257

Biochimica et Biophysica Acta, 381 (1975) 257--268

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 27581 REGULATION OF THE AMOUNT AND OF THE ACTIVITY OF PHOSPHOFRUCTOKINASES AND PYRUVATE KINASES IN ESCHERICHIA COLI

D. KOTLARZ,a H. GARREAUb and H. BUCa Ddparternent de Biologie Moleculaire, Institut Pasteur, 75015 Paris, and bINSERM, unitg 75, Facultd Necker-Enfants Malades, 156 rue de Vaugirard, 75015, Paris (France)

(Received July 2nd, 1974)

Summary Two isozymes of fructose-6-phosphate kinase and two isozymes of pyruvate kinase have been detected in Escherichia coli under a wide variety of growth conditions. Their kinetic behavior has been characterized with respect to different effectors and substrates. The conclusions reached on one hand by Malcovati and Kornberg (Biochim. Biophys. Acta (1969) 178, 420--423), on the other hand by Fraenkel, Kotlarz and Buc (J. Biol. Chem. (1973) 248, 4865--4866) have been found to be true in aerobiosis as well as in anaerobiosis. The biosynthesis of the four proteins is sensitive to the nature of the carbon sources as well as to the shift from aerobic to anaerobic conditions. Kinetics of depression after a shift to anaerobiosis have been followed and found to be of the order of the doubling time.

Introduction Two key enzymes which regulate the Embden--Meyerhof degradative pathway, fructose-6-phosphate kinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40), have been shown to be subjected to allosteric regulation in Escherichia coli K12 [1--6]. More precisely, two isoenzymes of each of these two proteins are present in this bacterium [7--9]. Fructose-6-phosphate kinase activity is mainly due to P y r u v a t e k i n a s e - I a n d p y r u v a t e k i n a s e - I I c o r r e s p o n d t o the r e s p e c t i v e i s o e n z y m e s d e s i g n a t e d PK-I and P K - I I b y M a l c o v a t i a n d K o r n b e r g [ S ] , a n d P F r - F a n d P y r - A b y K o r n b e r g a n d M a l c o v a t i [ 1 1 ] . P h o s p h o f r u c t o k i n a s e - I a n d p h o s p h o f r u c t o k i n a s e - I I c o r r e s p o n d t o t h e d e s i g n a t i o n s PFK-I and P F K - I I o f F r a e n k e l et al. [ 7 ] . I n t h i s a r t i c l e , w e h a v e u s e d t h e orJglnal n o m e n c l a t u r e s o f F r a e n k e l e t al. [ 7 ] o n o n e h a n d , and o f M a l c o v a t i and K o r n b e r g [ 8 ] o n t h e o t h e r . M o r e r e c e n t l y , K o r n b e r g and M a l c o v a t i [ 1 1 ] p r o p o s e d t h e n o m e n c l a t u r e p y r - A f o r PK-II and p y r - F f o r PK-I.

258 the biosynthesis of a regulatory enzyme coded by the gene p f k A . This enzyme phosphofructokinase I exhibits a sigmoidal response with respect to fructose 6-phosphate, is activated by ADP and inhibited by phosphoenolpyruvate. Activity phosphofructokinase II which represents less than 5% of the total activity in the wild type E. coli is 10-fold increased in mutants suppressed at an unliked locus called p f k B [10]. Phosphofructokinase II is devoid of allosteric characteristics [7]. On the other hand, Malcovati and Kornberg [8,9,11] have shown that two types of pyruvate kinase activities can be distinguished according to their elution pattern from a DEAE-cellulose column by a linear gradient of 0--0.5 M KC1. The first peak of pyruvate kinase activity which is eluted, pyruvate kinase-I, exhibits a sigmoidal response versus phosphoenolpyruvate concentration and is activated b y fructose-P2, while the second one, pyruvate kinase-II, is activated by 5'-AMP and exhibits less sigmoidal kinetics with respect to the substrate, phosphoenolpyruvate. The purpose of this communication is to study the presence or absence of these four isozymes under different conditions of growth and the amount of derepression of their biosynthesis under aerobic or anaerobic conditions, as well as the kinetics of derepression after a shift from aerobiosis to anaerobiosis. Materials and Methods A prototrophic derivative of E. coli K10 (Hfr C, str s, BI-), has been used in all experiments.

(1) Cultures The cultures were grown at 37°C in minimal salt medium 63 [ 12], supplemented with thiamin to a final concentration of 1 pg/ml. The total a m o u n t of the carbon source was kept constant (final concentration of 0.4 g/100 ml for six-carbons sugars and 0.8g/100 ml for the three other substrates, glycerol, pyruvate and acetate). Aerobic conditions corresponded to an air supply of 10 1/min in 10-1 containers and to a shaking rate of 600 rev./min. For anaerobic growth, nitrogen was supplied at 2 1 per min plus 0.2 1/min of CO2 and the shaking rate decreased to 50 rev./min. Dissolved oxygen was monitored with an oxygen electrode and shown to decrease to zero within 2--3 min of nitrogen replacement. In all cases, the pH was maintained at neutrality. Growth was stopped b y the addition of chloramphenicol to a final concentration of 50 pg/ml. Under these conditions, protein synthesis stops within 1 min or less. Bacterial growth was followed by turbidity measurements at 600 nm in a Zeiss s p e c t r o p h o t o m e t e r PM QII. In every case, bacteria were collected in the exponential phase of growth. Bacteria were centrifuged and washed with a 5 mM potassium phosphate buffer, pH 7.5, 0.5 mM EDTA, 1 mM 2-mercaptoethanol. The pellet was then resuspended in the same buffer (4 ml for 1 g wet weight of bacteria} and the suspension submitted to sonic disruption. Cell wall and debris were centrifuged and the supernatant diluted 2-fold in glycerol. These crude extracts could be kept at --18°C for two weeks w i t h o u t any measurable loss of the enzymatic activities. They were used for all enzymatic measurements.

259

(2) Enzymatic assays Standard assays for aldolase were performed in a mixture containing 50 mM Tris--HC1, pH 7.5, 0.2 mM NADH, 3 pg/ml of triosephosphate isomerase, 3 pg/ml of glycerophosphate dehydrogenase. Fructose-P2 was omitted in the control blank and adjusted to a final concentration of 5 mM in the assay. Each of the t w o pyruvate kinase activities can be measured directly in the crude extracts according to the principle given in ref. 8. For that purpose we used the following mixture: 50 mM Tris--acetate, pH 7.0, 0.2 mM NADH, 5 pg/ml lactate dehydrogenase, 50 mM KC1, 5 mM MgC12,2 mM ADP. In four cuvettes we respectively added to that standard mixture: a, b, c, d,

Phosphoenolpyruvate Phosphoenolpyruvate Phosphoenolpyruvate Phosphoenolpyruvate

0 ; 1 mM; 1 mM; 1 mM;

5'-AMP 5'AMP 5'-AMP 5'-AMP

: : : :

0 ; 0 ; 1 mM; 0 ;

Fructose-P2 Fructose-P2 Fructose-P2 Fructose-P2

: : : :

0 0 0 1 mM

As it will be shown in the next section, the maximal enzymatic activity due to pyruvate kinase-I is equal to 1.2 times the difference V d - - V b while for pyruvate kinase-II it amounts to Vc -- Va. We have checked that ADP was at saturating concentration by adding up to 3 mM of ADP in every cuvette. Furthermore, no appreciable NADH oxidation takes place when we omit lactate dehydrogenase in Cuvette d which indicates that aldolase activity is negligible under the assay conditions. The standard assay mixture for measuring phosphofructokinase activities contained 0.1 M Tris--HC1, pH 8.2, 0.2 mM NADH, 10 mM MgC12,1 mM ATP, 3 pg/ml of triosephosphate isomerase, 3 pg/ml of glycerophosphate dehydrogenase, 30 pg/ml of aldolase. With 0.2 mM fructose 6-phosphate and 2 mM phosphoenolpyruvate, phosphofructokinase II is fully active [7], b u t phosphofructokinase I is completely inhibited [4]. By adding to the standard assay mixture 1 mM Fru-6-P and 1 mM GDP, total phosphofructokinase activities can be measured. The difference represents phosphofructokinase I activity. Fru-6-P is omitted for control measurements. The fact that both activities are indeed present in the extract will be justified in the next section. All enzymes activities were followed b y measuring the rate of decrease of absorbance at 340 nm in a Gilford 2000 spectrophotometer at 28 ° C.

(3) Control experiments It is crucial to check that enzymatic response in the crude extracts with respect to substrates or effectors are n o t affected by an eventual degradation of these derivatives. These controls were performed with extracts from bacteria grown either anaerobically on glucose or aerobically on pyruvate. The eventual degradation of effectors in the presence of the bacterial extract is checked as follows: in the pyruvate kinase assay medium, fructose-P~ and AMP are added. Aliquots are removed before and 10 min after the addition of bacterial extracts. Effectors concentrations are measured enzymatically in these aliquots, after precipitation o f proteins b y HC104 and neutralization

260

according to Marchand et al. [13]. A similar procedure is used to check the stability of GDP and phosphoenolpyruvate in the phosphofructokinase medium assay. Less than 5% degradation of any of these four effectors occurred in 10 min, the maximal time o f a routine enzymatic assay. Degradation of substrates by another metabolic pathway cannot affect the measurement of the catalytic activities of phosphofructokinase or pyruvate kinase isozymes. This point is established as follows: a single substrate is omitted in the assay medium which is incubated 10 min with the extract. Then the reaction is started by the addition of the missing substrate. The velocity which is measured has been found equal, within experimental error, to the one obtained in the absence of preincubation with the extract; this is true for pyruvate kinase activities measured in the presence of AMP and fructose-P2 and of phosphofructokinase activities measured in the presence of GDP or phosphoenolpyruvate; substrate concentrations remain therefore saturating during the time of an enzymatic assay.

(4) Protein synthesis Protein synthesis was measured b y following [14 C]isoleucine incorporation during growth (in the presence o f 5 mM non-radioactive isoleucine). Different volumes of bacterial suspension were pipetted out of the flask in order to treat a relatively constant number of bacteria (around 108 ). These aliquots were boiled for 10 min in a 10% solution of trichloroacetic acid, then cooled and filtered on Millipore filters. The filter was rinsed with cold 10% trichloroacetic acid, dried and placed in a toluene--POPOP mixture for the measurement of radioactivity which was counted in an Intertechnique SL40 scintillation spectrometer. Whenever we wanted to express specific activities of enzyme in I.U./mg of total protein in the extract, the biuret colorimetric method for determination of the protein concentration was used in parallel [14]. Fig. 1 shows that both methods gave results in close agreement and that the average content of protein per cell, which is proportional to the slope of the lines, is not significantly affected by the shift from aerobiosis to anaerobiosis.

(5) Purification of phosphofructokinase I Phosphofructokinase I can be purified to homogeneity. For the first steps of purification, we followed the method described by ref. 7. After warming at 65°C in presence of 10 mM Fru-6-P, the supernatant was adsorbed on a cibacron Sephadex G-200 column (5 mg of protein/ml of Sephadex) according to the m e t h o d given in refs 15 and 16. The column was washed with a solution of 0.4 M (NH4)2 SO4 in 0.1 M Tris--HC1, pH 8.2, 1 mM MgC12 until no material absorbing at 280 nm was detected in the eluate, and then rinsed with the buffer w i t h o u t (NH4)2 SO4. Phosphofructokinase I was eluted with a 0.2 mM solution of ATP. At this stage, the enzyme migrates as a single band on polyacrylamide gels either under native or denaturing conditions as in the previously described preparation [ 3]. This purified phosphofructokinase I preparation was used to obtain antibodies. A first injection o f 40 pg/rabbit of phosphofructokinase I, emulsified in Freund's adjuvant, was made in the t w o posterior footpads. One month later,

261 Counts per minutel'~C/ml of b o c t e r i o l suspension 250 I

E

500 I

350 (

.i,]ogeoj / /

g2 tD

o

I L

0

I 2

I 3

mg of totol p r o t e i n s / m l of crude extroct

Fig. 1. Incorporation of [ 14 C] i s o l e u c i n e ( •--) a n d c h a n g e in protein concentration in crude extracts (--0--) as a f u n c t i o n o f g r o w t h . [ 1 4 C ] I s o l e u c i n e w a s i n t r o d u c e d a t a n a b s o r b a n c e o f 0 . 2 a t 6 0 0 rim.

150 pg of phosphofructokinase I per rabbit were injected intraperitoneally and the same quantity intravenously the day after. Intraperitoneal and intravenous injections were repeated weekly four times. The blood was collected by cardiac puncture about 10 days after the last injection.

(6) Material Phosphoenolpyruvate, fructose 6-phosphate Fru-6-P, triosephosphate isomerase, aldolase, glycerophosphate dehydrogenase and lactate dehydrogenase were purchase from Boehringer; adenosine triphosphate (ATP), guanosine diphosphate (GDP), fructose 1,6-diphosphate (fructose-P:) from Sigma, [14 C] isoleucine (13 Ci/mole) from C.E.A., adenosine monophosphoric acid (5'-AMP) from E.D.C. Results

(1) Presence of the four isoenzymes We initially determined that cells grown aerobically or anaerobically on either glucose or pyruvate had the two types of pyruvate kinase activity described by Malcovati and Kornberg [8]. Using the same method of elution from DEAE-cellulose as described by these authors, no other pyruvate kinase activity was found in the eluates (cf. Fig. 2). For each Peak I and II of pyruvate kinase activity, the saturation curve for phosphoenolpyruvate in the presence or absence of fructose-P2 or 5'-AMP was determined. The concentration of phosphoenolpyruvate giving half the maximal velocity in the case of a sigrnoidal response, K1/2, is equal to 8 m M for pyruvate kinase-I and 0.7 mM for pyruvate kinase-II. In the presence of activators, these values were shifted to

262

150

1.5 E c

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100

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o

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60

70

F i g . 2. C h r o m a t o g r a p h y on DEAE-ceUulose of glucose grown extracts (aerobically, - ..... ; anaerobically, - - ) . Pyruvate kinase activity was assayed as described in Materials and Methods except phosphoenolpyruvate and ADP concentrations which were respectively equal to 5 and 2 raM. The first peak corresponds to pyruvate kinase-I, the second to pyruvate kinase-II.

Km = 0.15 mM for pyruvate kinase-I and Kra = 0.1 mM for pyruvate kinase-II. They were independent of the medium in which cells were grown (Table I). We noted, however, a great variability of the sigmoidicity in the saturation curve by phosphoenolpyruvate of pyruvate kinase-II depending upon the age of the extract, a fresher extract showing a more sigmoidal curve. We did not detect any desensitization o f pyruvate kinase-I. From Fig. 3, it appears that for a concentration of 1 mM of phosphoenolpyruvate, pyruvate kinase-I exhibits less than 1% of its maximal velocity. Adding 1 mM fructose 1,6-diphosphate brings the activity of pyruvate kinase-II to 80% of its maximal velocity. On the other hand, the presence of 1 mM AMP does not change the activity of pyruvate kinase-I but brings pyruvate kinase-II

TABLE

I

KINETICS CONSTANTS FOR PYRUVATE KINASE-I AND PYRUVATE EXTRACTS CULTIVATED UNDER VARIOUS CONDITIONS

KINASE-II

ACTIVITIES

OF

K m refers to the Michaelis constant for phosphoenolpyruvate o b t a i n e d i n e a c h c a s e a t s a t u r a t i o n e i t h e r in f r u c t o s e - P 2 o r i n 5 ' - A M P . K1/2 is t h e c o n c e n t r a t i o n of phosphoenolpyruvate required, in the absence of activator, to reach half-maximum velocity.

Pyruvate kinase-I

Pyruvate kinase-II

Km

K1/2

Km

K1/2

Aerobiosis glucose pyruvate

0.15 mM 0.14 raM

7.5 mM S mM

0.1 mM 0.11 mM

1.5--0.8 rnM 0.7 mM

Anaerobiosis glucose pyruvate

0.15 mM 0.15 raM

S raM 8.2 mM

0.1 0.1

1.2 1.5

ram mM

mM ram

263 o)

100

~ / x

50

/

c

o.

0

2

/ 4

1 1

x / ~ n o FOP

6

8

I0

mM

IPEPI

0

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50

ImM

×

7

~

0

e"/~"~+ 5' A M P

/

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L

O.t

0.2

I

I

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I

0.3

0.4

0.5

0.6

l

_

0.7 mM (PgPl

Fig. 3. K i n e t i c r e s p o n s e o f p y r u v a t e kinase-I a c t i v i t y w i t h r e s p e c t t o p h o s p h o e n o l p y r u v a t e in t h e p r e s e n c e o r a b s e n c e o f f r u c t o s e 1 , 6 - d i p h o s p h a t e ( u p p e r g r a p h ) o r of p y r u v a t e kinase-TI ( l o w e r g r a p h ) in t h e p r e s e n c e or a b s e n c e o f 1 m M 5 ' - A M P . I n c r e a s e d c o n c e n t r a t i o n s o f 5 ' - A M P ( u p to 5 m M ) d o n o t a f f e c t f u r t h e r v e l o c i t y o f p y r u v a t e k i n a s e - I I . Crosses a n d o p e n circles r e f e r t o t h e e f f e c t of aging u p o n t h e p y r u v a t e k i n a s e - I I a c t i v i t y , (o, f r e s h e x t r a c t ; X, e x t r a c t s t o r e d f o r o n e d a y at 4 ° C).

to its maximal activity. We are, therefore, justified in using the procedure described in Materials and Methods and in taking 1.2 × (Vd -- Vb ) and (Vc -Va), respectively, as a measurement of the relative enzymatic activities of pyruvate kinase-I and pyruvate kinase-II for the same amount of total protein. The anti-phosphofructokinase I antiserum gives positive precipitin reaction with phosphofructokinase I b u t does n o t cross-react with either purified phosphofructokinase II [7] or with crude preparations of pyruvate kinase-I or pyruvate kinase-II. With the anti-phosphofructokinase I antiserum insolubilized on Sepharose 4B according to the procedure described in ref. 17 the binding properties of the t w o phosphofructokinase and pyruvate kinase were studied. Phosphofructokinase I binds tightly to a Sepharose-anti-phosphofructokinase I suspension, as seen in Fig. 4, while phosphof~uctokinase II, as well as pyruvate kinase-I and pyruvate kinase-II, do n o t bind at all under any conditions assayed: range of pH from 6 to 8.5, change in ionic strength or in the buffer, presence of 1 M urea, at 4 or 25°C, presence or absence of substrates. Since a purified preparation of phosphofructokinase I is tightly b o u n d to the column, one can look in crude extracts for the properties of the fructose6-phosphate kinase which is n o t retained on the insolubilized antiserum. It appears that we always obtain a small fraction of the total phosphofructokinase

264

IO0 ~,e"O--A-I

a---e

z,,

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o

O

>.

50-

?, I0

-

I O. I

;-4~ 0.5

1.0

I .5.0

rnl of sephorose-onti PFK I suspension Fig. 4. R e t e n t i o n o f p h o s p h o f r u c t o k i n a s e I o n a n a n t i - p h o s p h o f r u c t o k i n a s e I s e r u m c o u p l e d o n S e p h a x o s e b e a d s . T o t h e s a m e v o l u m e o f a p h o s p h o f r u c t o k i n a s e I (o) o r o f a p h o s p h o f ~ u c t o k i n a s e II (0) e n z y m e p r e p a r a t i o n , c o r r e s p o n d i n g to t h e s a m e m a x i m u m a c t i v i t y , axe a d d e d i n c r e a s i n g v o l u m e s o f a s u s p e n s i o n o f a n t i - p h o s p h o f r u c t o k i n a s e I s e r u m c o u p l e d on S e p h a x o s e . E x p e r i m e n t s axe p e r f o r m e d in T r i s ~ c e t a t e b u f f e r , 0 . 0 5 M, p H 7.0, c o n t a i n i n g 1 m M o f M g C l 2. S a m p l e s axe g e n t l y s h a k e n f o r 1 h at 4 ° C , c e n t r i f u g e d , a n d a c t i v i t i e s a r e m e a s u r e d in t h e s u p e r n a t a n t . A c o n t r o l e x p e r i m e n t (~) is m a d e w i t h S e p h a x o s e b e a d s c o u p l e d t o n o r m a l r a b b i t s e r u m . P y r u v a t e k i n a s e - I p y r u v a t e k i n a s e - I I a c t i v i t i e s , w h e n a s s a y e d in t h e s a m e m a n n e r in t h e p r e s e n c e o f t h e a n t i - p h o s p h o f r u c t o k i n a s e I s e r u m , axe n o t a f f e c t e d .

activity, 3--6%, which is n o t retained. This fraction exhibits all the known characteristics of phosphofructokinase II (Michaelis--Menten kinetics towards fructose 6-phosphate, no affinity for a cibacron blue column, no precipitation at pH 4.4). Furthermore, for bacteria grown on glucose in aerobiosis or in anaerobiosis we have found no change in the sedimentation coefficient of purified phosphofructokinase I run on glucose gradient at 4°C (S = 7.5 S). We can therefore conclude that, up to now, no other isozymes than phosphofructokinase I, phosphofructosekinase II, pyruvate kinase-I and pyruvate kinase-II have been detected under growth conditions tested.

(2) Derepression Specific activities of pyruvate kinase-I, pyruvate kinase-II, phosphofructokinase I and phosphofructokinase II and aldolase have been measured in crude extracts of bacteria grown on fructose, glycerol, acetate and pyruvate under either aerobic or anaerobic conditions. Results are given in Table II. Under aerobic as well as under anaerobic conditions the derepression of most enzyme is sensitive to the nature of the carbon source. This is true for phosphofructokinase I, pyruvate kinase I and aldolase. Pyruvate kinase-II is slightly affected by this parameter under anaerobic conditions but not at all under aerobic growth where its synthesis seems to be constitutive (results in agreement with the conclusions given by Kornberg and Malcovati [8,11] and Maeba and Sanwal [5] ; cf. however ref. 9). Phosphofructokinase-II synthesis is n o t at all affected. For the allosteric enzymes, it appears therefore that it is the enzyme which is regulated by an allosteric effector present in the Embden--Meyerhof pathway (phosphoenolpyruvate in one case, fructose-P2 in the other) which is derepressed as the carbon source is changed. However, in both cases, as well as

265 T A B L E II S P E C I F I C A C T I V I T I E S IN C R U D E E X T R A C T S O F T H E E N Z Y M E S T E S T E D ( e x p r e s s e d in I . U . / m g of p r o t e i n ) p, r a t i o o f specific activities o b t a i n e d in glucose v e r s u s a c e t a t e m e d i u m f o r a g i v e n e n z y m e , k, r a t i o o f specific a c t i v i t i t i e s o b t a i n e d in a n a e r o b i c v e r s u s a e r o b i c c o n d i t i o n s . (1) P h o s p h o f m c t o k i n a s e d e r e p r e s s i o n Carbon source

Phosphofructokinase I Aer obiosis

Fructose Glucose Glycerol Pymvate Acetate p

glucose

-

-

pyruvate

0.4 0.31 0.23 0.17 0.11 1.8

P h o s p h o f r u c t o k i n a s e II

Anaerobiosis

k1

0.68

1.94

0.17--0.2

1.1

3.5 - - 4

Aerobiosis 0.014 0.016 0.015 0.011 0.012 ~1.5

Anaerobiosis

~2

0.013

1.2

0.014

1.3

1.1

(2) Pyruvate kinase derepression Carbon

Pyruvate kinase I

Pyruvate kinase II

source Aerobiosis Fructose Glucose Glycerol Pyruvate Acetate

0.4 0.3 0.22 0.07 0.08

Anaerobiosis

k1

0.49

1.6

0.08

1.1

Aerobiosis 0.215 0.2 0.22 0.19 0.27

Anaerobiosis

k2

0.52

2.6

0.36--0.47

2.0

glucose p

pyruvate

4.3

6

1

1.3

(3) Aldolase d e r e p r e s s i o n Carbon source

Aerobiosis

Anaerobiosis

k

Glucose Glycerol Pyruvate

0.075 0.067 0.048

0.155

2.06

0.073

1.52

1.56

2.12

glucose p

pyruvate

for aldolase, the change in the quantity of enzyme present in the cell as a function of the nature of the carbon source does not follow a simple law. One could have assumed that derepression is maximum if the sugar or the organic carbon source enters the Embden--Meyerhof pathway before the step which is catalyzed by a given enzyme and is poor in the opposite situation. Indeed, derepression seems to follow a kind of gradient being maximum for fructose and minimum for acetate. More quantitatively, one can say that p, the ratio of enzymatic activities

266 detected in glucose versus pyruvate decreases in the following order: p(pyruvate kinase-I) > p (phosphofructokinase I) > p (aldolase) Ii now, one looks at the relative derepression ~. observed after a shift from aerobic to anaerobic conditions, in a medium containing glucose or pyruvate, it appears that, again, phosphofructokinase II is not significantly affected, that pyruvate kinase-I is poorly derepressed while the effect is large for phosphofructokinase I, aldolase and largest for pyruvate kinase-II. It is again striking that among the allosteric isozymes, it is now the ones which are sensitive to the AMP/ATP ratio that are derepressed. This experiment illustrates, in particular, the fact that the biosynthesis as well as the allosteric regulation of one pyruvate kinase isozyme, pyruvate kinase-I, is sensitive to the catabolic needs of the ceil while the other, pyruvate kinase-II, responds in both respects to the energy level of the bacteria. On the other hand, phosphofructokinase I, the major fructose-6-phosphate kinase isozyme cumulates the two types of regulation and is derepressed, both under anaerobic condition and in energy-rich media. One can then wonder about the role of phosphofructokinase II, the enzymatic activity of which is always at least 20 times smaller than that of phosphofructokinase I. It should be recalled, however, that enzymatic assays are always done in vitro under maximal velocity conditions. In the absence of GDP or phosphoenolpyruvate, the dependence of the initial velocity of phosphofructokinase I is strongly cooperative with respect to fructose 6-phosphate, in a concentration range of this substrate which corresponds to physiological

o

% __

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3 o o

c_ E

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.

3--0-0

°

8 __&...A°4

>, N c

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l

2

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l

3

4

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Fig. 5. Kinetics of derepression of aidolase (o), phosphofructokinase I (o) and pyruvate kina.se-II (A) synthesis after a shift to anaerobic conditions indicated b y the arrow. T h e doubling time after the shift is measured b y the incorporation of [ 14C] isoleucine introduced three generations before the shift, and the specific activities are obtained by dividing enzymatic activities by the n u m b e r of c p m incorporated. G r o w t h is performed o n 2 % glucose, the doubling time after the shift being 180 rain.

267

conditions [6]. Phosphofructokinase II could therefore provide a kind of leak similar in its effect to the base level found for the ~-galactosidase system under conditions of repression. According to the results given b y Thomas et al. [18] it seems that there exists a gradient of derepression for phosphofructokinase activity as the oxygen tension is lowered, a result which will be analogous to the gradient we have observed as a function o f the nature of the carbon source. Kinetics of the derepression. A similar t y p e of gradual effect in the derepression is found when one passes from aerobic to anaerobic conditions. Samples are taken at various times during aerobic growth and then at times +5, +10, +30, +90, +120, +150 and +200 min after the shift. Under our conditions, the anaerobic doubling time was equal to 180 min. In each sample, total protein synthesis and specific activity of enzymes were measured. The results are plotted in Fig. 5. It is clear that all the enzymatic activities increase gradually. Within experimental error, maxima are reached after one doubling time. The nitrogen pressure within the flask is modified within 2--5 min. The rate limiting step in the derepression may therefore be linked to DNA replication. Conclusion There is a correlation b e t w e e n the extent of derepression of a given isozyme of pyruvate kinase or phosphofructokinase and the nature of the allosteric signal which regulates its enzymatic activity. Phosphofructokinase I and pyruvate kinase-I are sensitive to the concentration of specific metabolites of the E m b d e n - - M e y e r h o f pathway and their biosynthesis depend strongly on the nature of the carbon source. Activations of phosphofructokinase I and of pyruvate kinase-II are strongly regulated b y the concentration of ADP or AMP and their a m o u n t in the cell is very sensitive to the aerobic or anaerobic conditions of growth. The extent of derepression of a michaelian enzyme, aldolase, used as a control, does also depend on the nature of the carbon source and on the dissolved oxygen tension. The significant feature is n o t therefore the absolute extent of the derepression of the three allosteric enzymes, b u t rather the relative derepression of pyruvate kinase-I versus pyruvate kinase-II and of phosphofructokinase I versus phosphofructokinase II. It appears that at the biosynthetic level as well as for the control of the catalytic activity, the response to the energy charge or to the change in the flux through the Embden--Meyerhof pathway is controlled through two different isozymes in the pyruvate kinase system and b y a single enzyme, in the phosphofructokinase system. One can perhaps notice that the regulatory signals are very similar for the three allosteric enzymes. Fructose 6-phosphate and ATP are the substrates of phosphofructokinase I, which recognizes phosphoenolpyruvate and XDP (the substrates of pyruvate kinase-I and of pyruvate kinase-II) as allosteric effectors. On the other hand, one of the p r o d u c t of the reaction catalyzed by phosphofructokinase, fructose-P2, is the activator of pyruvate kinase-I while the activator of pyruvator kinase-II, AMP has a stereochemical structure close to the other p r o d u c t of phosphofructokinase, namely ADP. It is tempting to propose

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as a working hypothesis that the structural genes of the four isozymes have a c o m m o n origin and that they derive from the partial fusion of two structural ancestor genes pyruvate kinaseo and phosphofructokinaseo which were devoid of allosteric regulation. Along this line of reasoning, one could propose that allosteric sites of a regulatory protein could perhaps be generated through the same mechanism of partial gene fusion, one of the two catalytic activities being lost after the folding, either because crucial amino acids present at one of the sites are no more coded or because proper alignment of substrates is now impossible in a tertiary structure which is not exactly folded in the correct manner. Immunological experiments performed with antibodies directed against the native quaternary structure of phosphofructokinase I have, however, failed to detect any cross-reactlvity between the four isozymes. One has therefore to wait for the determination of the primary structures in order to know whether this hypothesis is correct or not. Acknowledgements Our research was supported b y grants from the Centre National de la Recherche Scientifique, D~le'gation Gdne'rale h la Recherche Scientifique et Technique, Commissariat h l'Energie A t o m i q u e and National Institute of Health. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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