allosteric properties of the isoenzymes of pyruvate kinase from rat kidney cortex

128 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 66710

ALLOSTERIC PROPERTIES OF THE ISOENZYMES OF PYRUVATE KINASE FROM RAT KIDNEY CORTEX*

L I D I A COSTA** L U I S J IMI~NEZ D E ASI~A, E N R I Q U E R O Z E N G U R T * * * E R N E S T O O. B A D E . . . . A.NO H E C T O R C A R M I N A T T I

Instituto de Investigaciones Bioqulmicas "Fundacidn Campomar" and Facultad de Ciencias Exactas y Naturales Obligado 2490, Buenos Aires 28 (Argentina)

(Received May 24th 1972)

SUMMARY

The pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.4o ) isoenzymes from kidney cortex have been studied further. Pyruvate kinase type I which is the major component, shows homotropic cooperativity towards the substrate phosphoenol pyruvate, the effect is independent of pH. This isoenzyme is markedly inhibited by alanine and other amino acids but its activity is not modified by ATP or fructose 1,6-diphosphate. The kinetic behavior of this isoenzyme is then clearly different from that of the other pyruvate kinases from rat tissues.

Pyruvate kinase type II exhibits sigmoid kinetics with respect to phosphoenol- pyruvate. The enzyme is activated by fructose 1,6-diphosphate and inhibited by ATP, alanine and cysteine. The allosteric properties of the enzyme are strongly affected by changes in the pH. It is concluded that this kidney cortex isoenzyme is very similar to the pyruvate kinase type L from liver.

INTRODUCTION

In a preliminary note we have reported the occurrence of two different forms of pyruvate kinase (ATP :pyruvate phosphotransferase, EC 2.7.1.4o) (types I and II) in rat kidney cortex 1. Both isoenzymes could be resolved on DEAE-cellulose chromatography. Pyruvate kinase I was found to be the major component; it showed a kinetic behavior somewhat different from other pyruvate kinases of mammals (see refs 2 and 3).

The present paper reports further studies on the kinetic properties of both types of kidney cortex pyruvate kinase. The results presented here reinforce our pre-

Abbrev ia t ion : P E P , p h o s p h o e n o l p y r u v a t e . * This work is pa r t of a Doctora l Thes i s by Lu i s J im6nez de Asfia as a r e q u i r e m e n t for t he

degree o f Ph. D. in B i o c h e m i s t r y a t the F a c u l t y of Sciences, Un ive r s i t y o f Buenos Aires, 1971. ** P re sen t address : Centro de Inves t igac iones Cardioldgicas, Buenos Aires, Argen t ina .

"~* P r e s e n t address : D e p a r t m e n t o f Biological Sciences, Pr ince ton , N.J . , U.S.A. . . . . P r e s e n t address : Cold Spr ing H a r b o r Labora to ry , Long Is land, N . Y . U . S . A .

Biochim. Biophys. Acta, 289 (1972) 128-136

ALLOSTERIC PROPERTIES OF PYRUVATE KINASE ISOENZYMES I29

vious conclusion that the minor component (pyruvate kinase II) resembles very close- ly isoenzyme L of liver. Furthermore pyruvate kinase I has different kinetic properties as compared to both liver isoenzymes (L and M), hence it could belong to a third group of pyruvate kinases present in mammalian tissues.

MATERIALS AND METHODS

The enzymes were isolated from rat kidney cortex as already described 1. The homogenate was centrifuged at IOO ooo × g for 60 min. The clear supernatant fluid was purified by (NH4)2SO a fractionation between 30-45% (pyruvate kinase II) and 55-70% (pyruvate kinase I) saturation. Both isoenzymes were further purified by chromatography on DEAE- and CM-cellulose columns, respectively, as previously described for liver pyruvate kinases4, 5. Pyruvate kinase I and pyruvate kinase I I were purified in the last steps 20 and 15 times, respectively. Other materials and methods were as previously described 4.

RESULTS

The fractionation of both types of pyruvate kinase found in the IOO ooo × g kidney cortex supernatant can be accomplished by chromatography on a DEAE- cellulose column. The major component (pyruvate kinase I) is eluted from the column with the equilibrating buffer (15 mM Tris-HC1, 25 ° mM sucrose, I mM EDTA and I mM dithiothreitol, adjusted to pH 7.5). I t represents 70-80% of the total enzymic activity. The column was washed with the above-mentioned buffer and the other isoenzyme (pyruvate kinase II) was eluted with the same solution to which a linear gradient of KC1 was superimposed. This second peak is eluted at 120 mM KC1. When the fractions obtained from this column were assayed at a low level of phospho- enolpyruvate (PEP) (0.8 mM) and in the presence or absence of fructose diphosphate (0.I mM), only the second peak was activated by this metabolite.

Furthermore both activities are also separated by (NHa)2SO 4 fractionation (see Methods).

The elution profile of i00 000 × g kidney medulla supernatant shows only one peak of pyruvate kinase activity, which is not adsorbed to the column. When all the fractions were tested in the presence of fructose diphosphate (at high or low level of PEP) this enzyme activity was not stimulated by the metabolite.

Effect of P E P concentration on pyruvate kinase I and pyruvate kinase I I activity Fig. IA shows plots of initial velocity of pyruvate kinase I I against P E P con-

centrations in the absence or in the presence of ATP or both, ATP and fructose all- phosphate. I t can be observed that the enzyme exhibits a marked cooperative effect towards PEP. These substrate interactions become more pronounced in the presence of ATP. In fact the slopes of the Hill plot increased from 2.9 to 3.4, respectively (see inset of the same figure). Conversely, when o.I mM fructose diphosphate is added simultaneously with the inhibitor, the response to P E P concentration is transformed into a hyperbolic curve. A similar experiment performed with pyruvate kinase I shows that the enzyme also displays sigmoid kinetics with respect to P E P (Fig. IB). However in contrast to the effects observed with the other isoenzyme (pyruvate

Biochim. Biophys. dcta, 289 (1972) 128-136

L. COSTA et al. 13o

~ 40 ~1, B

so L - -

a F r u 4 , 6 -

o.1 mM ~-~," I ~ / I - ~

.o_ f o t / ts : -

" 2 r a m A T e 0 5 ~ _ . . . . . . . . ~ ogLPE P_l

I I I I I , - , , ~ . . 0, 1 2 ~ 4 5 " 10

P E P ( r a M )

Fig. I. A. Effect of P E P concen t r a t ion on the reac t ion r a t e of p y r u v a t e k inase I I a c t i v i t y in the absence or in the presence of 2.o mM A T P or o.I mM fructose i 6 -d iphosphate . The i ncuba t ion m i x t u r e was as follows: IOO mM T r i s - m a l e a t e - t e t r a m e t h y l a m m o n i u m h y d r o x i d e buffer, p H 7.5, IO mM MgCI~, ioo mM KC1, 2. 5 mM ADP, o.2 mM N A D H , excess of commerc ia l l a c t a t e dehy- d rogenase and different concen t ra t ions of P E P as ind ica t ed in the figure. The i ncuba t ion was car r ied out a t 3 ° °C. B. Inf luence of P E P concen t r a t ion on the a c t i v i t y of p y r u v a t e k inase I in the absence or presence of 2.o mM A T P or o. i mM fructose 1 ,6-diphosphate , respec t ive ly .

kinase II), ATP and fructose diphosphate do not modify the kinetic behavior of pyruvate kinase I.

I t has been found (see below) that several amino acids are inhibitors of both type of pyruvate kinase activities. Alanine was chosen as model effector for the following experiment. The effect of alanine or both alanine and fructose diphosphate on the PEP saturation curve of pyruvate kinase II and pyruvate kinase I is shown in Fig. 2. In the presence of this amino acid, the substrate rate relationships for pyruvate kinase I I became more sigmoidal and the S0. 5 values for PEP increased. However, while fructose diphosphate reverses the alanine inhibition of pyruvate kinase II, this metabolite has no effect on the activity of the other isoenzyme.

Effect of different metabolites on pyruvate kinase I and pyruvate kinase I I activity Various amino acids were tested for their effect on both types of pyruvate ki-

nase activities from kidney cortex. The levels of PEP used were near the Km values for each enzyme. The data summarized in Table I show that the behavior of both isoenzymes against the amino acids is quite different. Pyruvate I kinase is inhibited by many amino acids. Tile other isoenzyme (pyruvate kinase II) is markedly inhibited

Biochim. Biophys. Acta, 289 (1972) 1128-t36

A L L O S T E R I C P R O P E R T I E S O F P Y R U V A T E K I N A S E I S O E N Z Y M E S I3Z

l! A

r i i i M÷FrU-I'6-P2

CO trol

o / / ' I"- alanine - I I I • " I

::ll:]13'~",,.u&.,,, ,~ I I I I - l l r E 00 1 2 3 4 5 20

coHtrot / y -

10-- . . . . . .

. , ~ - ' 1 rr~M atan~ne I .i / i # 2 / t l l ; ~ ,~ l l ~'/Lt ,°'h ,r ~w ~t h oyt ~ I ~ . d "~< , 1

; '- 1 2 3 4 5 " 10 PEP ( raM)

F i g . 2. A. Effect of P E P c o n c e n t r a t i o n on the reac t ion rate of p y r u v a t e k inase II a c t i v i t y in the absence or in the presence of a lan ine a n d / o r fructose 1 ,6 -d iphosphate as ind ica ted on the curves . B . Inf luence of P E P on the a c t i v i t y of p y r u v a t e k i n a s e I in the absence or in the presence of ala- n ine and /or fructose 1 ,6 -d iphosphate as ind ica ted on the curves . A s s a y condi t ions in A and B w e r e s imi lar to those described in F i g . I .

by alanine and cysteine and somewhat less by proline and valine. In this respect pyruvate kinase I and pyruvate kinase II are similar to liver M and L pyruvate kinases, respectively a-6.

Due to the fact that pyruvate kinase I is inhibited by many amino acids, that its affinity for PEP is very low and that fructose diphosphate does not have any stimulatory effect, a number of other metabolites were tested to see whether they would modify its activity. The following compounds at a concentration of 5 mM had no significant effect: glucose 6-phosphate, 3-phosphoglyceric acid, fructose 6-phosphate, ribose 5-phosphate, carbamyl phosphate, phosphocreatine, glucose 1-phosphate, inorganic phosphate, fumarate, citrate, malate, succinate, a-glycerophosphate and fl-glycerophosphate. The effect of these metabolites was tested at low or high level of PEP (o.5 and 2.5 mM) and with or without I mM phenylalanine.

The addition of Ca ~+ at 2.5 or 5.o mM concentration produces a marked de- crease in the activity of both types of kidney cortex pyruvate kinase. This inhibition is partially reversed by raising the Mg 2+ level as previously described for other pyruvate kinases. Furthermore in the presence of 2.5 mM Ca 2+, fructose diphosphate does not produce any stimulation in pyruvate kinase II activity even at IO mM Mg 2+.

Biochim. Biophys. dcta, 289 ( i 972 ) I 2 8 - I 3 6

1 3 2 L. COSTA et al.

T A B L E I

E F F E C T OF D I F F E R I ~ N T A M I N O A C I D S ON TILE A C T I V I T Y OF K I D N E Y C O R T E X 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 [

E x p e r i m e n t a l c o n d i t i o n s w e r e as d e s c r i b e d in F ig . i e x c e p t fo r P E P c o n c e n t r a t i o n w h i c h w a s 2.5 a n d 1.7 m M in t h e a s s a y o f 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 I1, r e s p e c t i v e l y . T h e f i g u r e s r e p r e s e n t p e r c e n t a g e o f e n z y m e a c t i v i t y .

Addi t ions Conch ( raM) Pyr*~vate kinase I Pyruvate kinase 11 ~

A l a n i n e 0 .5 54 62 .4 A l a n i n e 2 21 31 .8 P h e n y l a l a n i n e 0. 5 32 - - P h e n y l a l a n i n e 2 i o 85 C y s t e i n e 5 55 12 P r o l i n e 5 38 38.5 T r y p t o p h a n 2.5 39 99 I s o l e u c i n e 5 41 94 V a l i n e 2.5 44 63 T h r e o n i n e 5 48 95 T y r o s i n e 2.5 84 95 G l y c i n e 5 87 97 .5 H i s t i d i n e 5 73 94-5 L e u c i n e 5 86 I oo G l u t a m a t e 5 I o o 103 A s p a r t a t e 5 i o i 95 S e r i n e 5 99 89

* T h e i n i t i a l v e l o c i t i e s in t h e a b s e n c e o f a m i n o a c i d s w e r e 18 a n d 3 ° n m o l e s o f p y r u v a t e f o r m e d p e r m i n f o r 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 r e s p e c t i v e l y .

Effect of pH on kinetic behavior of pyruvate kinase I and pyruvate kinase I I I t has been demonstrated that the homotropic cooperative effect of isoenzyme

L from liver is strongly dependent on the pH of the medium4, 7. Preliminary experiments had shown that fructose diphosphate did not activate any of the isoenzymes of kidney cortex at acidic pH values and that in the alkaline range it produced a strong stimulation only on pyruvate kinase I I activity1; for instance at pH 8.2 there was a 2o-fold increase at I mM PEP. Therefore, it was of interest to s tudy the kinetics of both isoenzymes at different pH values. I t can be observed in Table I I that the change in pH from 7.5 to 6.8 produces a marked effect on the substrate cooperative interactions of pyruvate kinase II . In contrast, the Hill-coefficient values for the other isoenzyme were practically unchanged in this pH range.

T A B L E [ I

EFFECT OF p H ON THE P E P SATURATION CURVE OF PYRUVATI~ KINASE I AND PYRUVATE KINASE 1I

A s s a y c o n d i t i o n s w e r e as d e s c r i b e d in F ig . I e x c e p t t h a t t h e p H v a l u e s o f t h e i n c u b a t i o n m i x t u r e w e r e as i n d i c a t e d in t h e t a b l e .

p H Pyruvate kinase I Pyruvate kinase I I

So. 5 (m~$I) n n So. 5 ( m : ~ ) nH

6.8 1.35 1. 9 0. 9 1.2 7.3 - - - - 1.4 1.9 7 ' 5 2 . 2 1 . 8 1. 7 3 . 0

Biochim. Biophys. Acta, 289 (1972) 1 2 8 - 1 3 6

ALLOSTERIC PROPERTIES OF PYRUVATE KINASE ISOENZYMES 133

The effect of ATP on the activity of both types of pyruvate kinase at different pH values is shown in Fig. 3 A. Two relevant features of these experiments should be pointed out. One is that pyruvate kinase I is not inhibited by ATP at the pH tested even at 5 mM (this result is not shown in the figure). The second feature is that this metabolite inhibits pyruvate kinase I I activity at both pH values, but in the acid range cooperative effect is clearly observed (nE -- 2). In contrast, at pH 7.3 the ATP inhibition follows the Michaelis-Menten kinetics. Since it has been found (Table II) that the S0. 5 for P E P is dependent on pH, the concentration of PEP used was near the So. 5 value at each particular pH. In Fig. 3 B the percentage of pyruvate kinase I I activity has been plotted against the inhibitor concentration at two different levels of PEP. At low substrate no cooperative effect of ATP was detectable. On the other hand at higher substrate levels, such as 4 raM, sigmoidal kinetics towards the inhibitor is clearly observed.

When alanine was used as inhibitor of pyruvate kinase I I instead of ATP, the results were fairly similar. For example, the Hill coefficient value for this amino acid increases from I. 4 at pH 7.3 to 2.1 at pH 6.8.

A PK-I (pH 7.3) I - . 1 ~ : - 1 lOO

- P.-= cp,-,67) I i. 1 _1

pK-1T ( pH 6.7) o "e. ~ 2 " ' '

: >50 " ~ ~ ~ ' - , ~ . _ 0 I°g [ATP] -

~ > I PK-Tr

I o I I I I I I IIl B

2 100

"6

u

~ 5o

PK-I (2mM PEP) I h ~_J

......... "Q ................... ~"" ~ 1

,'>, ~ PK-rr 71 0 log [ATP] I

- " , ~ _ PK-Tr (1.7 mM PEP)

I I I ~ I , l l ~ Oq 0.4 0.8 1.2 1.6 2.O 2.4 5

ATP (raM)

Fig. 3. A. Effect of A T P c o n c e n t r a t i o n on the reac t ion r a t e of b o t h t y p e s of p y r u v a t e k inase ac- t i v i t y (PK- I and PIZ-II) a t two dif ferent p H va lues as ind ica ted . The e x p e r i m e n t a l cond i t ions were s imi la r to those descr ibed in Fig. i excep t for the concen t r a t i on of A D P (0.6 mM), the level of P E P (which was different in each exper iment ) a nd the p H values . I n the a s say of P K - I the con- c e n t r a t i o n of P E P was 1. 4 a nd 1. 7 mM a t p H 6.8 and 7.3, respec t ive ly . In the a s say of p y r u v a t e k inase I I i t was 0.9 a nd 1. 4 mM a t p H 6.8 a nd 7.3, respec t ive ly . B. Va r i a t i on of p y r u v a t e k inase I and p y r u v a t e k inase I I ac t iv i t i e s as a func t ion of A T P c o n c e n t r a t i o n a t different levels of P E P . E x p e r i m e n t a l cond i t ions were a s i n A excep t for the p H va lue (7.5) and the concen t r a t i on of P E P as i nd i ca t ed on t he curves. Inse ts , Hi l l p lo ts of the same d a t a ca l cu la t ed as descr ibed e lsewhere ~.

Biochim. Biophys. Acta, 289 (1972) 128-136

134 L. COSTA ct al.

Effect of K + on pyruvate I I kbzase activity Since the results presented here suggested that pyruvate kinase II has a kinetic

behavior very similar to that of the isoenzyme L 4,7 from liver the properties of this kidney enzyme were further studied.

The kinetics of K + activation at 1. 7 mM of PEP was studied in the presence or absence of fructose diphosphate (Fig. 4A). The enzyme shows a marked cooperative effect toward this cation. In the presence of fructose diphosphate or at high concentration of PEP (IO mM) the sigmoidal dependence on K + is transformed into a hyperbolic curve (see also Fig. 4B).

201 A ~ . ~ , ~ . _ ~ - ~ O ~ . - ~ e

°Io : ! E log I c0 B

control (10 mM ~ 13--lt-'-"O o e P E P y , , , ~ , ÷~ 30 Fru'~l,6 P2

/ 04 z I! ! o,Io oiZ 06 1 ilg4[K+]

/ I I I ! I , , I ~l 10 20 30 40 50 60 "' 100

K+(mM;

b'ig. 4- E f f e c t o f K + c o n c e n t r a t i o n o n t h e r e a c t i o n r a t e o f p y r u v a t e k i n a s e I1 at t w o d i f f e r e n t l eve l s o f P E P in t h e a b s e n c e o r in t h e p r e s e n c e o f o. i m M o f f r u c t o s e 1 , 6 - d i p h o s p h a t e . I n E x p t s A a n d B , t h e c o n c e n t r a t i o n o f P E P w a s 1. 7 a n d lO m M , r e s p e c t i v e l y . O t h e r e x p e r i m e n t a l c o n c e n t r a t i o n s w e r e s i m i l a r t o t h o s e d e s c r i b e d in F ig . i e x c e p t t h a t t h e K + c o n c e n t r a t i o n w a s v a r i e d as i n d i c a t e d .

D I S C U S S I O N

Previous reports1, s 13 have shown that liver and kidney cortex, the main gluconeogenic organs in mammals, contain at least two forms of pyruvate kinase which can be separated by electrophoresis in different systems. In our laboratory it has been found that the two different forms of pyruvate kinase from kidney cortex (pyruvate kinase I and pyruvate kinase II) can be fractionated by (NH4)2S04 precipitation and ion-exchange chromatography 1. In the course of the purification by these methods, the behavior of pyruvate kinase I and pyruvate kinase I[ was shown to be similar to that of liver isoenzymes M and L, respectively 4,5. The results described in this paper confirm our previous findings that pyruvate kinase I shares

Biochim. Biophys. Acta, 289 (x972) I 2 8 - I 3 6

ALLOSTERIC PROPERTIES OF PYRUVATE KINASE ISOENZYMES 135

properties of two hepatic isoenzymes 1. The kinetics of enzyme activity as a function of P E P concentration has been found to exhibit a sigmoidal dependence, like the L isoenzyme from liver. I t should be mentioned that these studies were performed with partially purified preparations of pyruvate kinase I free of enolase (EC 4.2.I . I I) , which indicates that the cooperative effect found is not an artifact. In contrast to the properties of the hepatic pyruvate kinase type L 7, the kinetic behavior of kidney isoenzyme (pyruvate kinase I) does not depend on pH and its activity is not affected by fructose diphosphate or ATP. In addition it is inhibited by several amino acids like the M isoenzyme from liver 5.

Alanine, chosen as a model inhibitor of pyruvate kinase I, slightly increases the homotropic cooperative effect of P E P and the inhibition is not counteracted by fructose diphosphate.

According to the chromatographic separation, pyruvate kinase I represents about 7o-8O},o of the total pyruvate kinase activity of the kidney cortex. This fact would indicate that this isoenzyme is more directly related to the metabolic control of glycolysis and gluconeogenesis in this tissue. However the physiological signifi- cance of pyruvate kinase I is not clear due to the fact that S0. 5 value for P E P is high in comparison to the intracellular concentration of this metabolitel*, 15. Furthermore fructose diphosphate, which is a powerful activator of other pyruvate kinases3,n, ~ 27 has no st imulatory effect on pyruvate kinase I activity.

These facts raise important questions regarding the function of this isoenzyme. On the other hand the presence in the cell of another compound which would enhance the affinity of this isoenzyme for P E P cannot be discarded. I t should be mentioned that the addition of a boiled crude extract of kidney cortex to a partially purified preparation of pyruvate kinase I, at two levels of P E P (0.5 mM and 2.5 mM) does not increase its activity.

Preliminary experiments on a hypothetic interconversion between a form of pyruvate kinase I with a low affinity for P E P to a more active form have given nega- tive results so far.

Previous work carried out in this laboratory indicates that the other isoenzyme from kidney cortex (pyruvate kinase II) is similar to Type L pyruvate kinase, which has been considered characteristic of liver. In the present paper we study this kidney isoenzyme in more detail and the following experimental facts reinforce the above conclusion: (I) The enzyme displays sigmoidal kinetics with respect to PEP; (2) ATP, alanine and fructose diphosphate have marked heterotropic effects on the homotropic cooperativity of the substrate; (3) the kinetic behavior of the enzyme towards P E P and the allosteric effectors is strongly dependent on pH within the physiological range; (4) at pH 7-5 there is a sigmoidal velocity response towards K+. In the presence of fructose diphosphate or at saturating levels of PEP, the K+ concentration graph gives a Michaelian curve.

The results presented in this paper are in agreement with those previously reported by Llorente et al . 2~ if it is considered that pyruvate kinase II , the activity of which is modulated by ATP and fructose diphosphate, only represents a small frac- tion of the total kidney cortex pyruvate kinase.

Biochim. Biophys. Acta, 289 (1972) 128 136

136 L. COSTA et al.

ACKNOWLEDGMENTS

We are grateful to Dr Luis F. Leloir for helpful advice and to Dr Sara H. Goldemberg for her help in the English version of the manuscript. This investigation was supported in part by grants from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (Argentina) and the Universidad de Buenos Aires. L.C. is a fellow and L.J. de A. and H.C. are career investigators of the Consejo Nacional de Investigaciones Cientfficas y T6cnicas (Argentina).

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Biochim. Biophys. Aeta, 289 (1972) 128-136

allosteric properties of the isoenzymes of pyruvate kinase from rat kidney cortex

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