- Email: [email protected]
[12] Phosphofructokinase from Bacillus licheniformis
70
KINASES
[12]
the crude extract) depends on the nature of the carbon source and on the oxygen tension. Bacteria grown on glucose contain double the amount of PFK1 as do bacteria grown on glycerol. Additionally, following a shift from aerobiosis to anaerobiosis, the level of P F K I doubles. 7 A similar conclusion was reached earlier by Doelle's group, 2'5"26who independently proposed on other bases that two different enzymes were present in E. coli. 27,28 It has to be stressed, however, that strains expressing either PFK1 alone or P F K 2 alone have comparable growth rates under all conditions tested 29 and that an identical " P a s t e u r effect" has been observed with both types of strains? ° These two observations, as well as the existence among Enterobacteriaceae of various genera expressing widely different amounts of the two enzymes, lead to the assumption that each type of P F K can ensure, with a comparable efficiency, the regulation of fructose 6-phosphate transphosphorylation. ._,5A. D. Thomas, H. W. Doelle, A. W. Westwood, and G. L. Gordon, J. Bacteriol. 112, 1099 (1972). '-'~ H. W. Doelle and N. W. Hollywood, Ettr. J. Biochem. 83, 479 (1978). 27 H. W. Doelle, FEBS Lett. 49, 220 (1974). 2s K. N. Ewings and H. W. Doelle, E,r. J. Biochem. 69, 563 (1976). 29 j. p. Robinson and D. G. Fraenkel, Biochem. Biophys. Res. C o m m , n . 81, 858 (1978). z0 H. W. Doelle and S. Mclvor, FEMS Microbiol. Lett. 7, 337 (1980).
[12]
Phosphofructokinase
f r o m Bacillus l i c h e n i f o r m i s
Bv CHARLES K . MARSCHKE a n d ROBERT W. BERNLOHR Fructose 6-phosphate + ATP ~ fructose 1,6-bisphosphate + ADP
Phosphofructokinase (ATP : D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from the mesophile Bacillus licheniformis is subject to temperature- and ligand-induced alterations of specific activity. Also, it is unstable at the low protein concentrations required for assay.1 Therefore, the e n z y m e ' s environment must be controlled during purification and kinetic analysis to maintain the structural integrity of the molecule. This report details procedures for the purification and stabilization of B. licheniformis phosphofructokinase that allow the determination of valid molecular and kinetic properties of the enzyme. 1 C. K. Marschke and R. W. Bernlohr, Arch. Biochem. Biophys. 156, 1 (1973).
METHODS IN ENZYMOLOGY,VOL. 90
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
[12]
PHOSPHOFRUCTOKINASE FROM B. licheniformis
71
C, lture and Harvest of Cells. A genetically stable rough colonial form of B. licheni)brmis is used for all studies. This is maintained as a spore stock as previously described. 2 Vegetative cells of B. lichen~)rmis are grown from this spore stock by the addition of spores to a minimal medium (5 × 106 spores/ml) containing essential inorganic constituents, 30 mM glucose, 10 mM NH4CI, and 0.5 mM k-alanine (the k-alanine is present to promote germination). The inorganic constituents of this minimal medium are 0.61 mM MgSO4, 0.61 mM MgC12, 0.005 mM MnCI2, 0.34 mM CaCI2, and 65 mM phosphate (as the potassium salt). The glucose, NH4C1, and inorganic constituents are sterilized separately and added to the sterile phosphate solution. Cells are cultured in 1 liter of this minimal medium in a 2.8-liter Fernbach flask on a Gyrotory shaker (New Brunswick Co.) at 350 excursions per minute. Growth is continued at 37° until a turbidity of 150-170 Klett units (540 nm) is reached. Then this seed culture is added to I l liters of sterile minimal medium (without L-alanine) in a fermentation vessel (New Brunswick Scientific Co., Model FS-307). The growth of the vegetative cells is conducted at 37° with the impeller rotation set at ,450 rpm and the air flow through the sparger set at the maximum rate. The bacterial cells are harvested before oxygen or nutrient limitation occurs. The method of Tuominen and Bernlohr ~ is use'd except that the sedimented cells are washed in a 0.2 M potassium phosphate buffer (pH 7.6). Preparation of Cell Extracts. The sedimented and washed cells are suspended in 0.2 M potassium phosphate buffer containing 5 mM 2-mercaptoethanol (approximately 300 mg of wet cells per milliliter). These cells are disrupted by sonic treatment at 5° (Measuring & Scientific Equipment, Ltd.) or by passage through a French pressure cell operated at 12,000 psi (2°). Cellular debris is sedimented by a 45-min centrifugation at 105,000g in a Spinco Model L centrifuge using a fixed-angle rotor. The supernatant solution is removed and dialyzed against 0.1 M potassium phosphate solution containing 1 mM 2-mercaptoethanoi (pH 8.0). Assay System Phosphofructokinase is assayed at 30° using the procedure described by Racker? The auxiliary enzyme system, which couples the phosphorylation of fructose 6-phosphate (Fru-6-P) to NADH oxidation, is prepared by dialyzing a mixture of 1.5 mg of bovine serum albumin (Cohn's fraction V), 4 mg of aldolase (EC 4.1.2.7), 32 IU of triosephosphate isomerase (EC 2 F. W. Tuominen and R. W. Bernlohr, J. Biol. Chem. 246, 1733 (1971). 3 E. Racker, J. Biol. Chem. 167, 843 (1947).
72
KINASES
[12]
5.3.1.1), and 8 IU of glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) against 1.0 mM glycylglycine-NaOH (pH 8.5) containing 1 mM 2mercaptoethanol (2°). After extensive dialysis the auxiliary enzyme solution is diluted to 2.5 ml with distilled water; 0.1 ml of this solution is used per 1.0 ml of assay solution. The NADH oxidation is monitored at 340 nm with a Zeiss PMQ II spectrophotometer equipped with a continuous recorder. A constant 30° assay temperature is maintained by the use of a refrigerated circulating bath and a flow cell. The assay solution contains 20 mM imidazole-HC1 (pH 6.82 at 29°), 0.10 M KC1, 3.0 mM MgCI2, 1.0 mM 2-mercaptoethanol, and the two substrates at various concentrations. The rate of NADH oxidation is proportional to phosphofructokinase concentration, and the rate is constant with time unless either enzyme inactivation or activation conditions exist.l A progressive decay of activity will result from the addition of Mn 2+ or sodium pyrophosphate to the assay and also with a high ATP concentration at low Fru-6-P concentrations. A time-dependent increase of activity results from the combination of inactivated enzyme with activating ligands and temperature. This occurs, for example, at 30° by combining Mg 2+, ATP, and phosphoenolpyruvate (PEP) with a previously inactivated phosphofructokinase. Purification of Phosphofructokinase The dialyzed 105,000 g supernatant solution is diluted with a 0.2 M potassium phosphate solution containing 5 mM 2-mercaptoethanol (pH 8.0) to a protein concentration of 10-20 mg/m]. Solid ammonium sulfate is then added (24.3 g/100 ml of solution), and the precipitated protein is collected by centrifugation at 20,000 g for 15 min (2°). This procedure is repeated for subsequent ammonium sulfate fractionations at 3.3 g/100 ml increments. The individual protein samples are suspended in 0.1 M potassium phosphate buffer (pH 7.6) before dialysis against the same phosphate buffer containing 1 mM 2-mercaptoethanol (2°). The greatest specific activity of phosphofructokinase results from the 34.2-37.5 g/100 ml ammonium sulfate fraction (see the table). This fraction is dialyzed against 95 mM potassium phosphate buffer containing 1 mM 2-mercaptoethanol (pH 7.2) before adding it to a 4 × 13 cm hydroxyapatite (BioGel HT) column (4°). The hydroxyapatite column has been previously equilibrated with the same phosphate buffer. After a 170-ml wash of the column with the 59 mM potassium phosphate solution, the phosphate concentration is increased to 115 raM, which elutes the phosphofructokinase. The protein in the region of maximum specific activity is precipitated by ammonium sulfate, collected by centrifugation, and dialyzed against 0.1 M potassium phosphate
PHOSPHOFRUCTOKINASE FROM B. licheniformis
[12]
PURIFICATION OF PHOSPHOFRUCTOKINASE
Fraction Undialyzed cell extract Ammonium sulfate fraction Hydroxyapatite eluent (center of PFK fraction)
(PFK)
FROM B .
73
lichenifi)rmis
¢~.b
Protein (mg/ml)
Volume (ml)
PFK (IU/mg)
Total IU
PFK recovered (%)
17.5 6.4 0.12
342 23.5 30.0
0.14 0.71 9.6
840 110 35
100 13 4
PFK was isolated from 50 liters of middle exponential-phase cells grown on glucose and NH4CI. b PFK was assayed at pH 6.8 with 1.0 mM ATP, 1.0 mM Fru-6-P, 100 mM KCI, 3.0 mM MgCI~, 20 mM imidazole, and 1.0 mM 2-mercaptoethanol. buffer (pH 7.6) containing 1 m M 2-mercaptoethanol. This solution is further clarified by centrifugation at 20,000 g for 30 min. The 20,000 ~,, supernatant solution is free of contaminating fructose-l,6-bisphosphate l-hydrolase, adenylate kinase, and adenosine triphosphate phosphatase activities? Glucose-6-phosphate dehydrogenase is present as a low-level contaminant while phosphoglucoisomerase is an easily detected contaminant of this phosphofructokinase preparation. This preparation is judged sufficiently pure for the subsequent molecular and kinetic characterizations. Results of a representative purification are summarized in the table. The protein assay procedure of L o w r y et al. 4 is used throughout the purification for determining protein concentrations. Four percent of the activity is recovered in a fraction exhibiting a specific activity of 9.6 IU/mg. This purity is about one-tenth of that observed with homogeneous skeletal muscle p h o s p h o f r u c t o k i n a s e ? The purified phosphofructokinase can be stored at - 2 0 ° in phosphate buffer. H o w e v e r , greater stability results from adding glycerol to the phosphate buffer. A 50% solution of glycerol reduces inactivation from 3% per day to 0.3% per day at - 2 0 °. Properties
Time-Dependent Alterations in the Activity of Phosphofructokinase. Initial attempts to stabilize the phosphofructokinase in cell extracts by the addition of substrates resulted, surprisingly, in the recovery of negligible enzyme activity. However, normal activities are restored to these "inac4 o. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem, 193, 265 (1951).
74
KINASES
[12]
_ ~/O° 0.08 /
0o
3mM PEP 3raM ATP No MgCI2
0 0.06
\.---0 O
0.04
©
/ 0
A c"
m
< q
~
I
0
o 0.02 / 30 °
I--
3mM PEP 3mM ATP 5mM MgCI2
\ 300
< v
O.0C
~ 0
L 20
i 40
I 60
MINUTES FIG. I. Time-dependent alterations of enzyme activity. The temperature oscillations of phosphofructokinase activity can be observed by incubating the purified enzyme (5.4 IU/mg) at 0° in the presence of buffer (20 m M potassium phosphate, pH 8) and the indicated effector molecules. The experiment is initiated by mixing the 0° enzyme solution with a 0° solution containing ATP, PEP, and MgC]2 to attain the designated levels of effectors. Two micro]iters of the enzyme solution (protein = 0.56 mg/ml) are withdrawn at the indicated times and assayed for phosphofructokinase activity. The temperatures for incubation of the phosphofructokinase are as indicated by the arrows in the figure.
tive" extracts by dialyzing them against phosphate buffer at 4 ° or by raising the temperature of the extract to 30 ° for 15-30 min before assaying. This reversible inactivation is mediated by either ATP or phosphoenolpyruvate. H o w e v e r , ATP differs from phosphoenolpyruvate in that the ATP also causes an irreversible inactivation o f phosphofructokinase. 1 The reversible inactivation o f phosphofructokinase is most apparent upon combining ATP and phosphoenolpyruvate with Mg 2+ (Fig. 1). In the
[12]
PHOSPHOFRUCTOKINASE FROM
B.
lichen'tyormis
75
p r e s e n c e o f Mg 2+, PEP, and ATP, a rapid inactivation o f the e n z y m e o c c u r s at 0 °. This loss is r e v e r s e d b y i n c u b a t i o n at 30 °, and a n o t h e r c y c l e o f inactivation o c c u r s u p o n returning the p h o s p h o f r u c t o k i n a s e to 0 °. T h e omission o f Mg 2+ r e d u c e s the extent o f activity regain. T h e r e may' also be a slight d e c r e a s e in the rate o f inactivation with the omission o f Mg 2+ (Fig. 1). T h e kinetic o r d e r o f the activation with r e s p e c t to e n z y m e c o n c e n t r a tion is a m e n a b l e to analysis. Figure 2A d e m o n s t r a t e s that the initial rate o f e n z y m e activation is a nonlinear function o f the p r o t e i n c o n c e n t r a t i o n . H o w e v e r , the log(activity gain) is a linear function o f log[protein] with a slope n e a r 2 (Fig. 2B). This suggests that the reversible c y c l e s o f e n z y m e activity are due to a dissociation o f the m o l e c u l e into dimers with the s u b s e q u e n t association o f t h e s e subunits to r e s t o r e an active molecule. This interpretation is s u p p o r t e d by m o l e c u l a r weight estimates o f 68,000 and 135,000 for the inactive and active molecules, r e s p e c t i v e l y . T h e s e estimates are f r o m s u c r o s e density-gradient centrifugations o f the inactive and active p h o s p h o f r u c t o k i n a s e s . 1 If the association o f dimers requires Mg 2÷, this w o u l d explain the r e t a r d e d activation that o c c u r s in the absence o f a d d e d Mg '÷ (Fig. 1).
A
B
10.0
+10[
8.0
_
0O F
<
-1.0
6.0 Gg" > ~ ~--× >
4.0
o.<~~
2.0
c- z
U_l-. O~, o~
~ (O 10 20 30 PROTEIN(~Jg/ml)
o
20
t
10 LOG[PROTEIN]
I
20
F~G. 2. Molecular order of the activation of phosphofructokinase. The enzyme (,1.4 mg of protein per milliliter) is previously inactivated by incubation with 1 mM ATP, 1 mM phosphoenolpyruvate, 5 mM MgCI2, and 20 mM potassium phosphate (pH 7.6) at 0° for 3 hr. Then the indicated amount of the inactivated enzyme is transferred to a 1.0-ml cuvette containing 1.2 mM ATP, 5.0 mM fructose 6-phosphate, 3.0 mM MgC12, 100 mM KC1, ! mM 2-mercaptoethanol, and 20 mM buffer (pH 6.8) at 30°. The initial rate of activity gain (A) is determined, and the logarithm of activity gain is plotted against the logarithm of the protein concentration (B). A line with a slope of 2.0 is provided for reference.
76
KINASES 100-
[]
[12] +F6P -... []
80
~x
,
_9
60
l-(,.J
<
2
._1
O n... i-
40
z o 20-
©
z w
NO ADDITIONS (BUFFER + 2-ME)
(..) uJ a..
i
i
i
I
I
J
0
2
5
10
15
2o
MINUTES AT 30 o
FIG. 3. Pbosphofructokinase stability under assay conditions. The stability of phosphofructokinase at assay concentrations can be determined by adding purified enzyme (7.0 IU/mg) to the base solution (20 mM imidazole-HCl with 1 mM 2-mercaptoethanol; pH 6.82) containing the indicated reactant(s). After incubation of this solution at 30° for various times, the enzyme reaction is started by adding the remaining (omitted) reactants. Fructose 6phosphate (F6P), phosphoenolpyruvate, and ATP are present at 1 mM, and MgCI2 and KCI are present at 3 and 100 raM, respectively. In all cases, the complete assay reaction contains 20 mM buffer, I mM 2-mercaptoethanol (2-ME), 1 mM ATP, 100 mM KCI, and 3 mM MgCI2. The control NADH oxidation rate is 0.062 A340 per minute.
The temperature-dependent inactivation seen with B. licheniform& phosphofructokinase is also found with B. cereus, B. megaterium, B. mycoides, and B. subtilis 168 M, but not with Escherichia col B. 1 This phenomenon of temperature inactivation appears to correlate with stability of the phosphofructokinases from these bacteria, since 15% of the E. coli B activity and 30-35% of the Bacilhts sp. activity is lost during these experiments. It is also important to note that the molecular weights of these enzymes from E. coli and Bacillus sp. are 130,000 to 140,000.1 Stability of Phosphofructokinase under Assay Conditions. This enzyme is fragile enough to be rapidly inactivated during incubation under assay conditions (Fig. 3). Inactivation is arrested, essentially, by the addition of Fru-6-P. Although both KC1 and PEP are stabilizing ligands, they are much less effective than Fru-6-P (Fig. 3). The addition of ATP or ATP with MgC12 only slightly improves the stability. This general instability dictates
PFK FROM Streptococcus lactis
[13]
77
that the reaction be initiated by the addition of phosphofructokinase. Even with this precaution, a time-dependent decrease of the reaction rate occurs when a high ATP concentration is combined with a low Fru-6-P concentration. Therefore, the instability of this enzyme under assay conditions can lead to invalid kinetic characterizations. Kinetics of PhosphojJ'uctokinase. Saturation functions for ATP and Fru-6-P are hyperbolic with apparent Km values of 0.07 and 0.05 raM, respectively. 1 Several divalent cations support catalysis by this enzyme but magnesium is the preferred ion. The pH optimum is in the range of 8.2-8.7. Either NH4 + or K + activates the B. licheniformis phosphofructokinase, whereas Li + and Na + ions have no effect on the enzyme activity. The apparent dissociation constants for NH4 + and K + are 0.5 and l0 mM, respectively. The most effective inhibitor of this enzyme is phosphoenolpyruvate. This compound increases the Km of the enzyme for Fru-6-P. Phosphoenolpyruvate, which is an effective inhibitor at 5-20 tzM, has an apparent Hill coefficient of 3. Other inhibitors of this enzyme include ATP, citrate, calcium, and pyrophosphate. Sensitivity to inhibition by ATP and citrate is conditioned by the concentration of Fru-6-P and Mg z+, whereas the inhibition by phosphoenolpyruvate is specifically relieved by Fru-6-P. Calcium is a competitive inhibitor of Mg z+, and its apparent K~ is 0.2 mM. Pyrophosphate has the effect of inactivating the phosphofructokinase under assay conditions.
[13] P h o s p h o f r u c t o k i n a s e f r o m Streptococcus lactis By ALlSON M. FORDYCE, C. H. MOORE, and G. G. PRrTCHARD Fructose 6-phosphate + ATP---, fructose 1,6-bisphosphate + A D P + H +
Assay Method
Principle. Phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) activity can be coupled via the enzymes fructose 1,6-bisphosphate aldolase, triosephosphate isomerase, and glycerol 3-phosphate dehydrogenase to NADH oxidation? Activity can then be determined spectrophotometrically by measuring the decrease in absorbance at 340 nm. Phosphorylation of 1 /zmol of fructose 6-phosphate results in oxidation of 2 tzmol of NADH in this assay. E. R a c k e r , J. Biol. Chem. 167, 843 (1947).
METHODS IN ENZYMOLOGY, VOL. 90
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
KINASES
[12]
the crude extract) depends on the nature of the carbon source and on the oxygen tension. Bacteria grown on glucose contain double the amount of PFK1 as do bacteria grown on glycerol. Additionally, following a shift from aerobiosis to anaerobiosis, the level of P F K I doubles. 7 A similar conclusion was reached earlier by Doelle's group, 2'5"26who independently proposed on other bases that two different enzymes were present in E. coli. 27,28 It has to be stressed, however, that strains expressing either PFK1 alone or P F K 2 alone have comparable growth rates under all conditions tested 29 and that an identical " P a s t e u r effect" has been observed with both types of strains? ° These two observations, as well as the existence among Enterobacteriaceae of various genera expressing widely different amounts of the two enzymes, lead to the assumption that each type of P F K can ensure, with a comparable efficiency, the regulation of fructose 6-phosphate transphosphorylation. ._,5A. D. Thomas, H. W. Doelle, A. W. Westwood, and G. L. Gordon, J. Bacteriol. 112, 1099 (1972). '-'~ H. W. Doelle and N. W. Hollywood, Ettr. J. Biochem. 83, 479 (1978). 27 H. W. Doelle, FEBS Lett. 49, 220 (1974). 2s K. N. Ewings and H. W. Doelle, E,r. J. Biochem. 69, 563 (1976). 29 j. p. Robinson and D. G. Fraenkel, Biochem. Biophys. Res. C o m m , n . 81, 858 (1978). z0 H. W. Doelle and S. Mclvor, FEMS Microbiol. Lett. 7, 337 (1980).
[12]
Phosphofructokinase
f r o m Bacillus l i c h e n i f o r m i s
Bv CHARLES K . MARSCHKE a n d ROBERT W. BERNLOHR Fructose 6-phosphate + ATP ~ fructose 1,6-bisphosphate + ADP
Phosphofructokinase (ATP : D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from the mesophile Bacillus licheniformis is subject to temperature- and ligand-induced alterations of specific activity. Also, it is unstable at the low protein concentrations required for assay.1 Therefore, the e n z y m e ' s environment must be controlled during purification and kinetic analysis to maintain the structural integrity of the molecule. This report details procedures for the purification and stabilization of B. licheniformis phosphofructokinase that allow the determination of valid molecular and kinetic properties of the enzyme. 1 C. K. Marschke and R. W. Bernlohr, Arch. Biochem. Biophys. 156, 1 (1973).
METHODS IN ENZYMOLOGY,VOL. 90
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
[12]
PHOSPHOFRUCTOKINASE FROM B. licheniformis
71
C, lture and Harvest of Cells. A genetically stable rough colonial form of B. licheni)brmis is used for all studies. This is maintained as a spore stock as previously described. 2 Vegetative cells of B. lichen~)rmis are grown from this spore stock by the addition of spores to a minimal medium (5 × 106 spores/ml) containing essential inorganic constituents, 30 mM glucose, 10 mM NH4CI, and 0.5 mM k-alanine (the k-alanine is present to promote germination). The inorganic constituents of this minimal medium are 0.61 mM MgSO4, 0.61 mM MgC12, 0.005 mM MnCI2, 0.34 mM CaCI2, and 65 mM phosphate (as the potassium salt). The glucose, NH4C1, and inorganic constituents are sterilized separately and added to the sterile phosphate solution. Cells are cultured in 1 liter of this minimal medium in a 2.8-liter Fernbach flask on a Gyrotory shaker (New Brunswick Co.) at 350 excursions per minute. Growth is continued at 37° until a turbidity of 150-170 Klett units (540 nm) is reached. Then this seed culture is added to I l liters of sterile minimal medium (without L-alanine) in a fermentation vessel (New Brunswick Scientific Co., Model FS-307). The growth of the vegetative cells is conducted at 37° with the impeller rotation set at ,450 rpm and the air flow through the sparger set at the maximum rate. The bacterial cells are harvested before oxygen or nutrient limitation occurs. The method of Tuominen and Bernlohr ~ is use'd except that the sedimented cells are washed in a 0.2 M potassium phosphate buffer (pH 7.6). Preparation of Cell Extracts. The sedimented and washed cells are suspended in 0.2 M potassium phosphate buffer containing 5 mM 2-mercaptoethanol (approximately 300 mg of wet cells per milliliter). These cells are disrupted by sonic treatment at 5° (Measuring & Scientific Equipment, Ltd.) or by passage through a French pressure cell operated at 12,000 psi (2°). Cellular debris is sedimented by a 45-min centrifugation at 105,000g in a Spinco Model L centrifuge using a fixed-angle rotor. The supernatant solution is removed and dialyzed against 0.1 M potassium phosphate solution containing 1 mM 2-mercaptoethanoi (pH 8.0). Assay System Phosphofructokinase is assayed at 30° using the procedure described by Racker? The auxiliary enzyme system, which couples the phosphorylation of fructose 6-phosphate (Fru-6-P) to NADH oxidation, is prepared by dialyzing a mixture of 1.5 mg of bovine serum albumin (Cohn's fraction V), 4 mg of aldolase (EC 4.1.2.7), 32 IU of triosephosphate isomerase (EC 2 F. W. Tuominen and R. W. Bernlohr, J. Biol. Chem. 246, 1733 (1971). 3 E. Racker, J. Biol. Chem. 167, 843 (1947).
72
KINASES
[12]
5.3.1.1), and 8 IU of glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) against 1.0 mM glycylglycine-NaOH (pH 8.5) containing 1 mM 2mercaptoethanol (2°). After extensive dialysis the auxiliary enzyme solution is diluted to 2.5 ml with distilled water; 0.1 ml of this solution is used per 1.0 ml of assay solution. The NADH oxidation is monitored at 340 nm with a Zeiss PMQ II spectrophotometer equipped with a continuous recorder. A constant 30° assay temperature is maintained by the use of a refrigerated circulating bath and a flow cell. The assay solution contains 20 mM imidazole-HC1 (pH 6.82 at 29°), 0.10 M KC1, 3.0 mM MgCI2, 1.0 mM 2-mercaptoethanol, and the two substrates at various concentrations. The rate of NADH oxidation is proportional to phosphofructokinase concentration, and the rate is constant with time unless either enzyme inactivation or activation conditions exist.l A progressive decay of activity will result from the addition of Mn 2+ or sodium pyrophosphate to the assay and also with a high ATP concentration at low Fru-6-P concentrations. A time-dependent increase of activity results from the combination of inactivated enzyme with activating ligands and temperature. This occurs, for example, at 30° by combining Mg 2+, ATP, and phosphoenolpyruvate (PEP) with a previously inactivated phosphofructokinase. Purification of Phosphofructokinase The dialyzed 105,000 g supernatant solution is diluted with a 0.2 M potassium phosphate solution containing 5 mM 2-mercaptoethanol (pH 8.0) to a protein concentration of 10-20 mg/m]. Solid ammonium sulfate is then added (24.3 g/100 ml of solution), and the precipitated protein is collected by centrifugation at 20,000 g for 15 min (2°). This procedure is repeated for subsequent ammonium sulfate fractionations at 3.3 g/100 ml increments. The individual protein samples are suspended in 0.1 M potassium phosphate buffer (pH 7.6) before dialysis against the same phosphate buffer containing 1 mM 2-mercaptoethanol (2°). The greatest specific activity of phosphofructokinase results from the 34.2-37.5 g/100 ml ammonium sulfate fraction (see the table). This fraction is dialyzed against 95 mM potassium phosphate buffer containing 1 mM 2-mercaptoethanol (pH 7.2) before adding it to a 4 × 13 cm hydroxyapatite (BioGel HT) column (4°). The hydroxyapatite column has been previously equilibrated with the same phosphate buffer. After a 170-ml wash of the column with the 59 mM potassium phosphate solution, the phosphate concentration is increased to 115 raM, which elutes the phosphofructokinase. The protein in the region of maximum specific activity is precipitated by ammonium sulfate, collected by centrifugation, and dialyzed against 0.1 M potassium phosphate
PHOSPHOFRUCTOKINASE FROM B. licheniformis
[12]
PURIFICATION OF PHOSPHOFRUCTOKINASE
Fraction Undialyzed cell extract Ammonium sulfate fraction Hydroxyapatite eluent (center of PFK fraction)
(PFK)
FROM B .
73
lichenifi)rmis
¢~.b
Protein (mg/ml)
Volume (ml)
PFK (IU/mg)
Total IU
PFK recovered (%)
17.5 6.4 0.12
342 23.5 30.0
0.14 0.71 9.6
840 110 35
100 13 4
PFK was isolated from 50 liters of middle exponential-phase cells grown on glucose and NH4CI. b PFK was assayed at pH 6.8 with 1.0 mM ATP, 1.0 mM Fru-6-P, 100 mM KCI, 3.0 mM MgCI~, 20 mM imidazole, and 1.0 mM 2-mercaptoethanol. buffer (pH 7.6) containing 1 m M 2-mercaptoethanol. This solution is further clarified by centrifugation at 20,000 g for 30 min. The 20,000 ~,, supernatant solution is free of contaminating fructose-l,6-bisphosphate l-hydrolase, adenylate kinase, and adenosine triphosphate phosphatase activities? Glucose-6-phosphate dehydrogenase is present as a low-level contaminant while phosphoglucoisomerase is an easily detected contaminant of this phosphofructokinase preparation. This preparation is judged sufficiently pure for the subsequent molecular and kinetic characterizations. Results of a representative purification are summarized in the table. The protein assay procedure of L o w r y et al. 4 is used throughout the purification for determining protein concentrations. Four percent of the activity is recovered in a fraction exhibiting a specific activity of 9.6 IU/mg. This purity is about one-tenth of that observed with homogeneous skeletal muscle p h o s p h o f r u c t o k i n a s e ? The purified phosphofructokinase can be stored at - 2 0 ° in phosphate buffer. H o w e v e r , greater stability results from adding glycerol to the phosphate buffer. A 50% solution of glycerol reduces inactivation from 3% per day to 0.3% per day at - 2 0 °. Properties
Time-Dependent Alterations in the Activity of Phosphofructokinase. Initial attempts to stabilize the phosphofructokinase in cell extracts by the addition of substrates resulted, surprisingly, in the recovery of negligible enzyme activity. However, normal activities are restored to these "inac4 o. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem, 193, 265 (1951).
74
KINASES
[12]
_ ~/O° 0.08 /
0o
3mM PEP 3raM ATP No MgCI2
0 0.06
\.---0 O
0.04
©
/ 0
A c"
m
< q
~
I
0
o 0.02 / 30 °
I--
3mM PEP 3mM ATP 5mM MgCI2
\ 300
< v
O.0C
~ 0
L 20
i 40
I 60
MINUTES FIG. I. Time-dependent alterations of enzyme activity. The temperature oscillations of phosphofructokinase activity can be observed by incubating the purified enzyme (5.4 IU/mg) at 0° in the presence of buffer (20 m M potassium phosphate, pH 8) and the indicated effector molecules. The experiment is initiated by mixing the 0° enzyme solution with a 0° solution containing ATP, PEP, and MgC]2 to attain the designated levels of effectors. Two micro]iters of the enzyme solution (protein = 0.56 mg/ml) are withdrawn at the indicated times and assayed for phosphofructokinase activity. The temperatures for incubation of the phosphofructokinase are as indicated by the arrows in the figure.
tive" extracts by dialyzing them against phosphate buffer at 4 ° or by raising the temperature of the extract to 30 ° for 15-30 min before assaying. This reversible inactivation is mediated by either ATP or phosphoenolpyruvate. H o w e v e r , ATP differs from phosphoenolpyruvate in that the ATP also causes an irreversible inactivation o f phosphofructokinase. 1 The reversible inactivation o f phosphofructokinase is most apparent upon combining ATP and phosphoenolpyruvate with Mg 2+ (Fig. 1). In the
[12]
PHOSPHOFRUCTOKINASE FROM
B.
lichen'tyormis
75
p r e s e n c e o f Mg 2+, PEP, and ATP, a rapid inactivation o f the e n z y m e o c c u r s at 0 °. This loss is r e v e r s e d b y i n c u b a t i o n at 30 °, and a n o t h e r c y c l e o f inactivation o c c u r s u p o n returning the p h o s p h o f r u c t o k i n a s e to 0 °. T h e omission o f Mg 2+ r e d u c e s the extent o f activity regain. T h e r e may' also be a slight d e c r e a s e in the rate o f inactivation with the omission o f Mg 2+ (Fig. 1). T h e kinetic o r d e r o f the activation with r e s p e c t to e n z y m e c o n c e n t r a tion is a m e n a b l e to analysis. Figure 2A d e m o n s t r a t e s that the initial rate o f e n z y m e activation is a nonlinear function o f the p r o t e i n c o n c e n t r a t i o n . H o w e v e r , the log(activity gain) is a linear function o f log[protein] with a slope n e a r 2 (Fig. 2B). This suggests that the reversible c y c l e s o f e n z y m e activity are due to a dissociation o f the m o l e c u l e into dimers with the s u b s e q u e n t association o f t h e s e subunits to r e s t o r e an active molecule. This interpretation is s u p p o r t e d by m o l e c u l a r weight estimates o f 68,000 and 135,000 for the inactive and active molecules, r e s p e c t i v e l y . T h e s e estimates are f r o m s u c r o s e density-gradient centrifugations o f the inactive and active p h o s p h o f r u c t o k i n a s e s . 1 If the association o f dimers requires Mg 2÷, this w o u l d explain the r e t a r d e d activation that o c c u r s in the absence o f a d d e d Mg '÷ (Fig. 1).
A
B
10.0
+10[
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~ (O 10 20 30 PROTEIN(~Jg/ml)
o
20
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10 LOG[PROTEIN]
I
20
F~G. 2. Molecular order of the activation of phosphofructokinase. The enzyme (,1.4 mg of protein per milliliter) is previously inactivated by incubation with 1 mM ATP, 1 mM phosphoenolpyruvate, 5 mM MgCI2, and 20 mM potassium phosphate (pH 7.6) at 0° for 3 hr. Then the indicated amount of the inactivated enzyme is transferred to a 1.0-ml cuvette containing 1.2 mM ATP, 5.0 mM fructose 6-phosphate, 3.0 mM MgC12, 100 mM KC1, ! mM 2-mercaptoethanol, and 20 mM buffer (pH 6.8) at 30°. The initial rate of activity gain (A) is determined, and the logarithm of activity gain is plotted against the logarithm of the protein concentration (B). A line with a slope of 2.0 is provided for reference.
76
KINASES 100-
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80
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,
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60
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MINUTES AT 30 o
FIG. 3. Pbosphofructokinase stability under assay conditions. The stability of phosphofructokinase at assay concentrations can be determined by adding purified enzyme (7.0 IU/mg) to the base solution (20 mM imidazole-HCl with 1 mM 2-mercaptoethanol; pH 6.82) containing the indicated reactant(s). After incubation of this solution at 30° for various times, the enzyme reaction is started by adding the remaining (omitted) reactants. Fructose 6phosphate (F6P), phosphoenolpyruvate, and ATP are present at 1 mM, and MgCI2 and KCI are present at 3 and 100 raM, respectively. In all cases, the complete assay reaction contains 20 mM buffer, I mM 2-mercaptoethanol (2-ME), 1 mM ATP, 100 mM KCI, and 3 mM MgCI2. The control NADH oxidation rate is 0.062 A340 per minute.
The temperature-dependent inactivation seen with B. licheniform& phosphofructokinase is also found with B. cereus, B. megaterium, B. mycoides, and B. subtilis 168 M, but not with Escherichia col B. 1 This phenomenon of temperature inactivation appears to correlate with stability of the phosphofructokinases from these bacteria, since 15% of the E. coli B activity and 30-35% of the Bacilhts sp. activity is lost during these experiments. It is also important to note that the molecular weights of these enzymes from E. coli and Bacillus sp. are 130,000 to 140,000.1 Stability of Phosphofructokinase under Assay Conditions. This enzyme is fragile enough to be rapidly inactivated during incubation under assay conditions (Fig. 3). Inactivation is arrested, essentially, by the addition of Fru-6-P. Although both KC1 and PEP are stabilizing ligands, they are much less effective than Fru-6-P (Fig. 3). The addition of ATP or ATP with MgC12 only slightly improves the stability. This general instability dictates
PFK FROM Streptococcus lactis
[13]
77
that the reaction be initiated by the addition of phosphofructokinase. Even with this precaution, a time-dependent decrease of the reaction rate occurs when a high ATP concentration is combined with a low Fru-6-P concentration. Therefore, the instability of this enzyme under assay conditions can lead to invalid kinetic characterizations. Kinetics of PhosphojJ'uctokinase. Saturation functions for ATP and Fru-6-P are hyperbolic with apparent Km values of 0.07 and 0.05 raM, respectively. 1 Several divalent cations support catalysis by this enzyme but magnesium is the preferred ion. The pH optimum is in the range of 8.2-8.7. Either NH4 + or K + activates the B. licheniformis phosphofructokinase, whereas Li + and Na + ions have no effect on the enzyme activity. The apparent dissociation constants for NH4 + and K + are 0.5 and l0 mM, respectively. The most effective inhibitor of this enzyme is phosphoenolpyruvate. This compound increases the Km of the enzyme for Fru-6-P. Phosphoenolpyruvate, which is an effective inhibitor at 5-20 tzM, has an apparent Hill coefficient of 3. Other inhibitors of this enzyme include ATP, citrate, calcium, and pyrophosphate. Sensitivity to inhibition by ATP and citrate is conditioned by the concentration of Fru-6-P and Mg z+, whereas the inhibition by phosphoenolpyruvate is specifically relieved by Fru-6-P. Calcium is a competitive inhibitor of Mg z+, and its apparent K~ is 0.2 mM. Pyrophosphate has the effect of inactivating the phosphofructokinase under assay conditions.
[13] P h o s p h o f r u c t o k i n a s e f r o m Streptococcus lactis By ALlSON M. FORDYCE, C. H. MOORE, and G. G. PRrTCHARD Fructose 6-phosphate + ATP---, fructose 1,6-bisphosphate + A D P + H +
Assay Method
Principle. Phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) activity can be coupled via the enzymes fructose 1,6-bisphosphate aldolase, triosephosphate isomerase, and glycerol 3-phosphate dehydrogenase to NADH oxidation? Activity can then be determined spectrophotometrically by measuring the decrease in absorbance at 340 nm. Phosphorylation of 1 /zmol of fructose 6-phosphate results in oxidation of 2 tzmol of NADH in this assay. E. R a c k e r , J. Biol. Chem. 167, 843 (1947).
METHODS IN ENZYMOLOGY, VOL. 90
Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6