Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 6-Phosphogluconate dehydratase from Zymomonas mobilis

ANALYTICAL

BIOCHEMISTRY

136,

530-534 (1984)

Use of Differential Dye-Ligand Chromatography with Affinity Elution for Enzyme Purification: 6-Phosphogluconate Dehydratase from Zymomonas mobilis R. K. SCOPES AND K. GRIFFITHS-SMITH Department

of Biochemistry,

LA Trobe

University,

Bundoora,

Victoria

3083, Australia

Received October 4, 1983 Using differential dye-ligand chromatography and affinity elution with a substrate analog, 6phosphogluconate dehydratase (EC 4.2.1.12) has been isolated from extracts of Zymomonus mobilis in a one-step procedure with 50% recovery. The specific activity of freshly isolated enzyme was 245 units mg-r. The enzyme contains iron, and it is rapidly inactivated in oxidizing conditions. It is inhibited by glycerophosphates, most strongly by the D-cY-isomerwhich structurally corresponds to half of the substrate molecule.

The preceding paper illustrated the effectiveness of using differential dye-ligand chromatography for isolating 2-keto-3-deoxy-6phosphogluconate (KDPG) aldolase from extracts of Zymomonas mobilis (1). The other enzyme specific to the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) has been less well characterized, mainly because of its lability. By using the rapid procedure of differential dye-ligand chromatography we have now succeeded in isolating this enzyme. It has previously been purified to a limited extent from Pseudomonas putida (2,3) and its mechanism of action investigated (3). The enzyme reaction is essentially irreversible, consequently it could represent a control point in metabolism; also, it has features of metal ion activation that resemble aconitase (4). Extracts of Z. mob&s contain 6-phosphogluconate dehydratase at a specificity activity somewhat greater than the best purified preparation from P. putida. Clearly the specific activity of pure enzyme must be considerably higher. Initial studies quickly showed that the enzyme bound to many dye-l&and columns, but on elution with salt any activity recovered decayed within minutes. It was not until buffer conditions were found in which the enzyme 0003-2697184 $3.00 Q 1984 by Academic Pm, Inc. All rights of reproduction in any form reSmwd.

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was more stable that a purification procedure could be developed. The present paper presents the results leading to the development of a stabilizing buffer system, and the use of that buffer in purifying the enzyme to near homogeneity using differential dye-ligand chromatography and affinity elution. A brief structural and kinetic characterization of the enzyme is also included. MATERIALS

AND METHODS

Z. mobilis, strains ATCC 10988 or CP4 (ZM4), was grown and extracted as described previously (l), except that the extraction buffer consisted of 20 mM K-Me%’ pH 6.5, containing 30 mM NaCl, 5 mM MnCl*, 0.5 mM freshly dissolved ammonium ferrous sulfate, and 10 mM @-mercaptoethanol. This buffer was also used for the chromatographic procedure. 6-Phosphogluconate dehydratase activity was measured in 20 mM K-Mes buffer, pH 6.5, containing 2 mM MgClz and 50 mM NaCl, using 1 mM 6-phosphogluconate, 0.15 mM NADH, and 5 units ml-’ each of lactate ’ Abbreviations used: Mes, 4-morpholineethanesutfonic acid; SDS, sodium dodecyl sulfate. 530

DIFFERENTIAL

DYE-LIGAND

dehydrogenase and 2-keto-3-deoxy-6-phosphogluconate aldolase (1). The reaction rate was measured at 340 nm with activity units being expressed in micromoles per minute at 25°C. Atomic absorption spectroscopy was carried out on a Varian AA-275 using the Fe line at 248.3 nm. Iron was also analyzed using a reagent containing 1% sodium dodecyl sulfate, 0.1 M ascorbic acid, and 2 mM qLy-dipyridyl. The pink color in the presence of ferrous ions was measured at 525 nm. Other procedures were as described in the previous paper ( 1). RESULTS

Enzyme Stability

CHROMATOGRAPHY TABLE

531 I

STABILIZATION OF6-P-GLUCONATE DEHYDRATASE ACTIVITY

Additive None 12 mM f3-Mercaptoethanol 10 mM Dithiothreitol 5 mM Ammonium ferrous sulfate 5 mM MnQ

5 mM

zn acetate

20% Glycerol 20% sucrose 10 mM K-gluconate 20 mM Na-ascorbate Iron-cysteine-ascorbate’ 2 mM EDTA 10 mM sodium sulfide

Dehydratase activity (units ml-‘) 6.6 5.1 5.9 3.3 15.5 5.1 16.0 3.6 13.4 10.8 2.2 4.2 1.1

Previous attempts to purify this enzyme have led to the conclusion that, like many Note. The extract initially had an activity of 29 units other dehydratases, 6-phosphogluconate deml-‘, additions were made as indicated. The samples were hydratase can be activated by ferrous ions in left at 4°C for 20 h before reassaying for activity. the presence of reducing agent (2). Our early a As used to reactivate aconitase (5). attempts to reactivate enzyme with ferrous ions were sometimes successful, but inclusion remains active for much longer in these conof iron in buffers during purification had little ditions. It was found that in this buffer, neither success in retaining active enzyme. A number glycerol nor gluconate could further improve of other possible protective agents were tried. the stabiIity, nor was operating at close to 0°C Table 1 illustrates some of the results obtained. beneficial compared to ambient temperature. Although activity was lost overnight in all cases, the loss was less in the presence of manDye-Screening ganous ions, glycerol, gluconate, or ascorbate. Because of the instability of the enzyme in Other metal ions (results not shown) had no protective effect. Following these results small the absence of ferrous and manganous ions, trials were made using a dye-ligand column the screening of dye-ligands for adsorption of known to bind the enzyme, Scarlet HE-3Gthe dehydratase using phosphate buffer, as deSepharose. The buffer used was 20 mM K- scribed for KDPG aldolase (1) was not successful; little activity was recovered. A screenphosphate with additives as indicated in Table 2. As a result of this experiment it was decided ing of a selection of dyes which do not bind to use manganese in all buffers, and to switch large amounts of protein, using the buffer deto Mes buffer at the same pH to avoid for- scribed above indicated several useful adsormation of manganous phosphate precipitates. bents (Table 3). The enzyme bound to many It was also found that in the presence of manof the adsorbents tested in this buffer (inganous ions, reactivation by ferrous ions was cluding Cibacron Blue F3G-A which is the much more successful. This led to the devel- same as Procion Blue H-B); we decided that opment of the buffer used for the purification the Blue HE-G was the most suitable, as it procedure described under Materials and binds the dehydratase strongly without binding Methods, which contains manganous and fer- very much protein compared with most other rous ions, plus a reducing agent. The enzyme dyes. As a “negative” adsorbent for the first

532

SCOPES AND GRIFPITHS-SMITH TABLE 2 TRIAL

SMALL-SCALE DIFFERENT

Buffer additives 5 mM MnClr + 1 mM dithiothrietol IO mM K-ghrconate + 1 mM dithiothreitol 5 mM MnClr + 10 mrvt Naascorbate

ELUTIONSWITH BUFFERS

Activity recovered in 0.5 M NaCl wash (units)

20

8 33

Note. The adsorbent was Scarlet HE-3G-Sepharose CL4B, using 20 mM K-phosphate, pH 6.5, plus additives. Sixty units of activity was applied, all of which adsorbed to each column. The elution was effected by adding 0.5 M NaCl to the buffers.

column, both Turquoise MX-G and Scarlet MX-G have been equally successful. Pur&kation

Procedure

Cells (30 g) were extracted as described, and the extract at pH 6.5 was passed through two columns of 8 cm2 cross section, the first containing 50 cm3 of Scarlet MX-G (C.I. 17908, Reactive Red 8)Sepharose CL-4B (approx 4 mg dye per g wet wt), and the second 50 cm3 of Blue HE-G (C.I. Reactive Blue 187)~Sepharose CL4B (approx 0.5 mg dye per g wet wt). 150 ml of extract containing 8- 10 mg ml-’ protein was applied, and washed in using

a further 100 ml buffer. The Scarlet MX-G column was removed, and the Blue HE-G column washed further with the same buffer containing 20 IIIM Na2S0,. After the protein trace had been restored to the baseline, the original buffer containing 20 mM DL-a-glycerophosphate (adjusted to pH 6.5 with HCl), was used to affinity elute 6-phosphogluconate dehydratase. As will be shown below, o-glycerophosphates are strong competitive inhibitors of the enzyme. The active fractions were collected, concentrated by ultrafiltration, and stored frozen. A summary of a typical purification is given in Table 4. Properties 6-Phosphogluconate dehydratase belongs to the class of dehydmtases requiring ferrous ions for activation; this includes aconitase (6), Dgluconate dehydratase (7), Dmannonate dehydratase (8), and some tartrate dehydratases (9). Aconitase has been shown to be an ironsulfur enzyme, with the natural state possibly containing an Fe3S3 cluster ( 10). As 6-phosphogluconate dehydratase is a similar brown color to aconitase, it seemed likely that it also contains a labile iron-sulfur cluster. A sample passed through a small column of G-25 to remove excess ferrous ions, then dialyzed extensively, was analyzed for iron both by atomic absorption spectroscopy, and by a spectrophotometric method using c+dipyridyl. Both

TABLE 3 SOME RESULTSOFDYE-SCREENING

FOR 6-PHOSPHOGLUCONATE

DEHYDRATASE

Enzyme not bound

Enzyme partially retarded

Enzyme totally bound

Red MX-5B (3 1) Rubine H-BN (3 1) Scarlet MX-G (30) Turquoise H-A (16) Turquoise MX-G (30) Yellow H-A (27)

Brown MX-GRN (40) Navy H-4R (47) Scarlet MX3G (44)

Blue H-B (58) Blue HE-G (37) Blue MX-G (67) Blue MX3G (52) Orange MX-2R (64) Yellow MX-R (50)

Note. Buffer was K-Mes, pH 6.5, containing manganous and ferrous ions as described in the text: 20 mg crude extract proteins applied to each 2 cm3 column. Percentage of protein that was bound to each dye is indicated in parentheses.

DIFFERENTIAL

DYE-LIGAND

533

CHROMATOGRAPHY

TABLE 4 ~RIFICATlONOF6-PHOSPHOGLUCONATE

DEHYDRATASEFROM

Volume Extract Not adsorbed on Scarlet MX-G Not adsorbed on Blue HE-G Eluted with cu-glycerophosphate

150 180 200 22

methods gave 3.0 + 0.1 iron atoms per 63 kDa subunit (2 preparations of enzyme). Polyacrylamide-SDS gel electrophoresis followed by gel scanning indicates that the enzyme eluted from the Blue HE-G column is at least 98% pure, with the main band at 63 KDa (Fig. 1). On gradient polyacrylamide gel electrophoresis at pH 9 the enzyme ran close to bovine serum albumin dimer. This suggests that, like the gluconate dehydratase from Clostridium pasteurianum (7), (i-phosphogluconate dehydratase is a dimer of molecular weight about 130,000. The K, for 6-phosphogluconate was determined to be 0.04 -t- 0.01 mM in the buffer described above for the enzyme assay. This is a somewhat lower value than that of the P. putida enzyme (3). In view of the fact that DL-cu-glycerophosphate was such an effective eluting ligand in the purification procedure, the effect of glycerophosphates on the enzyme’s activity was tested. The apparent Ki of DL-cu-glycerophosphate was 0.4 mM, as a competitive inhibitor. Using L-a-glycero-

KDo

1. Densitometer scan of polyacrylamide-SDS gel of 6-phosphogluconate dehydratase eluted from Blue HEG column. FIG.

30 g

Zymomonas mobilis STRAIN ZM4

Total activity (units)

Total protein Ow4

8100 7000 40 4050

1450 1040 920 16.5

CELLS

Specific activity (units mg-‘) 5.6 6.7 245

phosphate, the Ki value was 1.0 tnM (Fig. 2). Consequently it was concluded that the D isomer is the more effective inhibitor, as would be expected, it being a structural analog of the carbon atoms 4 to 6 of the substrate. Using the rate expression for two simultaneous competitive inhibitors i and j: V

-=max 0

1 .?(l

+$(1+&j

the Ki for D-a-glycerophosphate is estimated to be 0.3 mM. Nevertheless, the L-isomer, and P-glycerophosphate (Ki = 1.8 mM) also bound quite tightly. Other inhibition constants determined were for phosphate (2.5 mM), 3phosphoglycerate (2 mM), and sulfate (ca. 20 mM). Gluconate (0.1 M) did not inhibit the enzyme. No other allosteric effectors for.this enzyme have been found; adenine nucleotides had no effect on the activity.

FIG. 2. Eadie-Hofstee plots for 6-phosphogluconate dehydratase. (0) In the absence of inhibitors; (A) in the presence of 1 mM DL-a-glycerophosphate; (m) in the presence of 5 mM L-or-glycerophosphate.

534

SCOPES AND GRIFFITHS-SMITH

DISCUSSION

The method illustrated in this and the previous paper (1) of differential dye-l&and chromatography for purifying proteins, is seen to be a very useful procedure. Although the 6phosphogluconate dehydratase purification took a long time to develop, this was entirely due to the lability of the enzyme; when conditions were found (the presence of both manganous and ferrous ions) to retain its activity during the chromatography, it took very little time to find two suitable dye-adsorbents for this enzyme. The first column took out 2530% of the protein from the crude extract, and the second (Blue HE-G), being rather specific for the dehydratase, bound all of it, but less than 10% more of the extract protein. Salt elution from the Blue column does not achieve much separation of the dehydratase from the rest of the adsorbed protein, so a scheme for affinity elution was developed. Using the substrate itself, the enzyme catalyzes the formation of product, which does not bind to the enzyme, so no elution is effected. D-WGlycerophosphate should be a substrate analog, but is not readily available as the pure isomer. Inhibition studies using the L-isomer and the DL-mixture confirmed that D-cy-glycerophosphate bound strongly, with a Ki of about 0.3 mM. However, L-a-glycerophosphate, &glycerophosphate, and inorganic phosphate also act as quite strong competitive inhibitors. In the purification procedure a “dummy substrate” wash (11) was included using sulfate, which does not interact with the enzyme strongly. This removes traces of phosphoglycerate mutase which would otherwise elute with a-glycerophosphate and contaminate the dehydratase. The more easily obtainable 50:50 (Y-,&glycerophosphate, which contains about 25% of the Da-isomer, elutes the enzyme equally as well as 100% a-glycerophosphate. The structural and kinetic properties of 6-

phosphogluconate dehydratase indicate that it is a relatively simple enzyme, apart from the involvement of iron. It is anticipated that further studies on this enzyme may indicate an activation-deactivation process similar to that described for mitochondrial aconitase (610). The dehydratase is rather labile in the absence of reducing agents. Even during the 2-3 h needed for this isolation procedure, about half of the activity is lost and cannot be accounted for. The 45-fold purification indicates that the enzyme makes up about 2% of the soluble protein, which agrees well with a quantitative estimation of a band at 63 kDa in the crude extract on sodium dodecyl sulfate gel electrophoresis. ACKNOWLEDGMENTS This work was supported by a grant from the Australian Research Grants Scheme.

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Scopes, R. IL (1984) And Biochem. 136, Kovachevich, R., and Wood, W. A. (1955) J. Biol. Chem. 213, 745-756. Meloche, H. P., and Wood, W. A. (1964) J. Biol. Chem. 239, 3505-35 10. Wood, W. A. (1971) in The Enzymes (P. Boyer, ed.), Vol. 5, 3rd edn., pp. 573-578, Academic Press, New York. Gawron, O., Waheed, A., Glaid, A. J., and Jaklitsch, A. (1979) Biochem. J. 139, 709-714. Villafranca, J. J., and Mildvan, A. S. (197 1) J. Biol. Chem. 246,772-779. Gottschalk, G., and Bender, R. (1982) in Methods in Enzymology (W. A. Wood, ed.), Vol. 90, pp. 283287, Academic Press, New York. Robert-Baudouy, J., Jimeno-Abendano, J., and Stoeber, F. (1982) in Methods in Enzymology (W. A. Wood, ed.), Vol. 90, pp. 288-294, Academic Press, New York. Rode, H., and Gilfhom, F. (1982) J. Bucferiol. 151, 1602-1604. Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-Johnson, W. H., and Munck, E. (1980) J. Biol. Chem. 255, 1793-1796. Scopes, R. K. (1977) Biochem. J. 161,253-263.