Glucose-6-Phosphate Dehydrogenase from Escherichia coli and from a "High-Level" Mutant

Transcription

1 JOURNAL OF BACrFOLOGY, Apr. 1972, p Copyright American Society for Microbiology Vol. 110, No. 1 Printed in U.S.A. Glucose-6-Phosphate Dehydrogenase from Escherichia coli and from a "High-Level" Mutant SANTIMOY BANERJEE AND D. G. FRAENKEL Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts Received for publication 22 December 1971 Glucose-6-phosphate dehydrogenase has been purified to near homogeneity from wild-type Escherichia coli K-12 and from a mutant previously found to contain substantially more of the enzyme. The two enzymes are the same in all characteristics studied thus far: specific activity, kinetics, specificity, and subunit size. Many of the enzymes of the central pathways of sugar intermediary metabolism in Escherichia coli are synthesized in an apparently unregulated manner: their levels do not vary much with the nutritional content of the growth medium. Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate: nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase, EC ] is one such enzyme. We have recently described the selection of a mutant strain which contains about six times as much glucose-6-phosphate dehydrogenase activity as the wild-type strain (5). The activity in the mutant is also constitutive. The mutation, zwfll, is closely linked genetically to the structural gene for the enzyme, and is cis dominant. Antibody titration indicated that a zwfll strain contains more normal enzyme (rather than the usual amount of an altered "improved" enzyme). However, to establish the identity, or nonidentity, of the enzyme in wild type and mutant, it is necessary directly to compare the enzymes from the two strains. In this paper, we report their purification and an initial comparison of their properties. No significant differences have yet been found. MATERIALS AND METHODS The two strains (5) of E. coli K-12 used were strain K-10 [wild-type glucose-6-phosphate dehydrogenase (zwf+)] and a derivative of it, strain DF82 [high-level glucose-6-phosphate dehydrogenase (zwfll), and also carrying a mutation in gluconate- 6-phosphate dehydrogenase (not relevant to the present work)]. The cells were grown in minimal medium 63 (19) with 1 gg of thiamine-hcl and 4 mg of glycerol per ml; the facilities of the New England 155 Enzyme Center at Tufts University were sometimes used. The cells were harvested in stationary phase and stored at -15 C. The standard assay reaction mixture (1 ml) for glucose-6-phosphate dehydrogenase contained 100 mm tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, ph 8.0, 10 mm MgCl2, 0.18 mm NADP+, and 1 mm D-glucose-6-phosphate. The reaction was initiated by the addition of enzyme, and formation of reduced NADP (NADPH) was followed at 340 nm with time in a Gilford model 2000 spectrophotometer at 25 C. One unit of activity is the amount of enzyme required to catalyze the reduction of 1 Amole of NADP+ per min at 25 C. Specific activity is expressed in units per milligram of protein. Protein was assayed by the Folin method (9), with bovine serum albumin corrected for moisture content (8) as standard. Polyacrylamide gel electrophoresis was done by the method of Davis (3). Bromothymol blue was used as marker dye. Electrophoresis was at 4 C with 3 ma per gel. Protein was stained with Coomassie Blue or amido schwartz, and destained with 7.5% acetic acid containing 0.5% methanol. Specific staining of glucose-6-phosphate dehydrogenase was by the method of Rattazzi et al. (15). Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was by the method of Weber and Osborn (20). After staining, the gels were scanned with a densitometer (Photovolt Corp., Varicord model 42B). Glucose-6-phosphate, NADP+, NAD+, NADPH, NADH, diethylaminoethyl (DEAE) cellulose (medium mesh, 0.8 meq per g), rabbit muscle phosphorylase, bovine serum albumin, and protamine sulfate, were from Sigma Chemical Co. Hydroxyapatite was from BioRad Laboratories. Bentonite was from Fisher Scientific Co. Boehringer and Sons were the supplier of yeast glucose-6-phosphate dehydrogenase, bovine liver glutamic dehydrogenase, rabbit muscle aldolase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

2 156 BANERJEE AND FRAENJKEL J. BACTERIOL. RESULTS The purification was developed by using the high-level strain and then was applied to the wild-type strain. Data for typical preparations are shown in Table 1. All operations were done at 0 to 4 C. Centrifugations were for 10 min at 13,000 x g unless noted otherwise. In the usual preparation, 80 to 100 g of cells (wet weight) was suspended in 250 ml of 0.01 M Tris-hydrochloride, 0.01 M MgCl2, ph 7.6, and treated, in 60-ml portions, for 15 min in a Raytheon ultrasonicoscillator. The debris was removed by centrifugation for 30 min at 20,000 x g (crude extract). Protamine sulfate, 2%, ph 7.0, was added to the point of precipitation of glucose- 6-phosphate dehydrogenase, the precipitate was removed by centrifugation, and the enzyme was then precipitated by further addition of protamine sulfate (16). The amounts of protamine sulfate used were determined by preliminary trial for each preparation and were typically 0.1 and 0.3 ml per ml for the two steps, respectively. The precipitate was washed once with 5 ml of water, and the enzyme was extracted with several 5-ml portions of 25 mm sodium phosphate, ph 7.6 (protamine extract). Solid ammonium sulfate was added to 30% saturation (209 mg per ml), the precipitate was discarded, and the enzyme was precipitated by further ammonium sulfate addition to 50% (129 mg per ml). The precipitate was dissolved in 10 ml of 10 mm Tris-acetic acid buffer, ph 7.6, containing 40 mm NaCl, and was dialyzed for 4 hr against 1 liter of the latter buffer (with one buffer change). The dialyzed fraction was applied, 1 mg of protein per 3 ml of column volume, to a column of DEAE cellulose (packed under atmospheric pressure and equilibrated with the same buffer as used in dialysis), and the column was washed with 1 volume TABLE 1. Fraction Purification of glucose-6-phosphate dehydrogenasea Wild-type High-level strain strain Specific Units Specific Units activity activity Crude extract ,400 Protamine extract ,900 DEAE cellulose Hydroxyapatite Bentonite a The wild-type strain was K-10 (zwf) and the high-level strain was DF82 (zwfll). Starting material was ca. 100 g of cells (wet weight). of the buffer containing 0.1 M NaCl. A linear gradient (4 volumes of the buffer with 0.1 M NaCl in the mixing chamber and 4 volumes of the buffer containing 0.25 M NaCl in the reservoir) was applied, and 5-ml fractions were collected. The fractions containing glucose-6- phosphate dehydrogenase activity (elution was between 0.16 and 0.19 M NaCl) were pooled, and the enzyme was precipitated by ammonium sulfate addition to 60% (390 mg per ml). The precipitate was dissolved in 5 ml of 0.03 M sodium phosphate buffer, ph 7.6, and dialyzed for 4 hr (DEAE-cellulose fraction). The latter fraction was applied, 1 ml of protein per ml of column volume, to a column of hydroxyapatite (equilibrated with the 0.03 M buffer). The column was then washed with 3 volumes each of 0.03, 0.06, 0.09, and 0.12 M sodium phosphate buffers, ph 7.6; the enzyme generally eluted with the 0.09 M buffer. The active fractions were combined and concentrated by vacuum dialysis in Schleicher and Schuell Co., collodion bags, no. 100 (hydroxyapatite fraction). A final purification step involved treatment of the hydroxyapatite fraction with bentonite, 8 mg per mg of protein. The enzyme remained in the supernatant fluid (bentonite fraction). The specific activities of the final preparations of enzyme from the two strains were similar and represent approximately 100-fold purification from the high-level strain and 450-fold purification from the wild type. The bentonite fractions were pure according to several criteria. Polyacrylamide gel electrophoresis showed a single band upon protein staining (Fig. 1, left-hand gel); activity staining showed that this band was the enzyme. Slightly less purified preparations (e.g., the wild-type enzyme in gels of Fig. 2) showed a major protein band, which had activity, and some material migrating faster, which did not have activity. Such slightly impure preparations were obtained from the wild-type strain when the amount of bentonite used in the final step was not optimal (because of the low recovery from this step not all preparations of wild-type enzyme were taken to the purity shown in Fig. 1). The final preparations also sometimes showed some protein migrating slower than the major component (e.g., Fig. 2, gels with enzyme from high-level strain). This material had activity and is probably aggregated glucose-6-phosphate dehydrogenase; SDS gel electrophoresis showed only a single component, and, if before normal electrophoresis such fractions were first diluted in the presence of substrate, the slower-migrating component was no longer found.

3 VOL. 1 10, 1972 GLUCOSE-6-PHOSPHATE DEHYDROGENASE 157 In polyacrylamide gel electrophoresis at ph 8.4 and 9.2, the enzymes from wild-type and high-level strains migrated similarly, and in gels containing samples of both preparations no separation was evident (Fig. 2). The same was true at ph 7.2, but migration was slower and the protein bands were more diffuse. SDS polyacrylamide gel electrophoresis of the pure enzymes showed a single component (shown for enzyme from wild-type strain in right-hand gel of Fig. 1), and in gels containing samples from both preparations there was no separation (not shown). The molecular weight of the component seen in the SDS gels (presumably the protomer) and determined (Fig. 3) by use of a series of internal standards (20), and is about 52,000 daltons. Double reciprocal plots of activity as a function of substrate concentration are shown in Fig. 4. From these plots, the Km values were: for glucose-6-phosphate, wild-type enzyme, mm, and high-level enzyme, mm; and for NADP+, wild-type enzyme, mm, and high-level enzyme, mm. NAD+ could replace NADP+, with 10 mm NAD+ giving 25% of the rate in the usual assay with NADP+. The Km for NAD+ was 1.67 mm. (This reaction is unlikely to represent contamination of the NAD+ with NADP+, since the same NAD+ was neither substrate nor inhibitor of glucose- 6-phosphate dehydrogenase from yeast.) Both wild-type and high-level enzyme gave the same results in tests of specificity and inhibition. The following substances, 1 mm, were not substrates when substituted for glucose-6- phosphate in the standard assay, and did not affect the activity with glucose-6-phosphate: D- glucose-i-phosphate, D-fructose- 1-phosphate, D-gluconate-6-phosphate, D-galactose-6-phosphate, D-ribose-5-phosphate, and D-glucose. Figure 5 shows enzyme activity as a function of ph. Both enzymes showed somewhat lower activity when 10 mm MgCl2 was omitted from the standard assay. In an experiment with the high-level enzyme, taking the rate without MgCl2 as 100, in the presence of the following salts at 10 mm, the rates were: MgCl2, 161; CaCl2, 139; MnCl2, 44; CoSo,, 44; CdCl2, 17; Cu(NO3)2, 0; and HgCl2, 0. The inclusion of 1.0 mm sodium ethylenediaminetetraacetate did not affect the rate in the assay without MgCl2. For both enzymes, addition of 1 mm adenosine mono-, di-, or triphosphate, or cyclic 3', 5'-AMP, to the standard assay did not affect the rate of reaction. In the assay without MgCl2, adenosine triphosphate was inhibitory at higher concentrations (e.g., 50 to 75% inhibi- FIG. 1. Electrophoresis of bentonite fraction from wild-type strain. A 5-1ig amount was run in polyacrylamide gel at ph 8.4 (left-hand gel), 20,gg was run in SDS gel (right-hand gel), and the gels were stained for protein. tion at 10 mm). NADPH (TPNH) inhibited the reaction, equally with the two enzymes (Fig. 6). Inhibition of the enzyme from E. coli B by NADH was shown by Sanwal (16). We have had somewhat variable results and are not certain whether NADH affects the enzyme from E. coli K-12. DISCUSSION Glucose-6-phosphate dehydrogenase has been studied in many organisms and tissues. The activity in E. coli was first described in 1953 (17), and Sanwal recently reported a kinetic exploration of a partially purified preparation from E. coli B (16). The preparation we report here of the enzyme from E. coli K-12 is at least 90% pure, according to gel electrophoresis, and its specific activity is about 100 units per mg of protein. K. G. D. Allen and G. R. Julian (personal communication) purified the enzyme from E. coli B and found a specific activity of 250 (same units). Specific activities

4 158 BANERJEE AND FRAENKET, J. BACTERIOL. t allhk.,... I i "umomm omo 'n- FIG. 2. Polyacrylamide gel electrophoresis of the two enzymes. The two sets of gels were run at ph 8.4 and 9.2. Samples were (a) 10,gg of bentonite fraction from wild-type strain, (b) 20 Mlg of bentonite fraction from high-level strain, and (c) both fractions run together. Staining was of protein. t 2o 4-6. Ph.skoryLase Bovine serum albumin C ;Glamic Jhy&rogonase AlJolas. i vgyc.raljehyde 3-P IL o Rolative mbility o zyd"ro3enase FIG. 3. Migration in SDS gels. Separate gels were run for each protein indicated, including 20 Mg of the high-level enzyme, and all proteins were run together in one gel for exact determination of relative migrations. The migration of the glucose-6-phosphate dehydrogenase is indicated by the arrow (it did not separate from glutamic dehydrogenase). of the several reported preparations of crystalline glucose-6-phosphate dehydrogenase are 68 for the enzyme from bovine udder (6), 315 for the enzyme from Leuconostoc mesenteroides (11), 314 for Candida utilis (4), 676 for brewer's yeast (10), and 750 from human erythrocytes (21). Critical comparison of these values may not be useful because protein measurements were made in different ways, subactive states have been described for some of the enzymes (7, 21), and ionic conditions are known to have a marked influence on structure and activity (1, 2). The subunit molecular weight for the enzyme we describe is about 52,000 daltons (Fig. 3). Subunits of similar size have been reported for other glucose-6-phosphate dehydrogenases [e.g., brewer's yeast (23) and human erythrocyte (14)], and the known molecular weight of some other glucose-6-phosphate dehydrogenases is a multiple of that value [e.g., Candida (4) and Leuconostoc (12)1. The E. coli enzyme has kinetic and specificity properties similar to those of other glucose-6-phosphate dehydrogenases, being rather 3pecific for glucose-6-phosphate and NADP+; the Km for NAD+ was about 102 higher than for NADP+.

5 VoL. 110, 1972 GLUCOSE-6-PHOSPHATE DEHYDROGENASE I/66P (V0 m) I/TPN (I/mM) FIG. 4. Activities as a function of substrate concentration. The standard assay was modified by changing the concentration of one substrate. Bentonite fractions: 0, wild-type enzyme; 0, high-level enzyme. I N E.095 a C Vt I %.005 _- S es ph FIG. 5. Enzyme activity as a function of ph. The buffers (0.2 M) were maleate-naoh (ph 6.2 to 6.8), Tris-hydrochloride (ph 7.3 to 8.8), and glycine- NaOH (ph 9.2 to 9.8). The ph values are of buffers, not of final reaction mixtures. The main purpose of the work reported in this paper was to compare glucose-6-phosphate dehydrogenase from wild-type E. coli K-12 with the enzyme from a "high-level" mutant. No significant differences have yet been found between the two enzymes. As expected from e asg 0.4I V. 0 E I Q.@ TPNH (mm) FIG. 6. Effect of NADPH (TPNH) on activity. Bentonite fractions from wild-type strain (0) and high-level strain (0) were assayed in standard assay with the addition of TPNH as indicated. (The TPNH concentration was determined from initial absorbancy measurement.) the earlier immunological- titrations of enzymes in crude extracts (5), considerably less purification was required to reach homogeneity starting with the high-level strain than with the wild-type strain; thus, the high level strain

6 160 BANERJEE AND FRAENKEL J. BACTERIOL. contains more enzyme. In all respects that were compared (specific activity, kinetics, specificity, and subunit size), the final preparations were similar. Thus, the zwfll mutation is likely to be in some function determining amount of gene product (such as a promoter) rather than in the gene product (the enzyme itself). Our recent genetic results (to be reported) are in accord with this conclusion; zwfll maps outside, but close to, the zwf locus as defined by deficiency mutations. A similar situation of hyperproduction of a constitutive enzyme caused by a linked mutation without an obvious effect on enzyme structure has recently been described by Pardee et al. (13) for nicotinamide deamidase of E. coli. However, the present data do not prove that the zwfll mutation does not affect the protein, and further comparison of the two enzymes is necessary. It is interesting that Yoshida recently showed that a variant of human glucose-6-phosphate dehydrogenase found in fourfold greater amount than normal had similar specific activity and kinetic characteristics to the normal enzyme but actually contained a single amino acid substitution (22). Also, Sirotnak has reported that many mutations leading to increased amounts of dihydrofolate reductase in Diplococcus pneumoniae are in the structural gene of the enzyme (18). ACKNOWLEDGMENTS We thank C. Biswas and H. Paulus for their advice. This work was supported by National Science Foundation grants GB7207 and GB D.G.F. is a Career Development Awardee of the National Institute of General Medical Sciences. LITERATURE CITED 1. Bonsignore, A., I. Lorenzoni, R. Cancedda, M. E. Cosulich, and A. DeFlora Effect of divalent cations on the structure of human glucose 6-phosphate dehydrogenase. Biochem. Biophys. Res. Commun. 42: Cohen, P., and M. A. Rosemeyer Human glucose 6-phosphate dehydrogenase: purification of the erythrocyte enzyme and the influence of ions on its activity. Eur. J. Biochem. 8: Davis, B. J Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121: Engel, H. W., W. Domschke, M. Alberti, and G. F. Domagk Protein structure and enzymatic activity. B. Purification and properties of a crystalline glucose 6-phosphate dehydrogenase from Candida utilis. Biochim. Biophys. Acta 191: Fraenkel, D. G., and S. Banerjee A mutation increasing the amount of a constitutive enzyme in Escherichia coli, glucose 6-phosphate dehydrogenase. J. Mol. Biol. 56: Julian, G. R., R. G. Wolfe, and F. J. Reithel The enzymes of mammary gland. II. Preparation of glucose 6-phosphate dehydrogenase. J. Biol. Chem. 236: Kirkman, H. N., and E. M. Hendrickson Glucose 6-phosphate dehydrogenase from human erythrocytes. II. Subactive states of the enzyme from normal persons. J. Biol. 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Chem. 246: Rattazzi, M. C Glucose 6-phosphate dehydrogenase from human erythrocytes: molecular weight determination by gel filtration. Biochem. Biophys. Res. Commun. 31: Rattazzi, M. C., L. F. Berini, G. Fiorelli, and P. M. Mannucci Electrophoresis of glucose 6-phosphate dehydrogenase: a new technique. Nature (London) 213: Sanwal, B. D Regulatory mechanisms involving nicotinamide adenine nucleotides as allosteric effectors. III. Control of glucose 6-phosphate dehydrogenase. J. Biol. Chem. 245: Scott, D. B. M., and S. S. Cohen The oxidative pathway of carbohydrate metabolism in Escherichia coli. I. Isolation and properties of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Biochem. J. 55: Sirotnak, F. M High dihydrofolate reductase levels in Diplococcus pneumoniae after mutation in the structural gene: biochemical and immunological evidence for increased synthesis. J. Bacteriol. 106: Sistrom, W. R On the physical state of the intracellularly accumulated substrates of,t-galactosidasepermease in Escherichia coli. Biochim. Biophys. Acta 29: Weber, K., and M. Osborn The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244: Yoshida, A Glucose 6-phosphate dehydrogenase of human erythrocytes. I. Purification and characterization of normal (B+) enzyme. J. Biol. Chem. 241: Yoshida, A Amino acid substitution (histidine to tyrosine) in a glucose 6-phosphate dehydrogenase variant (G6PD Hektoen) associated with overproduction. J. Mol. Biol. 52: Yue, R. H., E. A. Noltmann, and S. A. Kuby Glucose 6-phosphate dehydrogenase (zwischenferment). II. Homogeneity measurements and physical properties of the crystalline apoenzyme from yeast. Biochemistry 6: