[32] Pyruvate, orthophosphase dikinase from acebacter xylinum

192

KINASES

[32]

[32] P y r u v a t e , O r t h o p h o s p h a t e D i k i n a s e f r o m Acetobacter xylinum

By MOSHE BENZlMAN Pyruvate + ATP + Pi Mg2+~.PEP + AMP + PPI

Assay Methods Method 1 Principle. Pyruvate, orthophosphate dikinase activity in the forward direction (formation of PEP) is measured as the ATP-dependent formation of PEP from pyruvate and Pi. The PEP in deproteinized reaction mixtures is determined by the oxidation of NADH in the presence of pyruvate kinase and lactate dehydrogenase. To prevent the reverse reaction from occurring, assay systems are supplemented with exogenous pyrophosphatase or adenylate kinase. The carboxylation of the formed PEP to oxaloacetate, in crude extracts, is prevented by the addition of succinate, which strongly inhibits PEP-carboxylase activity? This assay method is equally applicable to crude and purified preparations. Reagents Tris.H~SO~, 1 M, pH 8.2 Sodium pyruvate, 0.1 M MgC12, 0.1 M ATP, 0.05 M, pH 7.0 Sodium succinate, 0.1 M, pH 8.2 Potassium phosphate, 50 raM, pH 8.2 NADH, 10 mM KC1, 1 M ADP, 10 mM, pH 7.0 Pyrophosphatase, crystalline (200 units/ml) Lactate dehydrogenase, crystalline (360 units/ml) Pyruvate kinase, crystalline (300 units/ml) Enzyme, diluted with 5 mM Tris.H2SO4 buffer (pH 7.4) containing 1 mM MgC12, 5 mM EDTA, and 0.5 mM dithiothreitol (TMEDbuffer) to obtain 0.5-2.0 units (forward direction) per milliliter (for definition of units, see below) ~M. Benziman, J. Bacleriol. 98, 1005 (1969).

[32]

PYRUVATE, ORTHOPttOSPHATE DIKINASE FROM

A. xylinum

193

Procedure. Reagents are pipetted into test tubes as follows: Tris.H~SO, buffer, 0.10 ml; MgCI~, 0.10 ml; Na suceinate, 0.20 ml; ATP, 0.10 ml; K phosphate, 0.20 ml; pyrophosphatase, 0.01 ml; diluted enzyme preparation, 0.05 ml, and distilled water to make a final volume of 1.00 ml. ATP is omitted from the control tube. The tubes are equilibrated at 30 ° and the reaction is started by the addition of 0.02 ml of the pyruvate solution. After incubation for 5 rain the mixtures are placed in a boiling water bath for 1 min, to stop the reaction and then cooled in ice. After centrifugation, 0.2-ml samples of the supernatant solutions are transferred to silica cuvettes (10 mm light path) containing 0.1 ml Tris.H2SQ buffer, 0.05 ml MgC12, 0.05 ml KC1, 0.05 ml ADP, 0.05 ml NADH, 0.01 ml lactate dehydrogenase, and water to give a final volmne of 1.0 ml. Absorbancy is measured at 340 nm. When absorbancy readings become constant, 0.01 ml of the pyruvate kinase suspension is added and the change in absorbancy is followed until no further change occurs (about 5 min). The difference between the initial and final absorbancies is used to calculate the amount of PEP present in the sample. Under these conditions, the rate of PEP formation is linear with respect to time up to an incubation period of approximately 10 rain, and directly proportional to the amount of enzyme up to 0.07 unit of pyruvate, orthophosphate dikinase. Method 2 Principle. The activity of pyruvate, phosphate dikinase in the reverse reaction (formation of pyruvate) is measured as the PPi-dependent formation of pyruvate from PEP and AMP. This is assayed spectrophotometrically in the presence of lactate dehydrogenase, by measuring the rate of decrease in absorbancy at 340 mn concomitant with the oxidation of NADH. The oxidation of NADH in the absence of PPi is used as a control, and this absorbancy change is subtracted from the observed value for the complete system. With crude extracts, where the correction for the control is large, the assay is done in a stepwise manner. The dikinase reaction is allowed to proceed for 5 rain in the absence of NADH, and lactate dehydrogenase and is stopped by heating the reaction mixture for 1 min in a boiling water bath. After centrifugation the pyruvate in the supernatant solution is determined with lactate dehydrogenase. Reagents Imidazole.HC1, 0.5 M, pH 7.0 PEP (tricyclohexylammonium salt), 0.1 M, pH 7.0 AMP, 0.02 M, pH 7.0

194

KI~ASES

[32]

Sodium pyrophosphate, 0.1 M, pH 7.0 MgCI_~ 0.1 M NADH, 0.01 M Lactate dehydrogenase, crystalline (360 units/ml) Enzyme, diluted with TMED-buffer (pH 7.4) to obtain 0.1-1.0 unit (reverse direction) per milliliter (for definition of units, see below)

Procedure. Reagents are pipetted into silica cuvettes (10 mm light path) as follows: imidazole.HC1 buffer, 0.10 ml; PEP, 0.02 ml; MgC12, 0.10 ml; AMP, 0.10 ml; NADH, 0.02 ml; diluted enzyme preparation, 0.05 ml; lactate dehydrogenase, 0.01 ml and water to make a final volume of 1.00 ml. A blank cuvette contains all components with the exception of NADH. Any changes in absorbancy at 340 nm, which may be caused by NADH-oxidase present in the enzyme preparation, are recorded for 1-2 min. The reaction is then started by the addition of 0.01 ml of the PPi solution and the linear rate of decrease in absorbancy at 340 nm is recorded thereafter. The control value is subtracted from the rate observed after the addition of PPi. The reaction is carried out at 20-22% Pyruvate formation, when coupled to the lactate dehydrogenase system is linear with time and with enzyme concentration up to 0.05 unit of pyruvate, orthophosphate dikinase. Method 3 Principle. The pyruvate phosphate dikinase reaction in the reverse direction can also be assayed spectrophotometrically by measuring the rate of ATP formation as the increase in absorbancy at 340 nm in a reaction coupled with hexokinase and glucose-6-P dehydrogenase. This assay cannot be used with crude extracts because of their NADPH-oxidase activity. It can, however, be used satisfactorily after step 1 of the purification procedure. Reagents Imidazole.HC1, 0.5 M, pH 7.0 P E P (tricycloammonium salt) 0.1 M, pH 7.0 AMP, 0.02 M, pH 7.0 Sodium pyrophosphate, 0.1 M, pH 7.0 MgC12 0.1 M NADP, 10 mM Glucose, 0.1 M Hexokinase, crystalline (400 units/ml)

[32]

PYRUVATE, ORTHOPHOSPHATE DIKINASE FROM A .

xylinum

195

Glucose-6-phosphate dehydrogenase (500 units/ml) Enzyme, diluted with TMED-buffer (pH 7.4) to obtain 0.1-1.0 unit (reverse direction) per milliliter (for definition of units, see below) Procedure. Reagents are pipetted into a silica cuvette (10-ram light path) as follows: imidazole.HC1 buffer, 0.10 ml; PEP, 0.02 ml; MgC12, 0.1 ml; AMP, 0.10 ml; NADP, 0.03 ml; glucose, 0.05 ml; hexokinase, 0.01 ml; glueose-6-P dehydrogenase 0.01 ml; diluted enzyme preparation 0.05 ml, and water to a final volume of 1.00 ml. The reaction is started by adding 0.01 ml of the PPi solution and is followed by the increase in absorbaney at 340 nm. The reaction is carried out at 20-22 °. ATP formation, when coupled to the hexokinase-glucose-6-P dehydrogenase system, is linear with respect to time and is directly proportional to enzyme concentrations up to 0.05 unit of pyruvate, phosphate dikinase. Units. Units of enzyme activity are based either on the forward or on the reverse reaction when carried out under the conditions described. When measured by the forward reaction, one enzyme unit is defined as the amount of enzyme that will catalyze the formation of 1 t~mole of PEP per minute. When measured by the reverse reaction, one enzyme unit is defined as the amount that will catalyze the formation of 1 ~mole of pyruvate or ATP per minute. Units for the forward and reverse reaction are not equivalent. Specific activity is expressed as units per milligram of protein. Protein is determined by the colorimetric method of Lowry et al.'-' with crystalline bovine serum albumin as standard.

Other Assay Methods

Various other assay methods for this enzyme have been reported. The reaction proceeding toward PEP synthesis has been followed spectrophotometrieally by measuring the increase in absorbancy at 340 nm in a coupled system in which lactate, NAD, and lactate dehydrogenase were substituted for pyruvate. 3 This metbod, however, restricts the concentration of pyruvate in the assay mixture. The forward reaction has also been measured either spectrophotometrieally by coupling with PEP-carboxytransphosphorylase and malate dehydrogenase,4 or by measuring 14C02 incorporation into aspartate in the presence of PEP carboxylase, 20. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 3 M. Benziman and A. Palgi, J. Bacteriol. 104, 211 (1970). 4H. J. Evans and H. G. Wood, Biochemistry 10, 721 (1971).

196

KINASES

[3~]

aspartate aminotransferase, and glutamate. '~ Enzyme activity in the reverse reaction has been assayed by measuring the formation of pyruvate determined eolorimetrieally as the dinitrophyenyl hydrazine derivative2 Titrimetrie methods have been described based on hydrogen ion release in the forward reaction or on its uptake in the reverse reaction.; Purification Procedure

Growth of Organism. The organism used in this study was the cellulose-synthesizing A. xyllinum wild type employed in earlier investigations reported from this laboratory. 8 It was grown on succinate and harvested as described elsewhere in this series2 All the following steps were carried out at 15-25 °. Step 1. Preparation of Crude Extract. Cells were suspended in 5 mM Tris'H.,SO4--1 mM MgC1._,-5 mM EDTA-0.5 mM dithiothreitol buffer (TMED buffer) at pH 7.4, to give a final concentration of 30 mg (dry weight) of cells per milliliter. Portions (25 ml) of this suspension were treated for 15 min in a Raytheon Model DF 101 magnetorestrictive oscillator at 200 W and 10 kHz/sec. The sonic extract was then centrifuged at 18,000 g for 15 rain, and the precipitate was discarded. Step 2. High Speed Centrifugation. The crude extract was centrifuged in a Beckman Model L-2 ultracentrifuge at 150,000 g for 60 rain. The resulting sediment was discarded. Step 3. DEAE-Cellulose Chromatography. The high speed supernatant fraction was applied to a 1.5 X 20 cm DEAE-cellulose (Whatman DE-52) column, preequilibrated with TMED-buffer, pH 7.4. The column was eluted with 80 ml of 0.15 M KC1 in TMED buffer pH 7.4 at a rate of 90 ml/hr to remove undesired proteins. Dikinase activity is not eluted by this treatment. The concentration of KC1 was next increased to 0.25 M, and 3-ml fractions were collected at a flow rate of 40 ml/hr. The peak of enzymic activity appeared after 15 ml of eluate were collected. Eightyfive percent of the original activity was recovered in three fractions, which were combined. Step 4. Ammonium Sul]ate Fractionation. Solid ammonium sulfate to 35% saturation (20.9 g/100 ml) was added to the combined fractions from the previous step. The salt was added slowly with stirring, and 15 rain was allowed for precipitation. The precipitate was removed by cenM. D. Hatch and C. R. Slack, Biochem. J. 106, 141 (1968). 6y. Milner and It. G. Wood, Proc. Nat. Acad. Sci. U.S. 69, 2463 (1972). R. E. Reeves, Bioct, em. J. 12~;, 531 (1971). '~M. Schramm and S. Hestrin, J. Gen. Microbiol. l l , 123 (1954). M. Benziman, tiffs series, Vol. 13 [22].

[32]

PYRUVATE, ORTHOPHOSPttATE DIKINASE FROM

A. xylinum

197

PURIFICATION PROCEDURE FOR PYRUVATE ORTHOPHOSPHATE DIKINASE

Step and fraction 1. Crude extract 2. Supernatant of high speed centrifugation 3. DEAE-cellulose pooled fractions 4. (NH4)~SO4 precipitate (0.35 to 0.55 saturation)

Volume (ml)

Protein (mg)

Activity (units)~

Specific activityb

Yield (%)

70 70

1000 450

19 18

0.019 0.040

100 95

9

30

16

0.520

85

7

15

13

0.880

68

a One unit is the amount of enzyme catalyzing the formation of 1 tLmole of PEP per minute under the conditions of Assay Method 1. b Units per milligram of protein. trifugation at 15,000 g for 15 rain and discarded. The supernatant solution was then brought to 55% saturation (12.9 g/100 ml), stirred, and centrifuged as before. The precipitate was dissolved in a small volume of T M E D buffer p H 7.4. This fraction had a specific activity of 0.88 in the forward reaction and 0.36 in the reverse reaction when assayed under standard conditions. I t represents a 46-fold purification over the crude extract in 68% yield as measured by activity in both the forward and the reverse directions. A s u m m a r y of the purification procedure is presented in the accompanying table.

Properties The 46-fold purified kinase is free of p y r u v a t e kinase, P E P earboxylase, adenylate kinase, enolase and contains only traces of A T P a s e and pyrophosphatase activities. Crude extracts lost 65% of their pyruvate, phosphate dikinase activity in about 5 hr when kept at 4 °. The purified enzyme retained about 85% of its activity for at least 2 weeks when k e p t at room temperature. Stoichiometry. One mole of P E P , AMP, and Pi is formed for each mole of pyruvate, ATP, and PPi utilized in the reaction. Effect o] pH. The optimum p H for P E P synthesis is p H 8.2; for the reverse reaction it is p H 6.5. Ratio o] Activities o] the Foru,ard to Reverse Reaction. When the enzyme is assayed in both directions at 30 ° the ratio of the rate of the forward to the reverse reaction is 5.1 at p H 8.2 and 0.45 at p H 6.5. Specificity. G T P , CTP, I T P , or TTP at 10 m M concentrations cannot replace A T P in the reaction with pyruvate. Arsenate can be substituted

198

XINAS~S

[32]

for phosphate. At equimolar concentrations, however, the rate of PEP formation in the presence of arsenate is 65% of that obtained with phosphate. In the presence of 5 mM phosphate the further addition of 5 mM arsenate does not affect activity. GMP, CMP, and ADP (5 mM each) do not substitute in the reverse reaction for AMP. Metal Ion Requirement. Mg 2÷ is required for activity. Mn 2÷ or Ca 2+ at 5 mM concentrations cannot replace Mg 2~ in either the forward or the reverse reaction. Substrate Affinity Constants. The apparent Km values for the components of the system are as follows: Forward reaction, pyruvate, 0.2 mM; ATP, 0.4 raM; Pi, 0.8 mM; Mg 2+, 2.2 mM; reverse reaction, PEP, 0.1 mM; AMP, 1.6 ~M; PP~ 0.06 m M ; Mg 2+, 0.87 raM. Inhibitors. PEP formation is competitively inhibited by AMP with respect to ATP (Ki = 0.2 mM). ADP, GMP, and CMP at 1-3 mM concentration do not affect the forward reaction. PPj inhibits PEP formation in assay systems in which excess adenylate kinase is substituted for pyrophosphatase; 0.5 mM PP~ giving 50% inhibition. PEP at concentrations up to 2 mM does not affect the forward reaction. The reverse reaction is competitively inhibited by ATP with respect to AMP (K~ = 0.22 mM). The reverse reaction is also inhibited to the extent of 65% by 2 mM pyruvate. P~ up to concentration of 10 mM does not affect the reverse reaction. The enzyme is inhibited by p-hydroxymercuribenzoate. Within the range of enzyme activities used, preincubation at 25 ° for 5 min in the presence of 2 t~M PHMB brought about an inhibition of approximately 50%. The inhibition may be reversed by dithiothreitol (1 mM), or glutathione (5 mM). Potassium fluoride (50 raM) inhibits the forward and reverse reaction to the extent of 80% and 60%, respectively. The inhibition of the forward reaction by fluoride is not affected by raising the P~ concentration to 50 mM. Physiological Function. 1° Although the enzyme readily catalyzes the reverse reaction, the major physiological role of pyruvate, phosphate dikinase in A. xylinum is gluconeogenic in that it promotes the formation of PEP from pyruvate. Dikinase formation is induced on the transfer of glucose-grown cells of A. xylinum to succinate- or pyruvate-containing media. If such media are supplemented with glucose, fructose, or glycerol, enzyme formation is suppressed by 96%, 76%, and 67%, respectively. When glucose is added to cultures of cells growing on succinate, the growth of the organism is not affected, but is accompanied by a virtual cessation of dikinase synthesis. The changes in the pyruvate, phosphate dikinase activity of cells, are accompanied by parallel changes in the ability of cells to convert pyruvate into cellulose. Gluconeogenesis in this ~oM. Benziman and N. Eizen, J. Biol. Chem. 246, 57 (1971).

[33]

PYRUVATE, ORTHOPHOSPHATE DIKINASE

199

organism appears to be subject to control exerted on the synthesis of the dikinse. The reciprocal effect of AMP and ATP on the activity of the dikinase may have physiological implications in the regulation of gluconeogenesis and carbohydrate oxidation in A. xylinum. 11,~2 Distribution. In addition to cellulose-forming A. xylinum cells, the enzyme has been found in celluloseless mutants of this organismF ° in photosynthetic grasses 5 in propionibacteria, 4,6 in Endamoeba histolytica and Bacteroides symbiosus/3 Whereas in the former three sources the physiological role of the enzyme is considered to be gluconeogenic,5,l°,14 a function in a glycolytic direction has been ascribed to it in E. histolytica and B. symbiosus. ~3 11H. Weinhouse and M. Benziman, Biochem. Biophys. Res. Commun. 43, 233 (1971). 1,~H. Weinhouse and M. Benziman, Eur. J. Biochem. 28, 83 (1972). 13R. E. Reeves, R. A. Menzies, and D. S. ttsu, J. Biol. Chem. 243, 5486 (1968). 14H. J. Evans, and H. G. Wood, Proc. Nat. Acad. Sci. U.S. 61, 1448 (1968).

[33] P y r u v a t e , O r t h o p h o s p h a t ¢ D i k i n a s e 1 o f B a c t e r o l d e s symbiosus and Propionibacterium shermanH

By

YORAM MILNER, GEORGE MICHAELS, and HARLAND G. WOOD Pyruvate -F ATP + Pi ,

Mg2+,NH4~ ) P-enolpyruvate ~- AMP + PPi

(1)

Assay Methods

Principle. The assay measures the formation of pyruvate in the reverse of Reaction (1) by the formation of the hydrazone derivative, which can be determined spectrophotometrically. This assay is used in our laboratory routinely coupled with controls without added AMP and occasionally without added pyrophosphate.

Reagents A. Mixture A which contains (in ~moles/ml): imidazole.HC1 (Sigma) buffer (pH 6.7), 80; (NH4)2S04, 40; P-enolpyruvate (tricyclohexyl ammonium salt, Sigma), 4.8, and MgC12, 24. The mixture is prepared in 30-ml lots and kept frozen until used. B. AMP solution (Sigma disodium salt), 60 raM, pH = 6.7. Stored frozen. C. Sodium pyrophosphate buffer, 30 raM, pH = 6.7, kept at 4 °. 1 [ATP:pyruvate, orthophosphate phosphotransferase, EC 2.7.9.1].