[16]

PHOSPHOFRUCTOKINASE FROM Escherichia coli

91

the enzyme with succinic anhydride also results in dissociation into subunits. The molecular weight of the subunit has been determined to be about 35,000 by the sedimentation equilibrium method and acrylamide gel electrophoresis. Based on tryptic peptide mapping and acrylamide gel electrophoresis, these subunits appear to be identical. Ultraviolet Light Absorption Spectra. The enzyme absorbs maximally at 275 nm at pH 7 and 290 nm in 0.1 N NaOH. pH Optimum. The enzyme has optimum activity at a broad range of 7.0-8.2. Kinetic Properties. The Km value for ATP at infinite fructose 6-phosphate concentration is 5.5 X 10-~ M. Unlike many phosphofructokinases, clostridial enzyme is not inhibited by ATP. The enzyme shows sigmoidal kinetics with respect to fructose 6-phosphate. ADP is a positive effector and alters this sigmoidal kinetics to Michaelis-Menten kinetics. GTP, UTP, ITP, and CTP also serve as substrates.

[16] P h o s p h o f r u c t o k i n a s e f r o m E s c h e r i c h i a c o l i 1

By VERNE F. KEMERER, CHARLES C. GRIFFIN, and LUDWIG BRAND ATP + fructose 6-phosphate--~ ADP + fructose 1,6-diphosphate + H+

Assay Methods

Coupled Assay 2 Principle. The routine assay employed to monitor the purification of phosphofructokinase is based on conversion of the reaction product, fructose 1,6-diphosphate (FDP) to a-glycerophosphate in the presence of aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), a-glycerophosphate dehydrogenase (EC 1.1.1.8), and fl-NADH. The rate of disappearance of NADH is followed spectrophotometrically at 340 nm. Two micromoles of NADH are oxidized per micromole of FDP formed. Reagents. The composition of the assay mixture is shown in Table I. Procedure. Components of the assay (Table I) are mixed in a cuvette (1-cm pathlength), and the oxidation of NADH is followed spectrophotometrically at 340 nm. With impure enzyme preparations, a background 1ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11. 2C. C. Griffin, B. N. Houck, and L. Brand, Biochem. Biophys. Res. Commun. 27, 287 (1967).

92

KINASES

[15]

TABLE I STANDARD ASSAY MIXTURE

Component

Volume (ml)

Final concentration (raM)

Tris Cl, 0.3 M, pH 8.2 MgC12.6H,O, I0 raM H20 F6P, 0.1 M Auxiliary enzyme mixture b NADH, 1.5 mM (in 0.3 M Tris C1 pH 8.2) ATP, 30 m M (pH7) Phosphofructokinase c

0.5 0.3 1.2 0.2 0.3 0.3 0.1 0.1

80 a 1 -6.7 -0.15 1 --

Includes amount added with NADH. b Aldolase and a mixture of a-glycerophosphate dehydrogenase and triosephosphate isomerase [crystalline suspensions in (NH~4~)2SO4,i Boehringer Mannhein or Sigma] are mixed with 10 mM Tris C1-2 mM EDTA (pH 8) buffer to yield a solution containing 1.5 rag of aldolase per milliliter and 0.5 mg of a-glycerophosphate dehydrogenase-triosephosphate isomerase per milliliter. This solution is freed from ammonium sulfate by dialysis against the same buffer. The auxiliary enzyme mixture occasionally contains a contaminating phosphoglucose isomerase (EC 5.3.1.9) activity that may significantly reduce the concentration of F6P in any lengthy preincubation. Phosphofructokinase is diluted in 0.1 M sodium phosphate-0.1 M ammonium chloride (pH 7.4). r a t e of N A D H o x i d a t i o n is o b t a i n e d for 2 - 3 m i n w i t h o u t A T P in t h e r e a c t i o n m i x t u r e . A d e n o s i n e t r i p h o s p h a t e is t h e n a d d e d to i n i t i a t e t h e p h o s p h o f r u c t o k i n a s e - c a t a l y z e d reaction. T h e b a c k g r o u n d r a t e w h i c h is due to c o n t a m i n a t i n g m a n n i t o l - l - p h o s p h a t e d e h y d r o g e n a s e a n d o t h e r o x i d o r e d u c t a s e s is s u b t r a c t e d f r o m t h e r a t e o b t a i n e d a f t e r a d d i t i o n of ATP. Units and Specific Activity. One u n i t of a c t i v i t y is defined as t h a t a m o u n t of e n z y m e w h i c h c a t a l y z e s t h e f o r m a t i o n of 1 ~mole of F D P p e r m i n u t e u n d e r t h e c o n d i t i o n s of t h e s t a n d a r d a s s a y . F o r c a l c u l a t i n g specific a c t i v i t y ( u n i t s / m g ) p r o t e i n c o n c e n t r a t i o n is e s t i m a t e d b y t h e microbiuret method2

pH-Stat Assay 4,5 Principle. A t p H 8.5 a n d a b o v e p h o s p h o f r u c t o k i n a s e c a t a l y z e s t h e s t o i c h i o m e t r i c release of a p r o t o n in t h e reaction. T h e p H - s t a t a s s a y , 3 S. Zamenhof, this series, Vol. 3 [103]. 4j. E. Dyson and E. A. Noltmann, Anal. Biochem. 11, 362 (1965). A. Ku and C. C. Griffin, Arch. Biochem. Biophys. 149, 361 (1968).

[16]

PHOSPHOFRUCTOKINASE FROM Escherichia coli

93

which avoids the use of auxiliary enzymes, is based on the volume of standard base required to maintain constant pH in an essentially unbuffered reaction mixture. This assay is more usually employed for detailed kinetic studies than for monitoring the purification of phosphofructokinase. Reagents. A typical reaction mixture contains the following components in a final volume of 4-8 ml. KC1, 80 mM MgC12, 10 X [ATP] ATP, variable F6P, variable

Procedure. Components are mixed in a thermostated titration vessel under a continuously renewing atmosphere of nitrogen. The vessel is fitted with calomel and glass electrodes and a titrant delivery tip for addition of standard base. After adjustment of the pH of the reaction mixture to 8.5, the reaction is initiated by addition of phosphofructokinase. The volume of standard base required to maintain pH 8.5 is recorded as a function of time.

Purification

All operations are carried out at 4 ° unless otherwise noted. Centrifugations are performed at 13,000 g for 15 min. Step I. Homogenization. One pound of well-thawed E. coli B (threequarter log phase, minimal medium, Grain Processing, Muscatine, Iowa) is homogenized at low speed in a 4-liter, jacketed Waring Blendor for 45 rain in the presence of 1500 ml of 50 mM glycylglycine-1 mM EDTA (pH 7.0) and 1 kg of acid-washed glass beads (Potters Industries, Carlstadt, New Jersey, No. P-010). After settling for 10-15 min, the supernatant is decanted and the residue is washed by homogenization for 5 min with 500 ml of the above buffer. The supernatant and wash liquor are combined and centrifuged. Step 2. Autolysis. Magnesium chloride is added to a final concentration of 4 mM. The mixture is heated to 30 ° with stirring and 6 mg of deoxyribonuclease I (EC 3.1.4.5) and 1 mg of ribonuclease I (EC 3.1.4.22) (Worthington, No. 2007 codes D, and No.. 5650 code RAF, respectively) are added. The mixture is maintained at 30 ° for 3 hr with gentle stirring. Aliquots are removed before and after autolysis to measure the absorbance at 260 nm after precipitation of protein and nucleic acid with 3.5% perchloric acid. Usually a 3-fold increase in soluble OD2~o is observed during hydrolysis.

94

KINAS~S

[15]

Step 8. Heat. The autolyzed preparation is heated as rapidly as possible to 50 ° and rapidly cooled to 6 ° in a jacketed, stainless steel vessel. After cooling the material is centrifuged. Phosphofructokinase is stable for at least 40 min at 50 °, somewhat unstable at 55 ° and immediately loses all activity at 60 ° . Step ~. Precipitation by Ammonium Sulfate. Ammonium sulfate (475 g/liter) is added with stirring over a period of 40 rain. After a 20-min equilibration, the suspension is centrifuged. The precipitate is dissolved in 0.1 M Tris SO,-0.1 mM ATP-10 ~M FDP-1.5 mM 2-mercaptoethanol (pH 7.5) and dialyzed for 12 hr against two 4-liter changes of the same buffer. Step 5. Acetone Fractionation. The dialyzate is cooled to 0 ° and onefourth of its volume of acetone (--15 °) is added over a 5-min period with rapid stirring. After an additional 15 min in a bath at --5 ° the preparation is centrifuged at --5 °, and a ~1 volume (based on the volume of the supernatant) of acetone is added. The suspension is cooled to --10 °, stirred for 15 rain and centrifuged at --10% The precipitate is suspended in 300 ml of 50 mM sodium pyrophosphate (pH 9.0) and gently stirred for 1 hr and centrifuged to remove undissolved material. Step 6. QAE-Sephadex Chromatography I. The clear supernatant from step 5 is applied to a 4.0 }( 40 cm column of QAE-Scphadex A25 equilibrated with 50 mM sodium pyrophosphate-50 mM ammonium sulfate-2 mM 2-mercaptoethanol (pH 9.0) at a flow rate of 100 ml/hr. The column is washed for 3 hr with the starting buffer, and the enzyme is eluted with a linear gradient from 50 mM to 0.60 M ammonium sulfate in a total volume of 3600 ml. The mean elution molarity is 0.2. Step 7. Acid Precipitation. Pooled phosphofructokinase activity from the column is concentrated by lowering the pH to 4.4 with 2 M acetic acid. After centrifugation, the precipitate is suspended in 30 ml of 0.1 M Tris S04-1 mM EDTA-0.1 mM ATP-10 t~M FDP-2 mM 2-mercaptoethanol (pH 8.0), dialyzed against 20 volumes of the same buffer for 3 hr and centrifuged. Step 8. QAE-Sephadex Chromatography II. The phosphofructokinase preparation is applied to a 2.5 X 30 cm column of QAE-Sephadex A25 equilibrated with 0.1 M Tris SO4-1 mM EDTA-2 mM 2-mercaptoethanol (pH 8.0) at a flow rate of 30 ml/hr. The column is rinsed for 2 hr with equilibrating buffer and the flow rate increased to 45 ml/hr. A 1000-m] linear potassium sulfate gradient from 0.0 to 0.4 M is used for elution. The mean elution molarity is 0.1. The pooled enzyme is concentrated to 10 ml in an Amicon ultrafiltration cell (Model UF52 with PM 30 membrane).

PHOSPttOFRUCTOKINASE FROM Escherichia coli

[16]

0.30

95

0.5 M KP 7.1

P

35 ¸

II II

I¢

I I ! ! I

024

i i

i! 0.18 0 @0 O

o

-6

o.,2l

o.Jo

E

150

0.08 I00 0.06 50

/' o.oo

•

~x-~ t

/I

0.04

0 Volume (ml)

FIG. 1. Hydroxyapatite chromatography of Escherichia coli phosphofructokinase. Activity (x--x) eluted from a 1.1 × 25 cm column with a 259-ml linear potassium phosphate (pH 7.1) gradient to 0.1 M. Activity collected from 73-120 ml was pooled. After 177 ml were eluted from column the buffer concentration was raised to 0.5 M potassium phosphate.

Step 9. Hydroxyapatite Chromatography. The material from step 8 is dialyzed against two 250-ml changes of 30 m M potassium phosphate-2 m M 2-mercaptoethanol (pH 7.1) for a total of 6 hr. It is applied to a 1.1 }( 25 cm column of h y d r o x y a p a t i t e 6 equilibrated with the above buffer at a flow rate of l0 ml/hr. After a 2-hr rinse with the equilibrating buffer, the enzyme is eluted with a 250-ml linear gradient from 0.03 to 0.1 M in potassium phosphate (pH 7.1). A typical elution profile for this column is shown in Fig. 1. The pure enzyme is concentrated in the Amicon ultrafiltration cell and dialyzed against 0.1 M potassium phosphate-1 m M E D T A - 0 . 3 2 m M dithiothreitol-0.1 m M A T P - 1 0 ~M F D P ( p H 7.1). Phosphofructokinase is stable for a minimum of 40 days at 4 ° in this buffer; it is stable for at least 4 months when frozen at --15 ° 6 G. Bernardi, this series, Vol. 22 [29].

96

KINASES

PURIFICATION OF

Fraction Crude extract Autolysis/heat (NH4)~SO4 Acetone QAE I Acid QAE II Hydroxyapatite

TABLE II Escherichia coli PHOSPHOFRUCTOKINASE

Volume Activity (ml) (units) 1980 1860 328 266 506 28.6 9.25 3.75

[16]

7600 5850 5560 4880 3480 2780 2300 1840

Protein (mg)

Specifc activity (units/mg)

21,000 14,400 14,400 6,960 152 93 12.7 7.0

0. 362 0. 406 0.386 0. 700 22.8 29.9 181 263

PurifiY i e l d cation (%) (fold) 100 77 73 64 46 37 30 24

1 1.1 1.1 1.9 63 82 500 728

in this buffer with 20% sucrose added. A summary of the purification procedure is outlined in Table II.

Properties 2,7-i1 Gel Electrophoresis. Purified phosphofructokinase migrates as a single, sharp band in 7.5% polyacrylamide gels. The mobility relative to bromophenol blue is 0.43 in the 0rnstein-Davis ~2 system (resolving gel p H 9.5) and 0.65 in the Robard-Chrambach 13 system (pH 7.8). Preparations with specific activities of 200-220 show several faint satellite bands. Cation Requirement. Escherichia coli phosphofructokinase requires Mg 2~ as do other ATP-dependent phosphotransferases. Presumably, the M g - A T P complex is the true substrate for the enzyme although the enzyme m a y require free Mg 2÷ as well. Inhibition by A T P is not observed if the Mg ~÷ concentration is high enough to complex all the ATP. In addition, phosphofructokinase exhibits a requirement for a monovalent cation. This requirement is satisfied by NH4 ÷ or, at higher concentrations, K ÷. Substrate Specificity. The enzyme can utilize a variety of nucleoside triphosphates as phosphoryl donors. D e o x y - A T P is as effective as ATP. 7D. E. Atkinson and G. Walton, J. Biol. Chem. 240, 757 (1965). 8D. Blangy, H. Buc, and J. Monod, J. Mol. Biol. 31, 13 (1968). D. Blangy, FEBS Lett. 2, 109 (1968). ~ C. C. Griffin, unpublished observation. 1, V. Kemercr and L. Brand, unpublished observations. 12B. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964). 18D. Rodbard and A. Chrambach, Anal. Biochem. 40, 95 (1971).

[16]

PHOSPttOFRUCTOKINASE FROM Escherichia coli

97

Km's for the other nucleotides are approximately an order of magnitude higher. Only F6P has been shown to be an acceptor in the reaction. The borohydride reduction products (hexitol 6-phosphates) are not substrates. Other analogs have not been examined. Metabolic E]]ectors. The catalytic activity of phosphofructokinase responds sigmoidally to increasing concentrations of F6P. At low concentrations nucleoside diphosphates like ADP and GDP convert this sigmoidal response to a hyperbolic one. At higher concentrations ADP acts as a product inhibitor, competitive with respect to ATP and noncompetitive with F6P as the variable substrate. Neither 5'-AMP nor cyclic-3',5'AMP are positive effectors for the E. coli enzyme. Phosphoenolpyruvate is a feedback inhibitor, but citrate has no effect. Fructose 1,6-diphosphate is not an activator of E. coli phosphofructokinase, and product inhibition by this ester cannot be observed at concentrations as high as 5-10 mM in the pH-stat assay. Pyruvate, phosphogluconate, and ribulose 5-phosphate have no effect provided that the concentration of magnesium is kept high. Molecular Weight. Phosphofructokinase from E. coli B has a molecular weight of 141,000-142,0009,1~ and appears to be composed of 4 subunits of identical molecular weight. Unlike the mammalian enzyme, the enzyme for E. coli does not appear to undergo any freely reversible aggregations and dissociations. The sedimentation constant remains essentially unchanged with enzyme concentrations from 0.5 to 5 mg/ml. Fluorescent Derivatives. Dansyl chloride (DNS, 1-dimethylaminonaphthalene 5-sulfonyl chloride) may be covalently attached to phosphofructokinase (ca. 1 mg/ml) in 50 mM sodium bicarbonate-1 mM EDTA-0.1 mM ITP-1 mM F6P (pH 9.2) at 4 °. The reaction is terminated after 90 min by addition of a 100-fold excess of 2-mercaptoethanol. After centrifugation, the conjugated enzyme is separated from reactants by desalting on a column of Sephadex G-25. With 10-, 20-, and 50-fold molar excesses of dansyl chloride, dansyl phosphofructokinase conjugates with molar ratios (DNS/phosphofructokinase) of 1.5, 2.7, and 8 have been prepared. Molar ratios are estimated from an extinction coefficient if 3.4 X l0 b for DNS at 350 nm. 15 The specific activity after dansylation is approximately 60% of the native enzyme. All three conjugates show similar excitation and emission spectra with excitation maxima at 350 nm and emission maxima at 515 nm. The dansylated enzymes exhibit kinetic properties similar to the native enzyme. As shown in Fig. 2, the derivatives are allosteric with respect to F6P and are inhibited by high concentrations of ATP. Phosphoenol14 C. K. Marschke and R. W. Bernlohr, Arch. Biochem. Biophys. 156, 1 (1973). 1~R. Chen, Anal. Biochem. 25, 412 (1968).

98

KINASES

iO0

I

[16]

0

x~

5

80

F

6O

N

4o

_90

i/

r

~

x

20

I

I

I

I0

20

50

I

40

Fructose 6-Phosphate(mM)

0

I

I

I

I

I0

20

~)

40

50

Adenosine Triphosphote (mM)

FIG. 2. (A) Effect of F6P concentration on the activity of native (C) O) and octadansyl (x--x) phosphofructokinases from Escherichia coli. (B) Effect of ATP on the activity of native (O O) and octadansyl (x--x) phosphofructokinases. The coupled enzyme assay was used. Reaction mixtures contained 240 ~moles of Tris Cl pH 8.2, 3 ~moles of MgC12, 0.44 ~mole of #-NADH, 450 ~g of aldolase, and 150 ~g of a-glycerophosphate dehydrogenase-triosephosphate isomerase in 2.9 ml at 29°. In (A) the ATP concentration was 4 mM and in (B) the F6P concentration was 1 mM. p y r u v a t e is m u c h less p o t e n t a n i n h i b i t o r for the d a n s y l a t e d enzymes. I n all cases A D P is able to relieve A T P i n h i b i t i o n c o m p l e t e l y a t concent r a t i o n s below 1 m M in the presence of 4 m M A T P a n d 1 m M F 6 P .

Acknowledgment This work was supported by Grant No. GM 11632 from the National Institute of General Medical Sciences and Grants No. P-610 from The American Cancer Society and AM 13883 from The National Institute of Arthritis and Metabolic Diseases.