3-Deoxy-D-arabino-heptulosonate 7-Phosphate Synthase

The phenylalanine-sensitive 3-deoxy-marabino-heptulosonate ?-phosphate synthase (7-phospho-2-keto-3deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20°C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-marabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K. M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism.

The first committed step in the biosynthesis of aromatic compounds is the condensation of P-enolpyruvate and erythrose-4-P to give 3-deoxy-D-arabino-heptulosonate 7-phosphate and inorganic phosphate (1). This reaction is catalyzed by DAHP' synthase (7-phospho-2-keto-3-deoxy-o-arabinoheptonate D-erythrose-4-phosphate lyase (pyruvate phospho-  Davis (27). In order to locate enzyme activity on gels, electrophoresis was carried out with gel system no. 6 of Maurer (28) in which the proteins are separated at pH 8.0. The gels were sliced into l-mm discs, the discs were crushed manually in 0.

Purification
All manipulations were performed at 4°C. All buffers contained 2 mM P-enolpyruvate.
Step I-Cells were resuspended in Buffer A (0.1 M potassium phosphate, pH 6.5) containing lo-" M phenylmethylsulfonyl fluoride; 4 ml of buffer were used for each gram of wet cells. The cells were disrupted in an Aminco French pressure cell at 20,000 p.s.i., after which the cell debris was removed by centrifugation for 30 min at 25,000 X R.
Step II-To the supernatant of Step I was added dropwise a 2% solution of protamine sulfate in Buffer A to give a final concentration of 0.14 mg of protamine sulfate per mg of protein. After an additional 30 min of stirring, the suspension was centrifuged for 90 min at 25,000 X R.
Step III-The supernatant from Step II was run directly onto a hydroxylapatite column (Bio-Gel HTP; 5.0 X 100 cm) that had been equilibrated with Buffer A. The column was washed with 750 ml of Buffer A, and the enzyme was eluted with a linear gradient obtained from 1200 ml of Buffer A and 1200 ml of 0.4 M potassium phosphate, pH 6.5. Fractions of 15 ml were collected at a flow rate of 100 ml/h. Fractions containing DAHP synthase (PHE) activity were pooled.
Step IV-Finely ground (NH&S04 was added to thdpooled fractions from Step III to give 50% saturation.
The precipitated protein was collected by centrifugation and discarded. To the supernatant was added finely ground (NH&SO4 to give 70% saturation. The precipitate was collected by centrifugation, dissolved in 0.6 M potassium phosphate, pH 6.5, and recentrifuged to clarify the solution if necessary.
Step V-The resulting solution from Step IV was run onto a column of Phenyl-Sepharose CL-4B (Pharmacia; 2.6 X 25 cm) equilibrated with 0.6 M potassium phosphate, pH 6.5. After the column had been washed with 100 ml of equilibration buffer, the enzyme was eluted with a linear gradient obtained from 200 ml of equilibration buffer and 200 ml of 0.05 M potassium phosphate, pH 6.5. Fractions of 4 ml were collected at a flow rate of 80 ml/h. Fractions containing DAHP synthase (PHE) were pooled and diluted with an equal volume of 2 mM P-enolpyruvate, pH 6.5.
Step VI-The solution from Step V was run onto a DEAE-Sephadex A-50 column (2.6 x 15 cm) equilibrated with 0.05 M potassium phosphate, pH 6.5, the column was washed with 100 ml of 0.1 M potassium phosphate, pH 6.5, and the enzyme was eluted with a linear gradient obtained from 200 ml of 0.1 M potassium phosphate, pH 6.5, and 200 ml of 0.3 M potassium phosphate, pH 6.5. Fractions of 4 ml were collected at a flow rate of 50 ml/h. In order to concentrate the protein, fractions containing DAHP synthase (PHE) were pooled, diluted with an equal volume of 2 InM P-enolpyruvate, pH 6.5, run onto a small (0.9 x 15 cm) column of hydroxylapatite, and eluted with 0.4 M potassium phosphate, pH 6.5.
Step VW--The concentrated enzyme from Step VI was layered onto a Sephadex G-150 column (2.6 x 100 cm) which had been equilibrated with 0.05 M potassium phosphate, pH 6.5. The enzyme was eluted with the same buffer, and fractions of 2 ml were collected at a flow rate of 30 ml/h. Fractions which contained DAHP synthase (PHE) were pooled.
Step VIII--The solution from Step VII was passed through a column (0.9 X 15 cm) of Blue Sepharose CL-6B (Pharmacia) equilibrated with 0.05 M potassium phosphate, pH 6.5. Under these conditions the enzyme does not bind to the column while the trace impurities do. The minor species could be eluted with 0.4 M potassium phosphate, pH 6.5. The fractions containing DAHP synthase (PHE) were pooled and were either used for further studies or concentrated as in Step VI and dialyzed against 0.05 M potassium phosphate, pH 6.5.
A summary of a representative purification run is given in Table I. On polyacrylamide gels under nondenaturing conditions the protein from Step VIII runs as a single band (Fig.  3-Deoxy-D-arabino-heptulosonate 7-Phosphate Synthase 4261 lA, inset). When the gel is sliced and assayed for enzyme activity, the activity coincides with the one protein band, as expected. The enzyme in Fig. 1 (I) is at least 97% pure. This value is calculated by integration of a densitometric scan of Elution was with the same buffer at a flow rate of 10 ml per h. The molecular weights are plotted uersus the ratio of the elution volume ( V,) to the void volume ( VO) an SDS gel (Fig. lB, inset). Two minor bands (II and 111 in Fig. 1) can be detected on SDS gels by applying a large amount of protein to the gels.

Ultraviolet
Absorption Coefficient of DAHP synthase (PHE) The enzyme has a maximum absorption at 279 nm. ,The ratio of the absorbances at 280 nm and 260 nm is 2.13. The value for E% at 280 nm is 10.87 with bovine serum albumin as a standard and the method of Lowry et al. (26) for protein determination.

Molecular
Weight and Subunit Structure The molecular weight of the native enzyme was estimated to be 150,000 + 20,000 by gel filtration on Sephadex G-200 with urease, catalase, lactate dehydrogenase, bovine serum albumin, and ovalbumin as standards (Fig. 2). From sedimentation equilibrium analysis of pme DAHP synthase (PHE), a molecular weight for the native enzyme of 136,000 was calculated assuming a partial specific volume V of 0.739 cm"/g at 2.65"C (Fig. 3). The value for V was calculated from the amino acid analysis according to the method of Cohn and Edsall (36).
The subunit molecular weight was determined by the method of Weber and Osborn (30). With bovine serum albu'min, ovalbumin, aldolase, trypsin, and lysozyme as standards, a monomer molecular weight of 35,000 was obtained for DAHP synthase (PHE) (Fig. 4). This value, taken together with the value for the molecular weight of the native enzyme, suggests a tetrameric quaternary structure. This was confirmed by cross-linking experiments with dimethylsuberimidate. Pure DAHP synthase (PHE) was cross-linked and subject&d to SDS gel electrophoresis.

Amino Acid Analysis of DAHP Synthase (PHE)
The amino acid analysis of pure DAHP synthase (PHE) is given in Table II . Subunit molecular weight determination of DAHP synthase (PHE) by SDS polyacrylamide gel electrophoresis. Ten micrograms of DAHP synthase (PHE) and of each of the indicated standards were denatured as described under "Materials and Methods" and subjected to electrophoresis for 12 h at 3.5 mA per gel. The molecular weights of the denatured proteins are plotted versus the mobilities relative to a bromphenol blue standard.

Stability of DAHP Synthase (PHE)
The pure enzyme shows a rather narrow pH optimum for activity around pH 6.5 to 7.0 (Fig. 6A). Furthermore, the stability of the enzyme activity at the pH optimum is dependent upon protein concentration and upon the presence of P; enolpyruvate.
The data in Fig. 6B show that the pure enzyme is quite stable in 2 mM P-enolpyruvate until the enzyme concentration is lowered to 1 pg/ml. In addition, when the Penolpyruvate concentration is lowered to 2 pM, loss of enzyme activity is accelerated. This inactivation is not reversible by 21  I  I  I  I  I  I  02  04  06  06  IO  12   RELATIVE  MOBILITY FIG. 5. Cross-linking of pure DAHP synthase (PHE) with dimethylsuberimidate and determination of molecular weights of the crosslinked enzyme species. Inset D, 60 pg of pure DAHP synthase (PHE) were cross-linked with dimethylsuberimidate as described under "Materials and Methods," and the resulting solution was subjected to SDS-polyacrylamide gel electrophoresis. Inset C, the noncross-linked enzyme. The gel shown in inset D was scanned and mobilities measured relative to bromphenol blue standard. Aldolase (40 pg) crosslinked in the same manner was the standard used for the plot of molecular weight versus relative mobility; o---O, DAHP synthase (PHE); W, aldolase. The loss of activity is slower at lower temperatures.

Stimulation
of Enzyme Activity by Co" Crude preparations of DAHP synthase (PHE) are stimulated about 40% by 2 mM CoC12 ( Table I). Most of the stimulatory effect is lost after the first chromatographic purification step, with the rest vanishing in the following steps. Enzyme purified through the DEAE-Sephadex step is totally independent of Co'+ for activity (Table I). Attempts to restore Co'+ activation by mixing fractions or adding fractions to the pure enzyme have thus far been unsuccessful.

Inhibition of DAHP Synthase (PHE)
Pure DAHP synthase (PHE) is inhibited by L-phenylalanine and by its two analogs ,&2-thienyl-i&L-alanine and L-OH FK. 6. Stability of DAHP synthase (PHE). A, pH dependence of the enzyme activity measured in 0.1 M potassium phosphate, 0.1 mM P-enolpyruvate, and 0.1 mM erythrose-4-P at the indicated pH values. Assay Method B (4) was used. B, pure DAHP synthase (PHE) in 0.05 M potassium phosphate, pH 6.5, was preincubated for the indicated times and assayed for activity in Method A (4). The preincubation mixture contained: W, 10 pg of enzyme/ml and 2 mM P-enolpymvate; M, 1 pg of enzyme/ml and 2 mM P-enolpyruvate; A-A, 1 pg of enzyme/ml and 2 pM P-enolpyruvate. The P-enolpyruvate and the erythrose-4-P concentrations were 0.1 mM each. The inhibitor (I) was L-phenylalanine (W), (Fig. 7); 50% inhibition is obtained with 13, 25 (assuming only the L-isomer is inhibitory), and 175 pM inhibitor, respectively, when both substrates are 0.1 mM and assay Method B is used.
Neither L-tyrosine nor D-phenylalanine inhibit the pure enzyme. DISCUSSION DAHP synthase (PHE) purified through Step VIII of our procedure is at least 97% pure. Two polypeptides, recognizable in the later stages of the purification procedures as discrete bands on SDS-polyacrylamide gels, accompany the enzyme through seven purification steps and can be removed only by passing the preparation over a column of Blue Sepharose. This last "purification" step results in a 20% decrease in the specific activity of the enzyme. Attempts to reactivate homogeneous enzyme preparations through the addition of material removed in the last step have been unsuccessful. Therefore, it seems unlikely that the proteins removed in Step VIII interact directly with the DAHP synthase (PHE) activity, although this possibility cannot be excluded.
During the purification procedure the method of choice for concentrating the enzyme was adsorption to hydroxylapatite followed by elution with phosphate buffers of high ionic strength. More purified enzyme preparations can also be concentrated by precipitation with 80% (NH&SO4 with little loss of activity (data not shown). In crude systems, however, (NH&SO4 precipitations are rather unsuitable for use with the enzyme, since substantial losses of activity are encountered through irreversible inactivation. The molecular weight of the native enzyme, determined by three different methods, is 140,000. This value differs somewhat from previously reported values of 160,000 (7) and llO,-000 (9, 17), obtained by gel filtration alone. The subunit molecular weight of DAHP synthase (PHE) is 35,000, which is in fairly good agreement with the previously reported value of 33,000 (17). Although Simpson and Davidson (17) suggested that the native form of the enzyme is a trimer, our data conclusively prove that DAHP synthase (PHE) of E. coli has four subunits.
The amino acid analysis of pure DAHP synthase (PHE) shows no unusual features. However, it does not agree well with previously published data (17). Some of the discrepancies can be eliminated by recalculation of the data of Simpson and Davidson, since their values appear not to add up to a subunit molecular weight of 33,000. Some gross differences remain, e.g. the values for glycine and alanine. These differences might be best explained by impurities in previously available enzyme preparations, since Simpson and Davidson (17) estimate that their enzyme is about 85% pure.
This inhibition is specific, since up to 1 InM tyrosine does not inhibit the enzyme. Many phenylalanine analogs have been tested as inhibitors for this enzyme (data not shown; Ref. 37). In agreement with previous findings, analogs with substitutions at either the a-amino or the car-boxy1 group of phenylalanine do not inhibit this enzyme (37), and thienylalanine is an effective inhibitor (38). There have been no previous reports that dihydrophenylalanine, a nonaromatic compound, inhibits this enzyme. Dihydrophenylalanine is excreted into the growth medium of Streptomyces (39) and is assumed to be synthesized via the common aromatic pathway (40j.
In comparing the previously purified tyrosine-sensitive DAHP synthase from E. coli (4) with pure phenylalaninesensitive isoenzyme, one observes some common molecular parameters as well as some strikingly different ones (Table