatory enzymes dif- fer from those of Michaelis–Menten-type enzymes [21]. Competitive inhibition of epimerase by 5¢-UMP is known [14–16,20,22]. Typical plots of residual activities of native and monomeric epimerase versus inhibitor concentration show that the profiles are widely differ- ent (Fig. 8). In the case of native epimerase, no inhibi- tion was observed up to 0.8 mm 5¢-UMP, as compared with 62.5% inhibition for the monomeric epimerase. At 20 mm 5¢-UMP, the monomeric epimerase showed 76.3% inhibition, the native epimerase showed 85% inhibition. Dixon plots (inverse of rate versus inhibitor concentration) of the monomeric and native epimerase were hyperbolic and parabolic (results not shown). The hyperbolic dependency indicated partial inhibition from a single binding site of the inhibitor in mono- meric epimerase. The parabolic dependency indicated two binding sites of the inhibitor in native epimerase that are regulatory in nature. These patterns are simi- lar to those of the nonregulatory and regulatory types of enzyme [21]. UDP and UTP are also competitive inhibitors of epimerase, but have weaker affinity than 5¢-UMP [15,22]. Thus, they are expected to remove the 5¢-UMP of native epimerase. Abolition of the lag in catalysis of the native epimerase after interaction with these inhibitors was correlated with their inhibitor con- stants [23]. The values of the residual lag, correspond- ing inhibitor concentration and K i for 5¢-UMP, UDP and UTP were 10%, 0.4 mm, and 0.15 mm, 13%, 6mm, and 0.37 mm, and 25%, 6 mm, and 0.60 mm, respectively. Hence, the ability of the inhibitors to remove the kinetic lag was inversely related to their K i , and they also showed specificity of such substitution according to their K i values. Selective inactivation of one catalytic site 5¢-UMP and l(+)-arabinose are the most effective reagents for reductive inhibition. A combination of uridine nucleotides such as UDP or UTP and reducing sugars such as galactose or glucose can also induce reductive inhibition, but with lower efficiency. Native or inhibitor-free epimerase was incubated with 0.2 mm UDP or UTP along with 2 mmd(+)-Gal or d(+)-Glc at 4 °C for 40 h at pH 7.5. Whereas native epimerase without any reagent retained 96% ± 2% of its activ- ity, incubation with any combination of reagents reduced the activity to 65% ± 5%, with a distinctly different pattern in the Michaelis–Menten plot. There was a hyperbolic dependency between 0 mm and 0.075 mm UDP-Gal, after which there was no signifi- cant change in the catalytic rates up to 0.35 mm sub- strate. This evidently indicates inactivation of the second site (Fig. 9A). Inhibitor-free epimerase incu- bated with various combinations of reagents demon- strated the same biphasic Michaelis–Menten dependency as that shown in Fig. 6, with 80% ± 3% recovery of residual activity (Fig. 9B). Discussion Allosteric regulation of the epimerase from K. fragilis has not been investigated with confidence before. That there is deviation from the Michaelis–Menten relation- ship during the reversible conversion of UDP-Gal to Fig. 8. Inhibitory profiles of native and monomeric epimerase by 5¢-UMP. The enzyme and substrate concentrations were 1.65 n M and 0.1 mM, respectively. 5¢-UMP has no effect on the coupling enzyme. The enzyme activity in the absence of the inhibitor is considered to be 100%. A. Brahma et al. Regulation of catalytic sites of yeast epimerase FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS 6733 UDP-Glc before product equilibration is attained has been known for a long time [24]. The allosteric kinetics of the epimerase were reported more recently [25,26]. However, when the subunit-sharing model of a single catalytic site of the dimeric enzyme was proposed, the relevance of allostericity could not be explained [3]. Now it is known that the epimerase contains two NAD + molecules per dimer, and the subunit-sharing model of the catalytic site is invalid [7,11,13]. Also, binding of 1 mol of 5¢-UMP per dimer as isolated, leading to inactivation of the enzyme, indicates regula- tory behavior, provided that the catalytic sites are functional [14]. These findings have revived interest in exploring the regulatory behavior of this enzyme. To avoid misinterpretation of the results, the composition and stoichiometry of the bound cofactor(s) and the stability of the dimeric structure of epimerase with regard to fast association–dissociation were ascer- tained. The epimerase from E. coli can accommodate NADH in place of NAD + when overexpressed from a plasmid [27]. As E. coli and yeast epimerase are similar in many respects, there remains a possibility that the yeast enzyme can recruit NADH instead of NAD + , leading to partial inactivation as well as functional het- erogeneity. To verify this, the native epimerase was treated with 8 m urea to dissociate the cofactor(s) [17]. The resulting solution had no characteristic NADH fluorescence to the limit of detection (< 0.01 mol per dimer). Incomplete recruitment of NAD + and reacti- vation of the enzyme during the assay after it has absorbed NAD + from the assay mixture can also cause functional heterogeneity. This was ruled out, as the enzyme preincubated with 0.05 mm NAD + for 15 min prior to the assay did not show enhancement of activity. Reductive inhibition of epimerase followed by quantification of dissociated NADH (as illustrated in Experimental procedures) showed that the stoichi- ometry was nearly 2.0 per dimer or 1 per catalytic site. Earlier, maximum recovery of 1.70 ± 0.10 mol of NAD per dimer was reported, based on dissociation of the nucleotide by trichloroacetic acid or heat, where partial coprecipitation of the holoenzyme with the apoenzyme is suspected [14]. The stability of dimeric structure of epimerase with regard to rapid association–dissociation was estab- lished from complete and reversible dissociation of the subunits after modification with pCMB, followed by reduction under nondenaturing conditions [19]. The kinetic parameters of the reconstituted enzyme were distinctly different from those of the native enzyme (Fig. 1A,B). The native enzyme could never attain this property of the reconstituted enzyme. This indicates that the equilibrium described in Eqn (1) is not valid for epimerase. As kinetic data cannot predict the number of cata- lytic sites of an enzyme, selective inactivation of one site of epimerase appeared to be the only answer to this question. Such a proposition remained elusive, as the catalytic sites were found to be identical with regard to several modification reagents. Reductive inhi- bition offered a unique opportunity to address this issue. It was proposed that reductive inhibition could reduce one NAD + of the enzyme–inhibitor complex E 3 , leading to E 3A . As a consequence, E 3 would be inactivated by 50%. In reality, such experiments A B Fig. 9. Michaelis–Menten plots of epimerase preincubated with substrate analogs. (A) Native epimerase. • — • , epimerase incu- bated without any substrate analog. , enzyme preincubated with 0.2 m M UDP + 2 mMD(+)-Gal. , enzyme preincubated with 0.2 m M UDP + 2 mMD(+)-Glc. (B) Inhibitor-free epimerase. • — • , dependencies of inhibitor-free epimerase preincubated without substrate analogs. , dependencies of inhibitor-free epimerase preincubated with 0.2 m M UDP + 2 mMD(+)-Gal. Regulation of catalytic sites of yeast epimerase A. Brahma et al. 6734 FEBS Journal 276 (2009) 6725–6740 ª 2009 Council of Scientific and Industrial Research, New Delhi. Journal compilation ª 2009 FEBS [...]... residual activity of 58% ± 2% (n = 6) This indicates that the catalytic sites of the enzyme are functional and are distinguishable on the basis of binding with the inhibitor Careful analysis of Fig 2 shows that inactivation of E3 by trypsin and reductive inhibition consistently deviated from 50%, in spite of modification of one catalytic site of two Such inequality between two identical catalytic sites is usually... concentrations of UDP-Gal (Fig 5) Analysis of the Michaelis–Menten dependency of inhibitor-free epimerase also indicated that its catalytic sites are operating independently with unequal efficiencies The nonidentical nature of the Michaelis–Menten relationships of inhibitor-free and native epimerase is an indication of the regulatory role of the bound 5¢-UMP in the latter The Michaelis–Menten relationship of the. .. indicating that the coupling enzyme was free from epimerase activity By varying the volume of enzyme added between 10 lL and 400 lL instead of water in the assay mixture and observing the linear progress curve of D0.00 5–0 .03 absorption unitsÆmin)1, the assay permits detection of as little as 0.25% of enzyme activity To study the effects of inhibitor, the assay mixture was incubated with 0–2 0 mm 5¢-UMP... such as the close proximity of epimerase catalytic sites and flexibility of the UDP-Gal-binding regions are in agreement with the allosteric relationship between them Biological significance It is pertinent to ask why the native epimerase is the only isolable form of the enzyme from yeast cells harvested near termination of growth The content of inhibitor-free epimerase is gradually reduced with the concomitant... 6737 Regulation of catalytic sites of yeast epimerase A Brahma et al Scheme 3 Distinction between coenzyme fluorescence and NADH fluorescence of epimerase The underlining indicates the enzyme surface Binding of NAD+ ⁄ NADH to the enzyme is noncovalent The hatched sign indicates weak spatial interaction between NAD+ and a cysteine of the enzyme, leading to NADH-like coenzyme fluorescence The subscript ‘free’... presence of interfering enzymes, could alter the enzyme kinetics [28] The monophasic Michaelis–Menten relationship of the monomeric epimerase demonstrated the absence of such artefacts and the requirement for a dimeric structure to explain the regulation The difference in Vmax values among different forms of the epimerase is a reflection of change of conformation (Table 1) [29,30] The functional distinction... 4–9 ) are the average of three sets, where the variation of results was within ± 10% Under the stated assay conditions, only the native epimerase (E1) showed an initial lag in catalysis The duration of lag (in seconds) was calculated from the time axis by extrapolating the linear portion of the progress curve of the coupled assay The initial rate could be defined when progress to 30 s was obtained from. .. 672 5–6 740 ª 2009 Council of Scientific and Industrial Research, New Delhi Journal compilation ª 2009 FEBS A Brahma et al remove reagents and small peptides The recovery of monomeric epimerase from the spin column was 95% in terms of activity and 60% in terms of mass calculated on the basis of the dimeric enzyme [11] Models of inhibitor-free and monomeric epimerase are shown in Scheme 2 Preparation of. .. at the active site of UDP-glucose 4-epimerase from Kluyveromyces fragilis J Biol Chem 270, 1138 3– 11390 21 Roberts DV (1977) Regulatory Enzymes and their Kinetic Behavior In Enzyme Kinetics pp 16 8–2 27 Cambridge University Press, Cambridge 22 Mukherjee S & Bhaduri A (1992) An essential histidine residue for the activity of UDP-glucose 4-epimerase from Kluyveromyces fragilis J Biol Chem 267, 1170 9– 11713... Completion of the reaction was indicated by complete inactivation of the enzyme The reduced cofactor was dissociated from the enzyme after incubation with 8 m urea at pH 7.5 for 10 min The concentration of the reduced cofactor was determined from the fluorescence intensity with respect to a 6738 Trypsin digestion Digestion of epimerase by trypsin was performed essentially as described for preparation of monomeric . UDP-galactose 4-epimerase from Kluyveromyces fragilis – catalytic sites of the homodimeric enzyme are functional and regulated Amrita. This indicates that the catalytic sites of the enzyme are functional and are distinguishable on the basis of bind- ing with the inhibitor. Careful analysis of Fig.