Crystallization and Properties of Phosphofructokinase from Clostridium pasteurianum*

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1 THE JOURNAL OF BIOLOGICAL CHE~~TRV Vol. 245, No. 13, Issue of July 10, pp , 1970 Printed in U.S.A. Crystallization and Properties of Phosphofructokinase from Clostridium pasteurianum* KOSAKU UYEDA AND SHIGERU KUROOKA~ (Received for publication, Februry 9, 1970) From the Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas and Basic Biochemistry Unit, Veterans Administration Hospital, Dallas, Texas SUMMARY Phosphofructokinase has been purified from Closfridium pasteurianum and prepared in crystalline form in the presence and the absence of ATP. The enzyme preparation is homogeneous as shown by sedimentation velocity, sedimentation equilibrium, and acrylamide gel electrophoretic techniques. The molecular weight of the clostridial phosphofructokinase is 144,000. The enzyme is dissociated to subunits with molecular weight of about 35,000 in guanidine or urea or by treatment with maleic anhydride. Peptide mapping of a tryptic digest of the enzyme, acrylamide gel electrophoresis of the enzyme in urea, and the electrophoresis of the maleylated phosphofructokinase indicate that all these subunits are identical. Based on these observations, it is concluded that clostridial phosphofructokinase consists of 4 identical subunits. Kinetic studies show that the enzyme exhibits sigmoidal kinetics with respect to fructose 6-phosphate at all levels of ATP. ADP normalizes this initial velocity pattern to yield Michaelis-Menten kinetics. Unlike mammalian phosphofructokinases, the clostridial enzyme is not significantly inhibited by ATP, phosphoenolpyruvate, or citrate. The activity of the enzyme is absolutely dependent on NH4+ and Mg++, an d a possible physiological significance of the former cation is discussed. mammalian tissues are inhibited by a high concentration of ATP and citrate and are allosterically activated by fructose-6-p, fructose 1,6-di-phosphate, ADP, AMP, and phosphate. However, phosphofructokinases from yeast and Escherichiu coli are inhibited only by ATP and stimulated by either AMP or ADP. Phosphofructokinases from Dictyostelium discoideum and Arthrobatter crystallopoietes are unique among the phosphofructokinases in that they are not inhibited by ATP. In view of the considerable variations among the phosphofructokinases, and because of our current interest in the regulation of carbohydrate metabolism in Clostridium pasteuriunum, it appeared desirable first to study phosphofructokinase in this anaerobe. This paper describes the preparation of the enzyme in crystalline form and presents physicochemical properties which have not been investigated among bacterial phosphofructokinases. Observations on kinetic properties of clostridial phosphofructokinase are also presented. MATERIALS AND METHODS Materials Microgranular DEAE-cellulose (DE-52) was purchased from H. Reeve Angel and Company (New York, New York). Guanidine HCl was the product of HEICO, Inc. (Delaware Water Gap, Pennsylvania), and urea (Ultra-Pure) was purchased from Mann. All other chemicals were purchased from commercial sources as described previously (12). Trypsin was the product of Worthington. All other enzymes were purchased from Boehringer Mannheim. Phosphofructokinase (ATP:n-fructose g-phosphate l-phosphotransferase, EC ll), which catalyzes the phosphorylation of fructose-6-p by ATP, plays an important role in regulation of glycolysis in a variety of cells. The enzyme has been studied extensively in mammalian tissues (see reviews, References 1 and 2), plants (3, 4), insect (5), yeast (6, 7), slime mold (8), and bacteria (9-11). The activity of the enzyme is regulated by various metabolites. The nature of the allosteric activation and inhibition, however, differs depending upon the source of the enzyme. For example, phosphofructokinases from * This work was supported in part by Research Grant GM from the National Institutes of Health, United States Public Health Service. 1 On leave from Dainippon Pharmaceutical Company, Osaka, Japan Assay of Phosphojructokinase Activity Phosphofructokinase activity was determined by measuring the rate of formation of either fructose-l,6-di-p (Assay A) or ADP (Assay B). For Assay A the reaction mixture contained, in a final volume of 1 ml: imidazole HCl, 50 mm, ph 7.0; bovine serum albumin, 100 pg; dithiothreitol, 10 mu; DPNH, 0.16 mu; fructose-6-p, 2.5 mu; ATP, 0.5 mu; NH&l, 10 mu; MgClz, 6 mu; aldolase, 0.5 unit; triosephosphate isomerase, 1.5 units; and a-glycerophosphate dehydrogenase, 0.5 unit. The reaction was initiated by the addition of phosphofructokinase, and the rate of the reaction was determined at 28 with a Gilford recording spectrophotometer. One unit of enzyme activity is defined as the amount of the enzyme that catalyzes the formation of 1 pmole of fructose-l,6-di-p per min under these conditions. Specific activity is expressed as units per mg of protein. For

2 3316 Clostridial Phosphofructokinase Vol. 245, No. 13 Assay B, the reaction mixture contained, in a final volume of 1 ml: imidazole HCl, 50 mm, ph 7.0; ATP, 1 mm; MgC&, 3 mm; fructose-6-p, 2.5 mm; DPNH, 0.16 mm; Penolpyruvate, 0.5 mm; bovine serum albumin, 100 wg; NH&I, 10 mm; KCl, 10 nlm; pyruvate kinase and lactic dehydrogenase, 2 units each. The reaction was initiated by the addition of phosphofructokinase, and the rate of the react.ion was determined at 28 with a Gilford recording spectrophotometer. Ultracentrifugation Studies Ultracentrifugation was carried out in a Spinco model E ultracentrifuge. The sedimentation velocity experiments were performed at 47,660 rpm in a 30-mm double sector cell with quartz windows. High speed sedimentation equilibrium experiments were performed with a 3.0-mm liquid column according to the procedure of Yphantis (13), using a 12-mm double sector cell with sapphire windows. The protein concentration was between 0.05 and 0.5 mg per ml. The time required to reach equilibrium, which was usually 24 hours, was determined by measuring fringe displacement until it became constant. The fringe displacement was measured with a Nikon two-coordinate comparator. Sucrose density gradient centrifugation was carried out with a SW-39 rotor at 31,000 rpm for 15 hours according to the procedure of Martin and Ames (14). Acrylamide Gel Electrophoresis Acrylamide gel electrophoresis of the enzyme was carried out according to the procedure of Davies (15). Electrophoresis was performed in 7.5% acrylamide gel, and the electrode buffer was in Tris-glycine at ph 8.3. The sample was layered with an equal volume of a mixture containing 0.2 M sucrose and the electrode buffer. A current of 4 ma per gel was applied for about 1 hour at room temperature. The gel was stained in 1% Amido black in 7% acetic acid for 2 hours. The stained gel was destained electrophoretically in 7% acetic acid solution. The destained gel was scanned in the acetic acid at 600 rnp using a Gilford model 240 recording spectrophotometer equipped with a linear transport. Amino Acid Analysis Dry weight of six duplicate samples was determined after exhaustive dialysis against 0.05 M potassium phosphate at ph 7.0 and drying to constant weight at 95 in a vacuum desiccator. Hydrolysis of the protein (1 mg each) was carried out at 110 in evacuated, sealed vials containing 3 ml of constant boiling HCl for periods of 24, 48, and 72 hours. The HCl was removed with a rotary evaporator; the residue was dissolved in Hz0 and brought to dryness to remove acid completely. The final residue was dissolved in 0.5 ml of sodium citrate buffer at ph 2.2 and analyzed with a Beckman model 120C amino acid analyzer. Cysteine and cystine were determined after performic acid oxidation of the protein according to the procedure of Moore (16). Tryptophan was determined spectrophotometrically by the method of Bencze and Schmid (17). Maleylation of Enzyme Treatment of the enzyme with maleic anhydride was performed as described before (18). The enzyme (3 mg), in 0.05 M potassium phosphate at ph 8.1, was reacted with 0.8 mg of maleic anhydride. The ph of the solution was maintained at 8.5 by the addition of 2 N KOH during the reaction. The reac- tion mixture was stirred continuously for 30 min at 24. The maleylated enzyme was then dialyzed against 1 liter of 0.05 M potassium phosphate at ph 8.1. Tryptic Digestion and Peptide Mapping The technique employed for peptide mapping is as described by Chernoff and Liu (19). An ammonium sulfate suspension of the enzyme was centrifuged, and the precipitate was dissolved in 1 ml of 0.05 M potassium phosphate at ph 8. The solution was heated for 5 min at loo, and the precipitate was washed three times with 1 ml each of 0.2 M ammonium bicarbonate, ph 8.4. The washed, denatured protein was suspended in 0.2 ml of the ammonium bicarbonate buffer and digested with 0.06 mg of trypsin for 23 hours at 37. ilt the end of this period no undigested protein was visible. The solution was then immediately subjected to chromatography. A sample containing 1.5 mg of peptides was applied on a sheet of Whatman no. 3 MM paper. The paper was developed for 21 hours at 25 in l-butanol-pyridine-acetic acid-hz0 (15 : 10 : 3 : 12) after equilibration for 3 hours in the atmosphere of the same solvent. The paper was allowed to dry overnight and then saturated with pyridineacetic acid-water (25: 1:250) at ph 6.5. Electrophoresis was carried out in the buffer mixture for 85 min at 2000 volts using a Savant high voltage electrophoresis apparatus. The paper was dried by blowing streams of warm air for at least 4 to 6 hours. Peptide spots were stained by spraying 0.2y0 ninhydrin in l-butanol and heating the paper for 10 min at After marking the peptide spots, the paper was dipped in Sakaguchi reagent (diacetyl and cr-naphthol in an alkaline solution containing 257, ethanol) to detect arginine-containing peptides. Other Methods Protein was determined by a modification (20) of the Lowry phenol reagent method with crystalline bovine serum albumin as a standard. Puri$cation of Phosphofructokinase Clostridium pasteurianum was grown in a medium containing sucrose-ammonium sulfate as described by Lovenberg, Buchanan, and Rabinowitz (21) and stored frozen at -90. All operations were carried out at 2-4 unless otherwise stated. 1. Crude Extract-Frozen cells of C. pasteurianum (230 g) were suspended in 500 ml of a mixture of 0.05 M potassium phosphate (ph 9) and 1 mu EDTA. The suspension was divided into three equal batches and subjected to sonic oscillation with a Bronwill sonic oscillator for 7 min each. The sonically treated extract was then centrifuged at 21,500 X g for 1 hour. The ph of the supernatant solution was adjusted to 7.0 with addition of 10 N KOH. Z?. Heat Treatment-The crude extract (520 ml) was heated for 10 min in a 58 bath, chilled to 5 immediately at the end of the period, and centrifuged for 30 min at 21,500 x g. S. Isopropanol Fractionation-The supernatant solution from the heat treatment was then transferred to an ice-salt bath at -5, and 420 ml of isopropanol (at -5 ) were added dropwise with continuous stirring. After 10 min the solution was centrifuged at 16,300 x g for 10 min and the precipitate was discarded. To the supernatant solution were added 570 ml of isopropanol as above, and the mixture was stirred for 10 min at -5. The precipitate was removed by centrifugation at 16,300 x g for 10 min and dissolved in 100 ml of a mixture of

3 Issue of July 10, 1970 K. Uyeda and S. Kurooka 3317 TABLE Puri$cation of phosphofructokinase from C. paste&unum - Fractionation step Total VOlUIlH nz1 1. Crude extract Heat treatment Isopropanol First ammoniumsulfate Second ammonium sulfate DEAE chromatography and concentration First crystallization Second crystallization Third crystallization I Total ctivit3 u&its Specific activity w/ml units/mg FIG. 2. Sedimentation velocity patterns of crystalline phosphofructokinase. The patterns were determined with 0.8 ml of the sample containing 4.8 mg per ml of protein in 0.05 M potassium phosphate and 0.2 mm EDTA at ph 8.0. The measurements were made with a Spinco model E ultracentrifuge at a rotor speed of 47,660 rpm at an average temperature of The photographs were taken at a 55 bar angle at (a) 16 min and (b) 86 min after reaching the speed. iooc 9oc 80C 7oc 60C FIG. 1. Crystalline phosphofructokinase from C. paste&unum. The crystals were prepared in the absence (a) and the presence (5) of ATP as described in the text M potassium phosphate (ph 8) and 1 mm EDTA. The protein concentration of the enzyme solution was diluted to 10 mg per ml with the addition of the above buffer. The final volume was 240 ml. 4. First Ammonium Sulfate-To the above enzyme solution were added 240 ml of saturated ammonium sulfate solution, and the solution was stirred for 15 min. The precipitate was removed by centrifugation at 21,500 x g for 10 min and dissolved in 10 ml of a mixture of 0.05 M potassium phosphate (ph S), 1 mm EDTA, and 14 mm 2-mercaptoethanol. 5. Second Ammonium Sulfate-The above enzyme solution was diluted with addition of 15 ml of a mixture of 0.05 M potassium phosphate (ph S), (1 mu) EDTA, and 14 mu 2-mercaptoethanol to adjust protein concentration to 7 mg per ml. The final volume of the solution was 25 ml. To the enzyme solution, 20.5 ml of saturated ammonium sulfate solution were added with stirring, and the solution was allowed to stand 10 min. The precipitate was removed by centrifugation at 21,500 x g for 10 min and dissolved in 5 ml of a mixture of 0.05 M potassium phosphate (ph 8) and 1 MM EDTA. 6. DEAE-cellulose Chromatography-In order to remove excess salt, the enzyme solution was applied to a Sephadex G-25 column (1.5 x 27 cm) which had been equilibrated with potassium phosphate-edta-2-mercaptoethaaol mixture as above : i r2, (cm12 FIG. 3. High speed sedimentation equilibrium data of crystalline phosphofructokinase. The protein concentration was 0.2 mg per ml in 0.05 M potassium phosphate at ph 8.0. An Yphantis multichannel centerpiece with sapphire windows was used. The centrifugation was at 27,690 rpm for 24 hours at an average temperature of 20. and was eluted from the column with the same buffer mixture. The enzyme fractions were combined (20 ml) and adsorbed on a DEAE-cellulose (microgranular DE-52) column (1.7 x 10 cm) which had been equilibrated with the same buffer mixture. The column was washed with 70 ml of the buffer mixture containing 0.05 M NaCl until the absorbance of the solution at 280 rnp was less than The enzyme was then eluted from the column with the buffer mixture containing 0.15 M NaCI, and 3.5~ml

4 3318 Clostridial PhosphoS,,uctoXinase Vol. 245, Xo I I 1 r / 1 0 I MIGRATION DISTANCE (cm) FIG. 4. Analyt,ical acrylamide gel electrophoresis of clost ridial pllosphofructokirlasc. The protein concentration was 150 pg, and t,he conditions for the electrophoresis were as described in the text. The Amido black-stained gel was scanned at 600 rnp (--) with a Gilford model 240 recording spectrophotometer equipped with a gel scanner-linear 1 ransport unit. The enzymic activity (-----) was determined by slicing an unstained gel into about 2.mm sectiolw, which were homogenized with a glass rod alld added to Assay Mixture A. 0.6 Il lm WAVELENGTH FIG. 5. Ultraviolet absorption spectra of clostridial phosphofructokinase. The spectra were determined in 0.05 II potassium phosphate at ph 7 and in 0.1 s NaOII with a Cary model 15 recording spcctrophotometer. The protein concentration was 0.98 mg per ml based on dry weight. fractions were collected on an automatic fraction collector. The enzyme usually appeared in the fractions between 6 and 20, and these fractions were combined. The enzyme was then concentrated to about, 5 ml with a Diaflo concentrator using XM-50 membrane (Amicon, Boston, Massachusetts). The enzyme was precipitated with addition of 6 ml of saturated ammonium sul- (mp) T.\BL-E A mine acid composition of clostiidial pkoi;phofr~rcto~ir~nse The values and the standard deviations are calculated from the data at 24,48, and 72 hours of hydrolysis; the determinations xere performed in duplicate. The values for serine, threonine, and tyrosine are those obtained by extrapolating to zero hours of hydrolysis. For all other amino acids, the values are averages. Tyrosine and tryptophan were determined as described under Jlaterials and Methods. The recovery of the total amino acid based on the dry weight of the protein was Amino acid residue Lysine... Histidine... Arginine... Aspartic acid.... Threonine.... Serine Glutamic acid... Prolinc... Glyci nc... Alaninc... Half-cystine.... Valine... Methionine... Isolencine... Leucine.... Tyrosine.... Phenylalanine... II Number of residues?noles/145, f i f zk i f zlz zt F f f f f 0.G 22.0?t 2,s 30.7 f 0.9 fate solution and centrifuged, and the precipitate was dissolved in I.5 ml of the buffer mixture. 7. First Crystallization-To the above enzyme solution were added about 160 mg of ammonium sulfate with continuous stirring. When slight turbidit,g appeared, 2 pmoles of ATI were added, and crystals began to form in a few minutes. The enzyme solution was then allowed to stand overnight at 2. S. Second and Third Crystallixation-The crystals of the cnz\-me were then removed by centrifugation and dissolvrd in 1.5 ml of the same buffer mixture. The enzyme was recrystallized with addition of ammonium sulfate according to the above procedure (Step 7). The recrystallization was repeated once more under the same conditions. The enzyme could also be crystallized without addition of <4Tl in Step 7, but the crystals formed much more slowly. The enzyme was then stored as a crystalline suspension at 2..\n example of the purification procedure is summarized in Table I. Fig. 1 depicts the photographs of crystals of clostridial phosphofrudjokinase obtained in t,he presence and the absence of ATI. The shapes of t,he crystals are different depending upon the presence or absence of ATE duriug crystallization. HESULTS General Properties of Enzyme Stabilily--Crystalline suspensions of clost,ridial phosphofructokimasc in 4593 saturation of ammonium sulfate are stable for at least 2 months. After 2 N-eeks of storage, however, the enzyme showed a change in the activity such that a considerable lag period was observed when assayed in the standard assay mixture

5 Issue of July 10, 1970 K. Uyeda and S. Kurooka 3319 s 500 E 400 E zi g.z -0 Ei.f 900 I- 800 I- 700 I- 600 l- 3oc!A 2oc /-,- I- I- ioo- I I I I I I r2 (cm21 FIG. 6. Sedimentation equilibrium data of clostridial phospho fructokinase in guanidine. The enzyme (0.2 mg per ml) was dialyzed in 7 M guanidine and 0.1 M 2-mercaptoethanol for 24 hours at 2.,_ The dialyzed enzyme was placed in a 12-mm double sector centerpiece with sapphire windows, and the centrifugation was at 39,460 rpm for 24 hours at 20. TABLE III Molecular weights of native and dissociated phosphofructokinase Native Experimental conditions are described in the text. enzyme Dissociated enzyme Urea (4 M)-METa (0.1 M) Urea (6 M)-MET (0.1 M) Urea (8 M)-MET (0.1 M) Guanidine (7 M)- MET (0.06 M) Maleyl phosphofructokinase a 2-Mercaptoethanol. Method Molecular weight Sedimentation velocity Sedimentation equilibrium Sucrose density centrifugation Sedimentation librium Sedimentation librium Sedimentation librium Sedimentation librium Sedimentation librium equi- equi- equi- equi- equi- -h.w = 7.8 S 144,000 i 2, ,000 35,700 36,400 35,000 34,000 35,000 (Assay A, Materials and Methods ). The lag period is eliminated if the enzyme is previously incubated with 10 mm dithiothreitol. The result suggests the presence of a readily oxidizable sulfhydryl group (or groups) which is essential for the activity. Homogeneily-The crystalline enzyme was subjected to ultracentrifugation. The sedimentation velocity pattern of the enzyme shows a single symmetrical peak (Fig. 2). The high speed sedimentation equilibrium study was carried out according to the procedure of Yphantis (13). The result of the centrifugation is depicted in Fig. 3, which shows that all the points in the plot of the log of the fringe displacement vs. (radial distance)2 fa.11 on a straight line, indicating the homogeneity of the preparation. Fig. 4 shows the result of an analytical acrylamide gel electrophoresis of the crystalline enzyme. A symmetrical peak was obtained by scanning the stained gel with a Gilford gel scanner. The peak coincided exactly with the enzymic activity, which was determined by slicing the identical gel (without staining with Amido black) and assaying the enzymic activity in Assay A ( Materials and Methods ). Thus, the purified enzyme is homogeneous by both ultracentrifugal and electrophoretic cri- teria. Physical Properties of Enzyme Molecular Weight-From the result of the sedimentation velocity experiment (Fig. 2), the sedimentation coefficient, s20,w, of the enzyme was estimated as 7.8 S. By a high speed sedimentation equilibrium technique, the molecular weight of the enzyme was determined as 144,000 f 2,000 based on the results L------o. ELECTROPHORESIS FIG. 7. Tryptic peptide map of clostridial phosphofructokinase. The conditions for tryptic digestion and for paper chromatography and electrophoresis are described in the text. The solid line indicates the ninhydrin spots; shading indicates Sakaguchi-positive spots. Dotted line shows weak questionable spots. obtained with three different preparations of the enzyme at protein concentrations of 0.05 to 0.5 mg per ml (Fig. 3). For the calculation of molecular weight, the value of 0.74 was used as the partial specific volume, which was estimated from the amino acid composition. By sucrose density gradient centrifugation, the molecular weight of the enzyme is about 149,000, using pyruvate kinase, aldolase, and citrate synthase as internal marker proteins. The molecular weight of the enzyme was not altered by the presence of ATP (5 mm), ITP (5 mm), fructose- 6-P (5 mm or 0.3 mm), or ADP (10 as shown by the sucrose density gradient centrifugation. This observation is in contrast Origin 0

6 3320 Clostridial Phosphofructolcinase Vol. 245, No : ~FIG. 8. Analytical acrylamide gel electrophoresis of maleyl phosphofructokinase and the enzyme in urea. The preparation of maleyl phosphofructokinase is described in the text. (A) The maleyl phosphofructokinase (70 pg) was subjected to 10% anionic gel electrophoresis for 45 min at 4 ma and stained with Amido black. (B) The enzyme was dialyzed against 8 M urea for 24 hours. The dialyzed enzyme was placed on a 7.5% acrylamide gel containing 8 M urea. The electrophoresis was for 1 hour at 4 ma and was destained electrophoretically. The destained gels were scanned with a Gilford gel scanner as described in Fig. 4. to mammalian phosphofructokinases, which have been shown to be dissociated by ATP, especially at low protein concentration (22)* Ultraviolet Spectra-The absorption spectra of the enzyme at ph 7 and in 0.1 N NaOH are shown in Fig. 5. The spectrum at ph 7 shows an absorption maximum at 275 rnp and a minimum at 250 mp. The corresponding maximum and minimum in NaOH are 290 rnp and 270 rnp, respectively. The extinction coefficient (10 mg per ml) at 290 rnp in 0.1 N NaOH is 4.8, ba,sed on the dry weight of the protein. The low extinction coefficient suggests a low content of aromatic amino acids, as confirmed by amino acid analysis (Table II). The enzyme contains little tryptophan, since the enzyme shows essentially no distinct absorption peak in the region of 290 rne.c in 0.1 N NaOH. Amino Acid Composition Chemical Properties The amino acid analysis of phosphofructokinase is given in Table II. All data except tryptophan were obtained according to the method of Moore, Spackman, and Stein (23), using a Beckman model 120 amino acid analyzer with an integrator. The enzyme contains a relatively low concentration of aromatic amino acids, which is in agreement with the observation that the enzyme has a low molar extinction coefficient at 290 rnp (Fig. 5). The partial specific volume (G) calculated based on the amino acid composition (24) is 0.74 ml per g. Subunit Structure The clostridial enzyme was treated with three different reagents which are commonly employed to disrupt the quaternary structure of proteins. Guanidinium Chloride-A solution of the enzyme (200 pg per ml) was dialyzed for 48 hours at 4 under argon against 0.05 M 0.12mM I I I I FIG. 9. The effect of fructose-6-p on the velocity of the reaction with ATP as the variable substrate. Assay B was used. The concentrations of fructose-6-p (F6P) used are indicated above the lines. The insert shows the double reciprocal plot of the data. Tris-HCl, ph 7.3, containing 7 M guanidine and 100 mm 2-mereaptoethanol. The dialyzed enzyme in guanidine was then subjected to a high speed sedimentation equilibrium according to the procedure of Yphantis (13), and the result is shown in Fig. 6. The plot of the log of the fringe displacement vs. (radial distance)2 shows the homogeneity of the dissociated protein. Assuming the value for partial specific volume, B, as 0.74, the molecular weight of the dissociated enzyme in guanidine is 34,000, as summarized in Table III. A sedimentation velocity experiment of the enzyme (5 mg per ml) in 6.5 M guanidine and 0.05 M 2-mercaptoethanol revealed a single boundary. The sedimentation coefficient, s~~,~, was estimated as approximately 2.0 S. Urea-The enzyme solution (100 to 400 pg per ml) was dialyzed for 24 hours at 2-4 against 4 M, 6 M or 8 M urea containing 0.1 IVY 2-mercaptoethanol under argon. The dialyzed enzyme in urea was then subjected to high speed sedimentation equilibrium centrifugation as above. The plots of the log of the fringe displacement vs. (radial distance)2 yielded straight lines in all cases. As summarized in Table III, the molecular weights of the enzyme in different concentrations of urea calculated from the plots are 35,000 to 36,400, which are in good agreement with the value obtained in 7 M guanidine. The result shows that clostridial phosphofructokinase consists of 4 subunits whose molecular weight is 35,000 =t 1,400. Maleylated PhosphofructohGnase-Reaction of proteins with maleic anhydride has been shown to result in dissociation of the protein, and the method has been employed extensively by Sia and Horecker (25) for their study on subunit structure of aldol- ATP, (mm).

7 Issue of July 10, a E 4 \ 1 E 3 : K. Uyeda and S. Kurooka 3321 TABLE Hill coeficients for fructose-6-p in the presence of di$erent concentrations of ATP and ADP IV Compound Hill coeficient ATP (0.5 mrvr). 3.6 ATP (1 mm) _ ATP (5 mm) 3.7 ATP (1 mrvr) + ADP (0). 4.2 ATP (1 mrvr) + ADP (0.5 mrvr) 4.1 ATP (1 mm) + ADP (1 mm) 3.2 ATP (1 miw) + ADP (5 miu) ATP (1 mm) + ADP (10 mna) FGP,(mM) FIG. 10. The effect of ATP on the velocity of the reaction with fructose-6-p as the variable substrate. Assay B was used. The concentrations of ATP used are indicated. The insert shows the double reciprocal plot of the data. The results of Fig. 10 and Fig. 12 were used for the calculation of Hill coefficients. The coefficients were calculated from the slopes of the plots of log (v/(v,,, - v)) vs. log (fructose-6-p). 16 ase and fructose diphosphatase. In order to obtain additional information concerning the subunit, clostridial phosphofructokinase was reacted with maleic anhydride as described under Materials and Methods, and the molecular weight and homogeneity of the protein were analyzed by sedimentation equilibrium techniques. The linearity of the resulting plots indicated the homogeneity of the maleylated phosphofructokinase, and the molecular weight of the protein was estimated as 35,000, which is again in close agreement with above results. 0 I Tryptic Peptide $1 upping The peptide map of the tryptic digest of a heat-denatured phosphofructokinase is shown in Fig. 7. The total number of peptides observed varied between 33 and 38. The varia.tion is due to a few weak questionable spots. Since the enzyme contains approximately 34 arginine and lysine residues per 35,000 molecular weight units, the observed number of peptides in the tryptic digest supports the idea that all 4 subunits are identical. More reassuring, perhaps, is the result in Fig. 7, showing that I5 arginine-containing spots were detected when the peptide map was sprayed with Sakaguchi reagent. This value is in close agreement with the expected number of 18 arginine-containing peptides if the subunits are identical. Thus, the observed numbers of total peptides and of arginine-containing peptides are in good agreement with the predicted values for 4 identical subunits. If the subunits are not identical, they must be very similar in amino acid sequence. Two additional lines of evidence in support of the conclusion that the subunits of clostridial phosphofructokinase are identical are as follows. When maleylated phosphofructokinase (molecular weight 35,000) was subjected to acrylamide gel electrophoresis in 10% anionic gel, the protein migrated as a single band, indicating that the chemically dissociated subunits are also identical or very similar in structure (Fig. &I). Furthermore, when the dissociated enzyme in 8 M urea was analyzed by disc electrophoresis in 8 M urea, a single band was obtained, as shown in Fig. 8B. These results support the above contention that the subunits in the phosphofruct,okinase are identical. 1 I I 1 2 ATP, FIG. 11. The effect of ADP on the velocity of the reaction with ATP as the variable substrate. The concentration of fructose-6-p was 1 mm, and the concentrations of ADP used are indicated. Assay A was used, and other experimental details are given under Materials andmethods. Kinetic (mm) Properties E$ect of ph-the enzyme showed maximum activity at ph 7 to 8.2. At ph 6 and 10 the activity was approximately 60% that of the optimum, and the enzyme was completely inactive at ph 5. Kinetics with Respect to ATP and Fructose-6-P--Plots of the initial velocity versus ATP concentration, obtained at several fixed concentrations of fructose-6-p, are shown in Fig. 9. The Lineweaver-Burk plots (Fig. 9, insert) were linear and gave a series of apparently parallel lines. The K, value for ATP at infinite fructose-6-p concentration is 5.5 x 10e5 M. As discussed below (Fig. ll), the higher concentration of ATP (5 mm) seems to show no or only slight inhibition. The extent of ATP inhibition

8 3322 Clostridial Phosphofructokinase Vol. 245, No F6P, (mm) FIG. 12. The effect of ADP on the velocity of the reaction with fructose-6-p (F6P) as the variable substrate. The concentration of ATP was 1 mm, and the concentrations of ADP used are indicated. Assay A was used, and other experimental detail are given under Materials and Methods TABLE K, values of different nucleotides Assay A was used except for the addition of different nucleoside triphosphates. Other experimental details are given under Materials and Methods. Compound ATP. UTP. CTP... ITP. GTP. V I( 6.5 X 1OP 2.5 x lo- 3.3 x 10-d 8.3 x lo- 8.3 x 10-d was not increased when Mg to ATP ratios were 1: 1 and 2: 1. The lack of inhibition by ATP is in contrast to phosphofructokinases of higher organisms. A possibility that the lack of ATP inhibition was caused by some alteration of the enzyme during purification was examined by testing the effect of ATP with the crude and partially purified enzymes. Neither the enzyme in the crude extract nor heat-treated enzyme (purification method Step 2) showed any significant inhibition by ATP (5 mm>. Moreover, the ATP inhibition is not apparent at ph 6.8, 7.4, and 8. Plots of the initial velocity against fructose-6-p concentration obtained at several fixed concentrations of ATP yielded a series of sigmoidal curves (Fig. 10). The Lineweaver-Burk plots yielded a family of lines which are concave upward. The plot of the log (z)/(v,,~ - v)) against the log of fructose-6-p concentration (Hill plot) gave straight lines; Hill coefficients of the lines were calculated from the slopes and summarized in Table IV. The Hill coefficient for fructose-6-p is close to 4 at all levels of ATP. E$ect of ADP-Fig. 11 shows the effect of different concentrations of ADP on the kinetics with respect to ATP. ADP at FGP,(mM) FIG. 13. The effect of different nucleoside triphosphates on the velocity of the reaction with fructose-6-p (F6P) as the variable substrate. The concentrations of nucleoside triphosphates were at 1 mm. Assay A was used, and other experimental details are given under Materials and Methods. 0 2 i 6 8 IO (NH:)-! (mm)- FIG. 14. The effect of ATP on the velocity of the reaction with a variable concentration of NH&l. The concentrat.ions of ATP used are as indicated, the Mg to ATP ratios were maintained at 2:l. Assay A was used, and other experimental conditions are given under Materials and Methods. 0.5 mm increases V,,, about 2 times, but at higher concentrations the compound inhibits the enzyme. Thus, ADP acts as a stimulator as well as an inhibitor depending upon the concentration. Since its effect is complex, the nature of the inhibition could not be determined accurately, but the Lineweaver-Burk plot seems to suggest that ADP is a noncompetitive inhibitor of ATP. In order to understand more fully the nature of ADP stimulation, its effect on fructose-6-p was next investigated. Fig. 12 illustrates the effect of variable ADP on the kinetics with respect to fructose-6-p. As can be seen, the apparent K, for fructose- 6-P is significantly lowered in the presence of higher concentrations of ADP, and the sigmoidal shape of the curves is more normalized. The Hill coefficients for these curves are calcula,ted from the plots of the log (v/(v,,, - v)) versus log (fructose-6-p) and summarized in Table IV. The coefficients were decreased from about 4 to 1.7 in the presence of 10 m ADP. Xpeci$city for Nucleoside Triphosphate-As shown in Table V, clostridial phosphofructokinase shows activity with GTP, ITP, UTP, and CTP. The K, values determined at an infinite concentration of fructose-6-p are also summarized in the table, -I

9 Issue of July 10, 1970 K. Uyeda and X. Kurooka and the result indicates that K, for ATP is at least 3 times lower than the other nucleotides examined. It is of interest to see what effect these nucleotides may have on the kinetics with respect to fructose-6-p. The result of this experiment is shown in Fig. 13 and indicates that UTP and CTP exhibit an effect similar to ATP, resulting in the sigmoidal curves for fructose-6-p. The effect, however, seems to be much less with GTP or ITP. Moreover, vmsx is at least 2.5 times higher with GTP or ITP as a substrate than ATP, UTP, or CTP. Effect of Cations-Phosphofructokinases from various sources show varying effects with different cations. Clostridial phosphofructokinase shows absolute dependence on NH,+. When the rate of the reaction is determined in the presence of varying concentrations of NH&I at several fixed concentrations of ATP-Mg++ (0.04 mu, 0.1 mm, 0.4 mu, and 1 MM), the corresponding Lineweaver-Burk plot of the results, as depicted in Fig. 14, shows a common intercept on the X-axis. The K, for NH4+ at 1 mu ATP was estimated as 1.8 x 10-d M. Under the same conditions, K, for K+ is approximately 2 x lo-+ M. Kf causes inhibition at higher concentration; 0.1 M and 0.2 M KC1 inhibits the phosphofructokinase 15% and 50%, respectively. Na+ neither activates nor inhibits the clostridial phosphofructokinase. Effect of Mg+f on the initial velocity of the reaction shows no cooperativity, and the metal ion probably participates only in chelation with ATP, which is perhaps the true substrate for the enzyme. E$ect of Other Compound-A possible stimulation or inhibition of clostridial phosphofructokinase activity by the following compounds was investigated: AMP, cyclic AMP, fructose 1,6- diphosphate, coenzyme A, pyruvate, glucose, P-enolpyruvate, citrate, IMP, glucose-6-p, and phosphate. Among these compounds, only AMP (5 mu) and cyclic AMP (5 InM) stimulated the enzymic activity about 30%. Fructose-l, 6-di-P at 1 and 5 mu did not affect the activity, but at 10 mm it inhibited about 20%. Thus, the effect of these compounds is small, and the concentration of the compounds required to influence the activity is so high that the effect probably has little physiological significa.nce. All the other compounds tested at 1 to 10 mm neither stimulate nor inhibit the enzyme under these conditions. DISCUSSION Phosphofructokinases from rabbit skeletal muscle (21, 26), heart muscle (5), and yeast (7) have been obtained in pure form. The skeletal and heart muscle enzymes (22, 27) have been crystallized, but only in the presence of ATP. So far, phosphofructokinase from C. pasteuriunum is the only phosphofructokinase which has been crystallized in the absence of the nucleotide. The specific activity of crystalline clostridial phosphofructokinase, 160, is comparable to those of muscle, 160 (22); heart, 125 (27); E. coli, 190 (9); and yeast, 116 (7). Studies on physico-chemical properties of clostridial phosphofructokinase revealed considerable difference from those of mammalian phosphofructokinase. For example, this clostridial phosphofructokinase does not show any concentration-dependent aggregation or disaggregation phenomenon. The molecular weight of the enzyme is about 144,000 at a protein concentration of between 10 pg and 5 mg as determined by sedimentation equilibrium, sedimentation velocity, and sucrose density gradient centrifugation. It has also been demonstrated by Parmeggiani et al. (22) that the muscle phosphofructokinase is dissociated in the presence of ATP. Such a dissociation phenomenon has not been observed with clostridial phosphofructokinase in the presence of ATP, fructose-6-p, ITP, or ADP. Phosphofructokinase exhibits complex allosteric properties. The degree of the complexity, however, depends on the source of the enzyme. For instance, mammalian phosphofructokinases are inhibited by ATP and citrate, and this inhibition is counteracted by cyclic AMP, AMP, ADP, phosphate, fructose-6-p, and fructose-l, 6-di-P. Yeast or insect phosphofructokinases are not inhibited by citrate, and only AMP and phosphate are effective in releasing ATP inhibition. ATP does not inhibit E. coli, D. discoideum, or A. crystallopoietes phosphofructokinase. The latter two enzymes seem to lack any of the allosteric properties described above. Our present investigation shows that clostridial phosphofructokinase is inhibited only 20% by high concentration (5 mu) of ATP. It appears, therefore, that the ATP inhibition of phosphofructokinase is probably not important in the regulation of glycolysis in C. pasteuriunum. Perhaps more significant in regulation is the observation that the activity of clostridial phosphofructokinase is affected by the concentration of fructose-6-p and the relative concentration of ATP to ADP. The latter shows strong activation. No other compounds so far tested have any effect on the activity of clostridial enzyme, including P-enolpyruvate, which has been shown to be an inhibitor of muscle (12) and E. coli (10) phosphofructokinases. Thus, the C. paste&unum phosphofructokinase exhibits perhaps the simplest regulatory mechanism among allosteric phosphofructokinases. A significant difference between the clostridial and muscle phosphofructokinase is the effect of ATP upon the affinity for fructose-6-p. Mammalian phosphofructokinase is inhibited strongly by high concentration of ATP. This inhibition can be attributed to the decreased affinity of the enzyme for fructose- 6-P, as shown by the sigmoidal nature of the initial velocity curve. At lower concentrations of ATP, however, the kinetic pattern is normal. In contrast, clostrid.al phosphofructokinase exhibits a sigmoidal curve with respect to fructose-6-p at all concentrations of ATP. CTP and UTP exhibit the same effect as ATP. GTP and ITP, however, show a strikingly different effect from the other nucleoside triphosphates, in that they not only decrease the K, from fructose-6-p but also increase the V max about 2 times. To our knowledge, no studies have yet been reported on the effect of different nucleoside triphosphates on the kinetics with respect to fructose-6-p by bacterial enzymes. ADP exhibit an interesting allosteric effect with respect to fructose-g-p, as shown in Fig. 12. An increasing concentration of ADP decreases the K, of fructose-6-p and also lowers the Hill coefficient for fructose-6-p from 4 to 1.7. A similar effect of ADP upon E. coli phosphofructokinase has been observed by Atkinson and Walton (28) and Blangy, But, and Monod (10). For the simplest interpretation of the results reported here, the following model can be suggested, based on the model of concerted transition theory proposed by Blangy et cd. (10). (a) The enzyme consists of 4 identical protomers, each protomer containing a binding site for each substrate. (6) The enzyme exists in at least 2 conformational states, which have different affinities for fructose-6-p. Assuming the enzyme follows compulsory order of addition, with ATP binding first followed by fructose-g-p, the enzyme-atp complex has low affinity for fructose-6-p. On the other hand, the enzyme complex with

10 3324 Clostridial Phosphofructokinase Vol. 245, No. 13 GTP or ITP shows high affinity for fructose-6-p. (c) ADP 5. WALKER, P. R., AND BAILEY, E., Biochem. J., 111, 365 (1969). causes the transition of the conformation from low to high 6. BETZ, A., AND MOORE, C., Arch. Biochem. Biophys., 120, 268 (1967). affinity states. This model accounts qualitatively for the 7. LINDELL, T. J., AND STELLWAGON, E., J. Biol. Chem., 243, results presented here. It is difficult, however, to give any 907 (1968). quantitative estimate of rate and thermodynamic parameters 8. BAUM~NN, P., AND WRIGHT, B. E., Biochemistry, 7,3653 (1968). for the various interactions between the substrates. Neither 9. GRIFFIN, C. C., Houcx, B. N., AND BRAND, L., Biochem. Biois it possible to decide whether the transition between these phys. Res. Commun., 27, 287 (1967). 10. BLANGY, D., But, H., AND MONOD, J., J. Mol. Biol., 31, 13 conformational states is concerted (29) or sequential (30). (1968). The sigmoidal kinetics observed for fructose-6-p with mam- 11. FERDINANDUS, J., AND CLARK, J. B., Biochem. J., 113, 735 malian phosphofructokinase occurs only below ph 7.5; above 8 (1969). the enzyme exhibits Michaelis-Menten kinetics. In contrast, 12. UYEDA, K., AND RACICER, E., J. Biol. Chem., 240, 4682 (1965). 13. YPHANTIS, D. A., Biochemistry, 3, 297 (1964). clostridial phosphofructokinase shows the sigmoidal kinetics 14. MARTIN, R. G., AND AMES, B. N., J. Biol. Chem., 236, 1372 with respect to fructose-6-p at ph values of 6, 7, and 8.1. (1961). The effect of the concentration of fructose-6-p on the initial 15. DAVIES, E. J., Ann. N. Y. Acad. Sci., 121,404 (1964). velocity of the reaction with respect to ATP varied such that a 16. MOORE, S., J. Biol. Chem., 238, 235 (1963). family of parallel lines were obtained. The effect of ATP 17. BENCZE, W. L., AND SCHMID, K., Anal. Chem., 29, 1193 (1957). 18. UYEDA, K., Biochemistry, 8, 2366 (1969). on the initial rate of the reaction with fructose-6-p did not yield lo LY. CHERNOFF, A. J., AND LIU, J C., Blood J. Hematol., 17, 54 straight lines, but rather curved lines which are concave upward. (1961). Similar kinetics yielding a series of parallel lines has been ob- 20. RABINOWITZ, J. C., AND PRICER, W. E., JR., J. Biol. Chem., tained with muscle (31, 32), yeast (33), and D. discoideum (8). 237, 2898 (1962). 21. LOVENBERG, W., BUCHANAN, B. B., AND RABINOWITZ, J. C., This type of pattern has been called ping-pang kinetics (34) ; J. Biol. Chem., 238, 3899 (1963). it is consistent with the reaction mechanism in which the first 22. PARMEGGIANI, A:, LUFT, J. H., LOVE, D. S., AND KREBS, E. G., product dissociates from the enzyme before the second substrate J. Biol. Chem (1966). binds, and may suggest a formation of phosphoryl enzyme as an 23. MOORE, S., SPA&MA;, D. H:, AN; STEIN, W. H., Anal. Chem., intermediate. Phosphofructokinases from muscle (12) and D. discoideum (8) have been shown to be stimulated by NH,+ but the physiological significance of the stimulation is completely unknown. Clostridial phosphofructokinase is absolutely dependent on NH4+ for its activity and shows low K, for the cation. It is tempting to suggest that, in an organism such as C. pasteurianum which fixes Nz, NH4+ stimulation of phosphofructokinase may be of some physiological significance. For example, it may act as a feed-forward effector (35) which increases the rate of ATP synthesis, which is essential for NP fixation, by stimulation of glycolysis through activation of phosphofructokinase. REFERENCES 1. STADTMAN, E. R., Advan. Enzymol., 28,41 (1966). 2. ATKINSON. D. E.. Annu. Rev. Biochem., 36, 85 (1966) DENNIS, D. T., AAD COULTATE, T. P., &o&m. Biop hys. Ada, , 129 (1967). 4. KELLEY, G. J., AND TURNER, J. F., Biochem. Biophys. RES. 34. Commun., 30, 195 (1968) , 1185 (1958). SCHACI-IMAN, H. K., in S. P. COLOWICK AND N. 0. KAPLAN (Editors), Methods in enzymology, VoZ. IV, Academic Press, New York, 1957, p. 70. SIA, C. L., AND HORECKER, B. L., Biochim. Biophys. Res. Commun., 31, 731 (1968). LING, K.-H., MARCUS, F., AND LARDY, H. A., J. Biol. Chem., 240, 1893 (1965). MANSOUR, T. E., WAKID, N., AND SPROUSE, H. M., J. Biol. Chem., 241, 1512 (1966). ATKINSON, D. E., AND WALTON, G. M., J. Biol. Chem., 240, 757 (1965). MONOD, J., WYMAN, J., AND CHANGEUX, J. P., J. Mol. Biol., 12, 88 (1965). KOSHLAND, D. E., NEMETHY, G., AND FILMER, D., Biochemistry, 6, 365 (1965). LAYZER, R. B., ROWLAND, L. P., AND BANK, W. J., J. Biol. Chem., 244, 3823 (1969). UYEDA, K., J. Biol. Chem., 246, 2268 (1970). VINUELA, E., SALAS, M. L., AND SOLS, A., Biochem. Biophys. Res. Commun., 12, 140 (1963). CLELAND, W. W., Biochim. Biophys. Acta, 67, 104 (1963). HESS, B., Biochem. Biophys. Res. Commun., 24, 824 (1966).

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