THE JOURNAL cm BIOLCXXCAL CHEMISTRY Vol. 248, No. 1, Issue of January 10, pp. 33-40, 1973

Printed in U.S.A.

Studies on the Mechanism of Action of Uridine

Diphosphate Galactose 4-Epimerase

II. SUBSTRATE-DEPENDENT REDUCTION BY SODIUM BOROHYDRIDE*

(Received for publication, June 14, 1972)

TIMPLE G. WEE$ AND PERRY A. FREY$

Frowz the Departnaent of Chenzistry, The Ohio State University, Columbus, Ohio 432~0

SUMMARY

Esherichia coti UDP-galactose 4-epimerase is inactivated by NaBH4 in a substrate-dependent reaction. Experiments with NaEFH4 show that this reaction produces enzyme- bound tritiated DPNH but little or no tritiated nucleotide sugars, which could be expected if a nucleotide ketosugar intermediate had been reduced.

Upon prolonged incubation of this enzyme with substrates the catalytic activity decreases and the amount of enzyme- bound DPNH increases, both at the same rate. This process approaches equilibrium in about 2 hours in the presence of 3.5 X 10e3 M UDP-glucose + UDP-galactose at pH 8.5 and 27”. Under these conditions the catalytic activity has decreased by about 6’7% and the system exhibits the following two properties not characteristic of native enzyme. Reduction with NaB3H4 produces tritiated UDP- glucose and UDP-galactose, and intermolecular hydrogen transfer between substrates can be detected. The data establish that under these special conditions a detectable part of substrate epimerization proceeds via free UDP-4-keto- sugar intermediates.

The interconversion of UDP-galactose and UDP-glucose, an essential step in the metabolism of galactose in many organisms, is catalyzed by uridinc diphosphate galactose 4-epimerase (EC 5, I. 3.2). The enzyme has been highly purified from Escl~erichia coli (2) and Saccharomyces fragilis (3) and found to contain 1 mole of tightly bound DPN+ per mole of enzyme. DPN+ bound to the E. coli enzyme is partially reduced in the presence of substrates (2). This fact and a number of other experimental data support a hydrogen transfer pathway proposed by Maxwell (4) in which the enzyme .DPN+ complex oxidizes the substrate- sugar moiety at carbon 4 to produce enzyme . DPNH complex and an enzyme-bound TJDP-4-ketoglucose intermediate. The

* This work was supported by United Stat.es Public Health Service Grant AM 13502 from the National Institute of Arthritis and Metabolic Diseases. The preceding paper of this series is Reference 1.

$ Phillips Petroleum Fellow 1971 to 1972. g To whom correspondence should be addressed.

intermediate is then reduced either to UDP-glucose or to UDP- galactose. This reaction pathway is supported by several other lines of experimental evidence (5-8) and is not contradicted by any experiment. The most convincing evidence is that UDP- 4-keto-6-deoxyglucose can reoxidize and reactivate enzyme. DPNH complex and that UDPI-ketoglucose can be trapped by chemical reduction with NaB3H4 (9, 10).

An unusual and intriguing property of this hydrogen transfer pathway is that hydrogen transfer from DPNH to UDP-4- ketoglucose is nonstereospecific. Inasmuch as ordinary pyridine nucleotide dehydrogenases catalyze stereospecific hydrogen transfers (II), it is important to exclude other possible reaction pathways or to prove this one without ambiguity. A pathway involving stereospecific reversible oxidation at carbon 3 of the sugar to produce a 3-ketosugar intermediate which epimerizes at carbon 4 by an enolization mechanism has been informally entertained and has received some experimental support (12).

The present studies were begun with the intention of obtain- ing more direct evidence concerning the hydrogen transfer path- way. Our results support the pathway proposed by Maxwell and resolve ambiguities cited earlier (1, 12).

MATERIALS AND METHODS

Enzymes-We purified UDP-galactose 4-epimerase from E. coli cells according to the procedure described by Wilson and Hogness (2). The specific activities of our best preparations were 13,000 units per mg of protein which is equivalent to the best activities obtained by Wilson and Hogness and which they estimated to correspond to a purity of better than 90% (2). We routinely obtained specific activities in the range 10,500 to 12,000 units per mg of protein. When stored for several months at 4” the specific activities of some preparations decreased to as low as 8,000 units per mg of protein, but others retained activity.

Wilson and Hogness purified this enzyme from an inducible strain, E. coli K-12 gal+ (Xdg), induced by D-fUCOSe in the growth medium. We purified the enzyme from a different strain, Buttin’s strain, which was kindly supplied to us by G. Ordal and D. S. Hogness. This strain is operator constitutive for the galactose operon, and, when grown to stationary phase without induction, it produces specific activities in crude ex- tracts equivalent to or in excess of those reported by Wilson and Hogness for the inducible strain. We grew these cells with vigorous aeration in 20-liter carboys to the stationary phase in

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the medium described by Wilson and Hogness (Z), except that we omitted n-fucose and increased the glycerol concentration to 0.22 Y.

UDP-[3-3H]glucose were prepared enzymatically from [4-3H]- or [3-3H]glucose. The method is outlined here for ‘UDP-[4-3H]- glucose.

Enzymes obtained from commercial sources: UDP-glucose dehydrogennse, UI)P-glucose pyrophosphorylase, lactic dchy- drogenase, hexokinasc, and inorganic pyrophosphatase from Sigma; glutamic dehydrogenase and phosphoglucomutase from Calbiochem.

Radiochenlicals-Sal33HI was a product of Amersham-Searle. UDP-[i”C]glucose ([l~-14C]glucose) was obtained from New England Nuclear a.nd purified by paper chromatography with Solvent A. After elutiou from the paper it was diluted with carrier UDP-glucose to the desired specific activity. [4-3H]- Glucose and &H]glucose were obtained from Amersham-Searle and purified by paper chromatography in Solvent A. After elution from the papers the sugars were diluted with glucose to the desired specific act’ivities.

Coenzymes and S&strates-The following materials were pur- chased from commercial sources: DPN+, DPNH, UTP, and glucose 1,6-diphosphate from Roehringer Mannheim; IJDP- glucose, UDP-galactose, and UDP-xylose from Calbiochem.

Assays-We assayed UDP-galactose ii-epimerase by the coupled assay procedure described by Wilson and Hogness (Z), except that we used Ricinc buffer (Calbiochem; N, N-bis(2-hy- droxyethyl)glycine) instead of glycine. One unit of activity, as defined by these authors, is the amount of enzyme which pro- duces 1 pmole of liDI’-glucose in 1 hour under the conditions of the assay, which are: 0.10 M sodium bicinate buffer at pH 8.5, 1.25 X lop3 M DYS+, 2.5 X 10e4 M UDP-galactose, 440 units of UDP-glucose dchydrogenase, and less than 0.2 unit of UDP- galactose 4-epimem~e in a total volume of 0.50 ml at 27”. The increase in absorbance at 340 nm was continuously monitored in a Norelco-Unicnm SP800 double beam spectrophotometer equipped with a scale espansion accessory and external recorder.

The concentrat,ions of UDP-glucose a.nd UDP-galactose solu- tions were measured spectrophotometrically with the extinction coefficients 9.8 X IO3 itf-’ cm-l and 9.6 X lo3 h1-l cm+ deter- mined by Wilson and Hogness (2). ‘UDP-glucose and UDP- galactose elutcd from paper chromatograms were assayed en- zymatically with I’DP-galactose 4-epimerase and IJDP-glucose dehydrogenase.

The reaction mixture contained initially 8.3 pmoles of [4-aH]- glucose (5.0 X lo6 cpm per pmole), 17.8 pnioles of UTP, 0.02 pmole of glucose 1,6-diphosphnte, 23 units of hexokinase, 22 units of phosphoglucomutase, 10 unit,s of inorganic pyrophos- phatase, 15 units of UDP-glucose pyrophosphorylase, 6 pmoles of cysteine, and 25 pmoles of MgC12 in a total volume of 2.14 ml of 0.01 M Tris-HCl buffer at pH 7.5. After 2 hours at 27” en- zymatic assa.ys indicated that 8 pmoles of TJDP-glucose had been produced and the reaction was terminated by heating at 100” for 1 min. The solution was filtered, concentrated to 1 ml, and passed through a column, 1.5 x 42 cm, of Rio-Gel P-2 equil- ibrated and eluted with 0.001 M KkHP04. Fractions containing UDP-glucose were pooled, concent.rated by rotary evaporation, and UDP-glucose was further purified by paper chromatography in Solvent A. After elution and rechromatography in the same system we obtained 6 pmoles of UDP-[4-3H]glucose of specific activity 4.3 X lo6 cpm per wmole.

Paper Chromatography-Paper chromatography was per- formed with water-washed Schleicher and Schuell 2043-B filter paper and the following solvents: Solvent A, ethanol-l nf am- monium acetate, pH 7, 7:3; Solvent B, pyridine-ethyl acetate- water, 5: 12:4; and Solvent C, 88% phenol in water. Nucleo- tides were located under an ultraviolet lamp and sugars were located between authentic markers which were visualized with alkaline AgN03.

Substrate-dependent Reduction of UDP-galactose /t-Epimerase by NaB&-The data given in Fig. 1 illustrate the effect of UDP-glucose on the rate at which NaBHc inactivates UDP- galactose 4-epimerase. Under the conditions of this experi- ment the catalytic activity is more than 50% destroyed within 2 min in the presence of 1.6 X 10m4 M NaBH4 and 3.8 X low4 M

UDP-glucose but is only slightly affected by NaUH4 or UDP- glucose alone. The effect of UDP-glucose in this experiment could be interpreted to mean that an enzyme.DPNH UDP- kctoglucose intermediate exists and that it is rapidly reduced by

During purifica,tion of the enzyme total protein was measured by the Warburg and Christian method in the early stages (13). In the later stages of purification the optical density at 280 nm was divided by 1.05 to obtain the protein concentration in milli- grams per ml as described by Wilson and Hogness (2). For ob- taining the difference spectrum betweennative and reduced UDP- galactose 4-epimerase the protein concentmtions were measured by t.he Lowry method (14) and adjusted to equivalence.

Radiochemical assays were performed by liquid scintillation counting in a Packard model 3310 Tri-Carb liquid scintillation spectrometer. The scintillation solvent. contained i g of 2,5- diphenyloxazolc, 300 mg of p-bis[Z-(5-phenyloxazolyl)]benzene, and 100 g of nnphtha.lene per liter of diosane solution. In each assay 1.0 ml of aqueous sample was combined with 15 ml of scintillation solvent. Radioactive areas on paper chromatograms were detected with a Nuclear Chicago strip scanner. In the intermolecular hydrogen transfer experiments the paper chromat- ogrnms were cut into l-cm strips. The strips were individually soaked overnight in 1 ml of water inside liquid scintillation vials. The vials were counted the following day after adding 15 ml of scint,illation solvent to each one.

0 0

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0 2 4 6 fil Ib 12 TIME (min.)

Prenaration of Tritiated Substrates-UDP-14-3H1&cose and

FIG. 1. Subst.rate-dependent inactivation of UDP-galactose 4- epimerase by NaBH+ The complete reaction mixture contained initially 5500 units per ml of UDP-galactose &epimerase, 1.6 X IO-4 M freshly prepared NaBH4, 3.8 X IO+ M UI>P-glucose, and 0.125 M Na+ bicinate buffer, pH 8.5. The solutions were placed at 27” and 10.~1 samples were withdrawn at the indicated times and diluted to 1.60 ml in ice-cold 0.02 M KzHPOa for activity as- says. O---O, complete ; 0 ---0 , minus UDP-glucose;

‘ . L 10 O---O, minus NaBHa.

RESULTS

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4.0 5

1.0 t

/ d ~~AeA&.q$A j : 0 5 IO 15 20 25 30

FRACTION NUMBER

FIG. 2. Bio-Gel P-2 chromatography of UDP-galactose i-epi- merase reduced by NaB31Ta in the presence of UI)P-glucose. A solution containing 5.6 mg of enzyme (8300 units per mg) and 2.2 pmoles of UDP-ghlcose in a total volume of 0.7-2 ml at, 25” was reduced with 0.02 ml of 0.013 M NaB3Hd (140 mci per mmole). After 10 min 7G2 Imits of activity remained and the solution was passed into a column (1 X G2 cm) of Bio-Gel P-2, 100 to 200 mesh, and elut.ed wit.h 0.001 M K,HPO,. Fract.ions of 1.1 ml were col- lected at. 5-min intervals. Cont.rol experiments showed t.hat the position of UDP-glllcose corresponds to Fractions 18 to 22. O--O, Asso (protein); O--O, Also (UDP-hexoses); A- --A, radioactivity.

Nal<Hd. Alternatively UDP-glucose could be acting to induce a change in the conformation of the enzyme which has the effect of rendering enzyme-bound DPN+ more susceptible to reduction by NaUH4. Implicit in both of the above is the assumption that catalytic activity is lost because enzyme.DPN+ complex becomes reduced to enzyme .Dl’NH complcs, an ineffective catalyst. This we verified in an experiment in which we measured the dif- ference spectrum between native enzyme and the same concen- tration of enzyme which had been treated with NaRH* in t.he presence of UDP-glucose and isolated by gel filtration on a col- umn of Sephadex G-25. We observed an absorption band cen- tered in the 340- to 345.nm range in the sample inactivated by NaBH4, which is consistent with the presence of bound DPNH.

Experiments utilizing NaB3H4 resolve the question of whether enzyme.DPNH arises from the chemical trapping of a keto- glucose intermediate or esclusively by direct NaRH4 reduction of enzyme 1 DPN+. Fig. 2 shows that treatment of enzyme. DPN+ with NaB3H4 in the presence of UDP-glucose incorporates large amounts of tritium into the protein and little or none into nucleotide sugars. In Fig. 2, UDP-galactose 4-epimerase was reduced with NaB3H., in the presence of UDP-glucose and passed through a column of Bio-Gel P-2 which had been shown to sep- arate the enzyme from nucleotide sugars and nucleotide sugars from small molecular weight molecules. The protein contained 6 X IO5 cpm of radioactivity, whcrcas the fractions correspond- ing to the position of nueleotide sugars contained only traces of radioactivity. In other esperimcnts we isolated IJDP-galactose and UDP-glucose by paper chromatography from similar re- action mixtures and failed to detect significant levels of radio- activity. From the results of these experiments and of our earlier experiments (1) we conclude that the substrate-dependent inactivation by Nal%H( in Fig. 1 does not involve reduction of an enzyme-bound UDP-ketoglucose intermediate.

The experiments of Nelsestuen and Kirkwood establish t,hat reduced tritiated epimerase prepared under conditions com- parable to those of Fig. 2 contains tritiated DPNH and that at least 76% of the radioactivity in tritiated DPNH corresponds

to [4-P-3H]DPNH (9). We have verified that virtually all of the radioactivity in reduced tritiated epimerase is [4-/3-3H]DPNH. We dissociated the reduced tritiated complex, obtained under conditions similar to those of Fig. 2, by heating at 100” for 5 min at pH 9 in the presence of carrier DPNH as described by Nelsestuen and Kirkwood (9). We then separated the carrier DPNH from denatured protein on a column of Sephadex G-25. We found that 97.5% of the total radioact,ivity was eluted with DPNH and only 2.50/, with the protein. Samples of this triti- ated DPNH were used in hydrogen t,ransfer experiments to determine the stereochemistry of labeling. We used the pro- cedure of Levy and Vennesland (15) to oxidize a sample with ol-ketoglutarate in the presence of NHk+ and glutamic dehy- drogenase. After recrystallization to constant specific activity the glutamic acid contained 90% of the radioactivity expected if all of the radioactivity had originally been in the form of [4-/3-3H]DPNH. In the second experiment we used another sample of tritiated DPNH to reduce pyruvate in the presence of lactic dehydrogenase, a reaction in which hydrogen in the 4-a position of DPNH undergoes transfer (16). After adding carrier lactic acid we prepared the phenacyl ester of lactic scid (17) and recrystallized it to consta,nt specific a,ctivity. It con- tained 1.470 of the theoretical amount if all of the radioact.ivity had originally been present as [4-ol-3H]I)I’MI.

Bs first reported by Nelsestuen and Kirkwood the reduction by NaBHe is highly stereoselective for the @ side of carbon 4 in enzyme-bound DPN+. It may be stereospecific, for we are un- able to decide whether the small amount of radioactivity de- tected in the 4-a position corresponds to some slight nonspeci- ficity or to nonenzymatic nonstereospecific hydrogen transfer between [4-/-3H]DPNH and adventitious traces of DPN+ such as that reported by Ludowieg and Levy (18).

Slow Bppearance of CiDP-4-ketoglucose-.hIaitra and Ankel found, and we confirmed, that tritiat,ed IIDP-glucose and I;DP- galactose are produced when UDP-galactose 4-epimerase is placed in the presence of substrates for :1 prolonged period of time and then treated with NaB3H4. 1\Init.ra and Ankel showed that the nucleotide sugars are labeled at carbon 4, and we showed that the preliminary incubation is required and is accompanied by a substantial loss of catalytic activity (1, 10).

In a typical experiment we combined 5 mg of enzyme (11,000 units per mg of protein) with 4.3 pmoles of UDP-glucose in a total volume of 1.2 ml of 0.17 M sodium bjcinate buffer at pH 8.5 and 27”. After 145 min the specific activity had decreased to 5200 units per mg of protein. At this point we added 25 ~1 of 0.083 M NaB3H1 (200 mCi per mmole) which reduced the catalytic activity to 184 units per mg of protein within 10 min. Protein was separated from nucleotide sugars by gel filtration over a column, 1 X 40 cm, of Sephadex G-25 equilibrated and eluted with 0.003 M KzHP04. The protein and UDP-hexose bands were both radioactive. The protein contained 2.5 X lo5 cpm per mg of protein and, upon resolution in the presence of carrier DPNH as described above, all of the radioactivity was found to be associated with DPNH. Fractions containing UDP-glucose and UDP-galactose were pooled and further puri- fied by paper chromatography with Solvent A. After elution from the paper chromatograms the UDP-hesoses contained 2.6 X lo5 cpm per pmole.

Hydrogen Transfer From UDP-glucose to Enryrne-bound DPN+-The delayed trapping experiments clearly show that UDP+ketoglucose slowly appears during prolonged incubation of enzyme with substrates (1, 10). The required preliminary incubation period is, however, much longer than the time re-

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quired for complete equilibration of UDP-glucose with UDP- galactose. It is accompanied by a substantial loss of enzyme activity, which suggests that the appearance of UDP-4-keto- glucose may be associated with the formation of some abortive complex (1).

The question arises whether this slow appearance of UDP-4- ketoglucose is the result of direct hydrogen transfer from carbon 4 of t,he glucosyl moiety to enzyme-bound DPN+. A reasonable alternative pathway could involve hydrogen transfer from car- bon 3 followed by slow isomerization of UDP-3-ketoglucose t,o UDP-4-ketoglucose via an enediol intermediate. Nelsestuen and Kirkwood detected direct transfer of tritium from UDP- [4-aH]quinovose to the 6 side of enzyme-bound DPNH when it was permitted to react with this enzyme for 24 hours (9). This is consistent with the former pathway. However, inasmuch as the product,ion of UDP-4-ketoglucose appears to be an abortive reaction, we decided to establish whether the hydrogen transfer is specific, that is whether tritium would also be transferred to DPNH by UDP-[3-3H]glucose.

To evaluate this possibility we performed a delayed trapping experiment with UDP-[3-3H]glucose as the substrate and un- labeled NaBH4 as the trapping reagent. DPNH was then re- solved from the protein and subjected to radiochemical analysis. In Fig. 3 a solution of enzyme and UDP-[3-aH]glucose (1.8 X lo6 cpm per pmole) was subjected to delayed trapping with NaBH4 and then isolated from excess radioactive UDP-hexoses by gel filtration. We found 1.8 X 10’ cpm per mg of protein in the protein peak, or 2.0 x lo6 cpm per ,umole assuming a molecular weight of 79,000 and specific catalytic activity of 13,000 units per mg of protein for the pure enzyme (2). In Fig. 4 we resolved the complex of Fig. 3 by heating it with carrier

30 40 50 FRACTION NUMBER

FIG. 3. Reduction of UDP-gnlactose 4-epirnerase with NaBHa in thcx presence of U1)P-[3-3Tl]glucose. The reaction mixt,ure contained initially 5 mg of enzyme (9310 units per mg), 2.88 pmole of UDP-[3-8H]glucose (1.8 X 100 cpm per pmole), and 7.5 pmoles of K&P04 in a total volume of 1.35 ml. After 2 hours at 25’ the activity of the enzyme had decreased to GO00 units per mg. The solution was then reduced with 25 ~1 of 0.03 M NaBHa; after an additional 10 min the activity of the enzyme was 500 units per mg. The solution was passed into a column (1.5 X 42 cm) of Sephadex G-25 and eluted with 0.003 M KJIPOd at 4”. Fractions of 1.5 ml were collected at, 5-min intervals. O-0, Azso (protein); U--U, A?63 (UDP-hexose) ; A- - -A., radioac- tivity.

DPNH. Upon passage through a column of Sephadex G-25, which separates DPNH from protein and nucleotide sugars, most of the radioactivity was associated wit’h fractions contain- ing UDP-hexoses and none could be detected in fractions con- taining DPNH. That Fractions 38 through 46 of Fig. 4 con- tained UDP-glucose and UDP-galactose was verified in control experiments. In addition, these fractions were pooled, concen- trated, and shown by enzymatic assays to contain both UDP- glucose and UDP-galactose. Therefore the major detectable radioactivity found in the protein of Fig. 3 was associated with UDP-hexoses, which Nelsestuen and Kirkwood showed are tightly bound by the enzyme .DPNH complex (9). I f a sig- nificant amount of radioactivity had been associated with DPNH we would have detected it in Fig. 4, because in Fig. 5 we repeated the experiment of Figs. 3 and 4 with UDP-[4-%]glucose as the substrate under otherwise identical conditions. We easily detected radioactivity in both UDP-hexoses and DPNH. There- fore transfer of hydrogen to produce UDP-4-ketoglucose is spe- cific for transfer from carbon 4 of the glycosyl moiety of sub- strates to enzyme-bound pyridine nucleotide.

Exckange of EnxymeeDPNH - UDP-[14C]hexose with Unlabeled UDP-glucose-In order to understand the chemical trapping experiments it is necessary to know whether UDP-4-ketoglucose, produced during prolonged reaction of enzyme with substrates and detected by delayed trapping with NaB3H4 is present in an enzyme-bound form or free in solution. As shown here and in earlier work (1) tritiated UDP-hexoses produced in delayed trapping experiments with NaB3H4 are not enzyme-bound. That is, when the tritiated complexes produced in such experi- ments are separated from substrates by gel filtration, the protein contains tritiated DPNH, and tritiated UDP-hexoses are found only in fractions containing substrates. Inasmuch as it is known that enzyme-DPNH complex binds UDP-hexoses very tightly (9), it appears that UDP-4-ketoglucose is not enzyme-bound under the conditions of the delayed trapping experiments. How- ever, in such experiments it ma.y also be that UDP-4-ketoglucose is reduced by NaB3H4 while bound to the enzyme .DPNH com-

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FRACTION NUMBER

FIG. 4. Dissociat,ion of DPNH and UDP-[3-JH]glucose from the protein of Fig. 3. Fractions 22 and 23 from Fig. 3 were pooled. After adding G @moles of carrier DPNH the solu-

Con was heat,ed at 100” for 5 min. The solution was chilled, passed into a column (1.5 X 42 cm) of Sephadex G-25, and eluted with 0.003 M &HP04 at 4”. Fractions of approximately 1 ml were collected at 5-min intervals. O--O, Azso (protein); n---m, A 340 (DPNH) ; A- - -A, radioactivity.

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zu 30 40 50 60 FRACTION NUMBER

FIG. 5. Dissociation of [3H]DPNH and UDP-[4-3H]glucose from reduced t,ritiated complex. Reduced enzyme.DPNH complex was prepared as described in the legend to Fig. 3 except that UDP-[4-3H]glucose was substituted for UDP-[3-3H]glucose. Fractions containing the radioact.ive protein complex were pooled wit,h ci pmoles of carrier DPNII, heated at 100” for 5 min, passed into a column (1.5 X 42 cm) of Sephadex G-25, and eluted with 0.003 M K2HP04 at 4”. Fractions of 1.4 ml volume were collected at 5.min intervals. 0-0, ASO (protein); U----O, Aaao (DPNH); A- --A, radionct.ivity.

plex but, subsequent to being reduced, it exchanges with free substrate molecules which are not radioactive. That substrate molecules tightly bound to enzyme .DPNH complex can ex- change with free substrate molecules is established in the follow- ing experiment.

We prepared a.11 enzyme .I)PNH .UDP-[14C]hexose complex by combining 5.3 mg of enzyme with 3.2 pmoles of UDP-[%]glucose (1.15 X lo6 cpm per pmolc) in a total volume of 1.35 ml of 0.074 M sodium bicinate buffer at pH 8.5 and 27”. TJpon reducing the solution with 25 ~1 of 0.03 M N&H4 the catalytic activity de- creased from 9,600 to 250 units per mg of protein. After the protein was isolated from radioactive substrates by gel filtration over Sephadex G-25 it contained 1.2 X 10” cpm per pmole and the catalytic activity was 630 units per mg of protein. The specific radioactivity content was calculated assuming a molecu- lar weight of 79,CO0 and catalytic activity of 13,000 units per mg of protein for pure enzyme (2); it corresponded to approxi- mately 1 mole of 14C per molt of enzyme. In Fig. 6, Part A, a sample of this protein was placed at 27” for 15 min and then again subjected to Sephadcs G-25 chromatography. Nearly all of the radioactivity rem&cd bound to t,he protein. In Fig. 6, Pad B, another sample was combined with 2.3 X lop3 M UDP-glucose for 15 min at 27” aud then rcchromatographed over Sephadex G-25. Under these conditions 90% of the radioactivity was eluted with UDP-glucose and only 10% with protein.

We conclude that free substrate molecules can readily es- change with substrate molecules bound to enzyme .l)PNH com- plex. This esperiment lcavcs open the possibi1it.y that UDP-4- ketoglucose may be enzyme~bound uuder the conditions of de- layed trapping with Na13aII+ however, in the following experi- ments we show it is a free intermediate.

Properties of Abortive Complex---As noted earlier, the delayed chemical t,rapping experiments suggest that prolonged interaction of substrates with UDP-galactosc 4-epimerase may produce an abortive coml~les associated with the slow appearance of UDP- 4-ketoglucose in a form susceptible to reduction by NaBH& (1).

..A---- 4%~ ; --- 0

35 40 FRACTIOk NUMBER

I

FIG. 6. Exchange of UDP-glucose with UDP-[14C]hexose tightly bound to the enzyme.DPNH complex. A, a sample of enzyme.DPNHGUDP-[l%]hexose complex (preparation described in the text,), consisting of 1.8 mg of protein in 1.8 ml of 0.003 M KzHPOI, was placed at 27” for 15 min, passed into a column (1.5 X 42 cm) of Sephadex G-25, and eluted with 0.003 M KnHP04 at 4”. Fractions of 1.4 ml volume were collected at 5-min intervals. B, a 2.7-mg sample of the above complex was combined with 5.1 &ole of -UDP-glucose in a total volume of 2.2 ml of 0.003 M KgHP04 and nlaced at 27” for 15 min. It was then chilled and subjected to gel-filtration as in A. Fractions of 0.8 ml vol- ume were collected at 5-min intervals. O---O, A~80 (protein); U--U, A263 X 0.2 (UDP-hexoses); A---A, radioact,ivity.

Nelsestuen and Kirkwood recently detected such a complex and studied some of its properties (9). They found that it appears slowly in the presence of subst,rate, that it contains DPNH in excess of that attributable to the catalytic intermediate described by Wilson and Hogness (2), that it binds substrate molecules very tightly, and that its appearance is associated with reduced catalytic activity. We have sought to define more clearly the nature of the abortive complex and the possible relationship between its appearance and the nppearaucc of Ul)P-4-keto- glucose in a form reducible by NaEH4. We obtained the data given in Fig. 7. In Part A we combined UDP-galactose 4- epimerase w&h 3.5 X lop3 M UJ>P-glucose at $1 8.5 and 27” and measured it,s catalytic activit,y and A345 over a period of 10 hours. Data for only the first 6 hours are given in Fig. 7. We observed a biphasic increase in Az45 and a corresponding biphasio decrease in specific cat,alytic activity. In the first phase, which was very much faster than the second, the catalytic activity decreased to a nearly constant value and As45 increased to a nearly constant value. The initial phase, which appeared to approach equilibrium, was followed by a second phase in which

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the activity gradually decreased and As45 gradually increased over a period of 10 hours. The second phase was so slow that several days would have been required for it to reach equilibrium or proceed to completion (i.e. fully reduced inactive enzyme). Similar data were obtained in Fig. 7, Part B, with UDP-xylose,

a known substrate (19). The observed changes were more ex- tensive with UDP-xylose than with UDP-glucose.

We are here concerned primarily with the first phase depicted in Fig. 7. First, we note that the activity does not closely ap- proach zero and the increase in As45 is smaller tha.n that expected if all the enzyme-bound pyridine nucleotide were present as

DPNH (theoretical maximum AZ.,5 is 0.12 in Part A). Second, when analyzed by the burst kinetics method the data of Fig. 7,

Part A, show that the observed first order rate constant for loss in catalytic activity is 0.038 mill-l and the corresponding value for increase in A345 is 0.033 min?. Given the quality of the data these values are iu reasonable agreement and suggest a quantitative correltion between activity loss and appearance of

DPNH in excess of that present at zero time. DPNH present at zero time is believed to be a constituent of a catalytic inter- mediate (2).

There are several models which might be advanced to account for the first phase reaction in Fig. 7. There is one which, be-

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- ,,

-(

FIG. 7. Efl’ect of sltbst,rates on catalytic activity and A345 of UDP-galactosc 4-epimerase. Enzyme was incubated with UDP- xylose or UDP-glucose and 0.1 M sodium bicinat.c buffer at pH 8.5 and 27”. At the indicated t.imes the residual catalytic activ- ity and Aan5 were measured. Absorbance measurements were corrected for the absorbance of the enzyme in the absence of substrat,es. Dat,a were collected for 10 hours in Part A and 20 hours in Part R but only 6 hours are given here. A, enzyme (2.2 mg per ml) plus 3.5 X 10-S M UDP-glucose. B, enzyme (1.0 mg per ml) plus 1.95 X 10-s M UDP-xylose. O-0, A34:. O--O, catalytic activity.

cause of its simplicity and experimental verifiability, is particu- larly attractive. The model assumes UDP-4-ketoglucose is an enzyme-bound intermediate in the normal catalytic pathway. It can, however, be replaced slowly in the enzyme .DPSH com- plex by substrate molecules in a reaction similar to the exchange reaction depicted in Fig. 6. This produces free VDP-4-keto- glucose and an abortive complex which contains DPNH and 1 molecule of UDP-hexose. Since the a.ctivity does not approach zero and enzyme-bound pyridine nucleotide is not fully reduced,

I -

1 I XYESE ’ A

DISTANCE FROM ORIGIN ( cm 1 I I I

1500 S

DISTANCE FROM ORIGIN ~crn) 0

FIG. 8. Intermolecular hydrogen transfer catalyzed by UDP- galactose 4-epimerase. The react.ion mixture cont,ained initially 11 mg of enzyme (9.F X lo3 units per mg of prot,ein), 4.0 pmoles of UT>P-[4-3H]glucose (1.0 X lo5 cpm per rmole), 4.5 pmoles of UIIP-xylose, and 300 rmoles of sodium birinate bluffer, pH 8.5, in a volume of 2.6 ml and at. 27”. At zero time, after 10 hours, and after 20 hours 0.8-ml aliquots were heated to boiling and then centrifuged. Supernatant fluids wcrc brought to 0.1 M HCl and heated at 100’ for 15 min. The solutions were deionized with mixed bed ion exchangers (Dowes I-CO:j- and Dowex 50- H+), filtered, and the sugars purified by paper chromatography in Solvent C which separates xylose and galaclose from glucose plus arabinose. Areas containing xylose and glucose plus arab- inose were separat.ely eluted and rechromatographed in Solvent I3. Chromatograms were cut into strips and subjected to radio- chemical analysis. Part A gives data on xylose and Part B gives data on glucose plus arabinose after rechromatography in Sol- vent B. The open bars are data. obtained at. 20 hours, shaded bars at 10 hours, and cross-hatched bars at zero time. The activ- it.y of t,he enzyme did not fall below 3470 units per mg of pro- tein during the 20-hour period.

-I

-I

Ll

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39

Es UDPgol

UDP-hexose

E-HeUDP-4- ketoglucose

= UDP- hexose

E’UDPglu

m

the post,ulated exchange is detectably reversible. That is, free UDP-4-kctoglucose can replace substrate bound to the abortive .complex by reverse exchange. This model is subject to direct esperimentul verification as described below.

Free I’DP-4-ketosugars and Intermolecular Hydrogen Transfer- ‘The model predicts that, under conditions in which a large amount of abortive complex is permitted to form and react re- versibly over a long period of time, intermolecular hydrogen transfer between substrate molecules will be observed. The data presented in Fig. 8 establish this fact. As shown in Fig. 8, when UDP-[4-aH]glucosr and UDP-xylose are combined with a large amount of enzyme and permitted to react for a prolonged period of time tritium can be detected in xylose and arabinose. ‘The appearance of tritium in pentoses is progressive, being essen- tially absent at zero time, easily detected after 10 hours, and approximately doubled after 20 hours. The amount of radio- activity in peutoses after 20 hours is about 5% of that present in hexoses.

As further verification of this model we have performed an- .other experiment designed to distinguish between this and an alternative model, the alternative being that the data of Figs. 7 and 8 would also have been obtained if DPNH instead of, or in .addition to, UDP-4-ketoglucose were able to dissociate reversibly. We combined 1.2 mg of UDP-galactose 4-epimerase with 2.5 pmoles of UDP-[4-3HJglucose (1.3 x 10” cpm per pmole) in a total volume of 0.8 ml of 0.11 M sodium bicinate at pH 8.5 and 27”. L&fter 2 hours the activity of the enzyme had decreased from 9 x lo3 to 3 x IO3 units per mg of protein. At this time 1 pmole of carrier DPNH was added and the solution was passed through a column, 1.5 x 42 cm, of Sephadex G-25 equilibrated and eluted with 0.003 M K&PO4 at 4”. Spectrophotometric and radiochemical assays showed that protein, UDP-hexoses, and DPNH were widely separated, that both the protein and UDP-hexoses were radioactive, and that fractions containing DPNH contained no detectable radioactivity. I f the data of Figs. 7 and 8 had resulted from reversible dissociation of DPNH, we would have found approximately 1 X 104 cpm associated with DPNH in this csperiment. We actually found 1.8 X lo4 cpm associated with the protein and none in DPNH.

Having excluded t,hc possibility that reversibly dissociating DPNH could account for our results, the model proposed in the foregoing section is confirmed.

We int,erpret those properties of UDP-galactose 4-epimerase pertinent to the present study in terms of the reaction scheme in Fig. 9. Complexes Z and ZZZ are Michaelis complexes and Complex ZZ is an intermediate on the normal catalytic pathway. Complex ZZ contains DPNH and UDP-4-ketoglucose but may exist in more than one form as discussed below. The normal course of epimerieation proceeds through these three complexes.

UDP-4-ketoglucose +

E-H, UDP-hexose

FIG. 9. l%eactions of UDP-galnctose 4-epimerase. In this scheme E refers to native enzyme. DPN+ and E-H refers to enzyme. DPNH.

Complex IV is the abortive complex and is produced when UDP- hexoses replace UDP-4-ketoglucose from Conlplex ZZ in a rela- tively slow exchange reaction analogous to that observed in Fig. 6. Its appearance is accompanied by the appearance of free UDP-4-ketoglucose. The interconversion of Conlplexes ZZ and IV is detectably reversible under our conditions and proceeds to equilibrium in the first phase shown in Fig. 7. Fig, 9 does not account for the secondary very slow reaction detected in Fig. 7. There are several ways in which the scheme could be extended to account for this, however, we have no evidence bearing on it. Whatever the explanation, it will not negate the essential features of Fig. 9.

Fig. 9 accounts for our results as follows: NaUH4 can reduce E .Dl’N+ or Complexes Z and ZZZ (9), but it either cannot reduce Complex ZZ or it reduces it much more slowly (1). In delayed trapping experiments Complex 1 V and free UDP-4-ketoglucose are also present; free UDP-4-ketoglucose can then also be re- duced by NaRH4 to UDP-glucose and UDP-galactose (1, 10). Intermolecular hydrogen transfer is accounted for by the fact that the conversion of Complex II to Complex 1V is reversible and produces free UDP-4-ketosugar intermediates which, upon reversal, do not necessarily return to the same enzyme molecules from which they were released. Under conditions where Com- plex IV has not accumulated, or is present in only minute con- centration, hydrogen transfer is exclusively intramolecular (9, 20). Under the special conditions of Fig. 8 it is partially inter- molecular.

According to Fig. 9 conversion of Complex ZZ to Complex IV is substrate-dependent. We verified this in unpublished experi- ments. ht one-tenth the concentration of UDP-glucose em- ployed in Fig. 7, Part A, the decrease in catalytic activity was much slower and less extensive than that in Fig. 7. Under such conditions the change was only marginally detectable.’

Substrate-dependent reductive inactivation by NaBH., in Fig. 1 suggests that Compkxes I and ZZZ are more rapidly reduced by NaBH4 than is enzyme.DPN+ itself. UMP- dependent inactivation by Nal3Hb has also been reported (21). The present data do not establish this fact, however, because Nelscstuen and Kirkwood showed that substrates protect the enzyme. I)PNH complex against autoxidation and reactivation (9), presumably by maintaining it in the form of Complex IV which is stable. Data from our laboratory are in agreement with this. The possibility exists that both factors may be important. For example, we have found recently in accurate rate measure- ments that enzyme .DPN+ cannot be reduced even transiently by high concentrations of NaBH&N, but this reagent easily and rapidly reduces it in the presence of UMP.’

Complex ZZ of Fig. 9 must exist in at least two forms if UDPI- ketoglucose is an intermediate. That is, UDP-4-ketoglucose

1 J. Davis and P. A. Frey, unpublished experiments.

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cannot be reduced to both UDP-glucose and UDP-galactose through the same transition &ate. The different forms for Complex II could involve different enzyme conformations, differ- ent conformations of UDP-4-ketoglucose, two perhaps closely related binding sites for UDP-4-ketoglucose, or some combina- tion of these. To our knowledge there is no experimental basis for deciding which of these possibilities, or which combination of them, is responsible for Complex 11 reacting by two kinetic pathways to produce either Complex I or Complex III. We note in this connection, however, that one of several ways to rationalize the exchange observed in Fig. 6 is on the basis of a two-site model.

We should like to comment further on the very tight binding of UDP-sugars by the enzpme.DPNH complex. Others have discussed the conformational differences between enzyme .DPN+ and enzyme.DPNH implied by this fact (9), and it has been reported that the optical rotatory dispersion and circular di- chroism of yeast UDP-galactose 4-epimerase change upon re- ducing prosthetic DPN+ to DPNH in the presence of UXP (22). The related enzyme deoxythymidine diphosphate glucose oxidoreductase also contains tightly bound DPNf which medi- ates hydrogen transfer (23, 24). Glaser et al. found that the reduced form of this enzyme binds deoxythymidine nucleotides very tightly and suggested that this phenomenon plays an im- portant role in catalysis, in that reaction intermediates are prevented from escaping from the active site during the catalytic process (25). We believe this is also a correct interpret,ation of the fact that reduced UDP-galactose 4-epimerase binds UDP- sugars very tightly.

The present work gives additional information about the tight binding of IJDP-sugars by enzyme.DPKH. Our data suggest that enzyme .DPNH complex binds UDP+ketoglucose more tightly than UDP-hesoses by a factor of about 100. This interpretation is based upon the data of Fig. 7, where it can be seen in Part A that, as equilibrium between Complexes II and IV is approached (2 to 3 hours), free UDP-4-ketoglucose at a concentration smaller than that of the enzyme, i.e. <2 x 10-j M, is able to compete effectively with 3.5 X 10-a M UDP-hexoses for binding to enzyme-DPNH complex. That it competes effectively follows from the fact that the enzyme appears to be partitioned into approximately 33% active forms and 67% Complex IV. This estimate is based upon activity data and the assumption that only Complex IV is inactive. This extra t,ight binding of the intermediate by enzyme-DPNH, as compared with the already very tight binding of substrates which are closely related in structure, suggests to us that the transition state for hydrogen transfer may bear a closer structural relation- ship to UDP-4-ketoglucose than to the substrates and that one of the important factors in the mechanism of catalysis may involve conformational distortion of the substrate toward the structure of the transition state for hydrogen transfer. The potentialities for this type of catalytic process have been pre- sented elsewhere (26, 27). Impressive evidence in support of this as a catalytic mechanism of general importance derives from studies of the interactions of transition state analogs with en- zymes. In these studies, which are appearing with increasing frequency, it is often found that compounds closely related in structure to known or suspected reaction intermediates are bound very much more tightly by the respective enzymes than are the corresponding substrates. They are, therefore, very potent inhibitors. A classical example is proline racemase which is strongly inhibited by pyrrole 2-carboxylate and thiophene 2- carboxylate, planar molecules which presumably resemble an

intermediate on the racemization pathway (28). Other examples are given in the review by Wolfenden (27). In the present work the properties of the enzyme and the intermediate permit us to compare the relative affinities of substrates and UDP-Cketo- glucose for the enzyme .DPNH complex without recourse to structural analogs. The dat,a are consistent with UDP-4-keto- glucose being more closely related to the structure (or structures) of the transition state (or states) than UDP-hexoses if the enzyme.DPNH complex binds transition state (or states) more tightly t,han substrates. This interpretation is limited by the apparent fact that the enzyme is itself undergoing conformational changes in the transition state (or states), that is in the process of interconversion between enzyme .DPN+ and enzyme .DPNH (9).

The reaction pattern of Fig. 9 appears to be the simplest way of accounting for our results. A more detailed or extended scheme will incorporate the essential features of Fig. 9.

Acknowledgments-We wish to acknowledge the expert techni- cal assistance of Miss Kyung-Ja Oh and Mr. Kevin Quiggle who assisted in the enzyme purifmation.

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Timple G. Wee and Perry A. FreyBOROHYDRIDE

4-Epimerase: II. SUBSTRATE-DEPENDENT REDUCTION BY SODIUM Studies on the Mechanism of Action of Uridine Diphosphate Galactose

1973, 248:33-40.J. Biol. Chem. 

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