Biochimica et Biophysica Acta, 293 (I973) I I 8 - I 2 4 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s
BBA 66788 TWO I N T E R C O N V E R T I B L E FORMS OF L - T Y P E P Y R U V A T E K I N A S E FROM RAT L I V F R
T m J. C. V A N B E R K E L ,
j. F. K O S T E R AND W. C. H O L S M A N N
Department of Biochemistry I, Rotterdam Medical School, Rotterdam (The Netherlands) (Received J u l y 24th, i972 )
SUMMARY
I. Reduced L-type pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.4o ) from rat liver can be converted into an oxidized form by incubation with oxidized mercaptoethanol and oxidized glutathione. This interconversion can be completely reversed by incubation with reduced mercaptoethanol. 2. The kinetic and allosteric properties of the reduced and oxidized forms are described. 3. The results are discussed in view of a possible regulation of the enzyme.
INTRODUCTION
Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.4o ) catalyses the last step of glycolysis. Its activity is important in the regulation of the dynamic balance between gluconeogenesis and glycolysis in the liver. In the liver two types of pyruvate kinase are present 1. The L-type pyruvate kinase shows an allosterie response to one of its substrates phosphoenolpyruvate (PEP). Its activity is influenced by Fru-I,6-P2, Glc-I,6-P~ and other phosphorylated hexoses 2-5. These compounds increase the apparent affinity for the substrate PEP. On the other hand ATP and alanine act as allosteric inhibitors 3,6. It has been generally accepted that the L-type pyruvate kinase, localized in the rat liver hepatocytes 7, must be inhibited during gluconeogenesis in order to increase the EPEP] necessary for gluconeogenesis. In a previous paper 4 we pointed out that the L-type pyruvate kinase cannot be regulated by a fluctuating [Fru-I,6-P~l in the liver. The ATP concentration in vivo only fluctuates between 2 and 3 mM (refs 8 and 9) and, therefore, some authors 6,1° assumed that during gluconeogenesis alanine is the most important inhibitor of pyruvate kinase activity. However, during gluconeogenesis the Ialaninel in tile liver is lowered (65%) due to an increased flux into the gluconeogenic pathway n. These properties would lead to an uninhibited enzyme under gluconeogenic conditions, which is rather Abbreviation: PEP, phosphoenolpyruvate.
INTERCONVERTIBLE FORMS OF L - T Y P E PYRUVATE KINASE
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unlikely. Another possibility is that the reduction state of the enzyme plays an important role in its regulation. We investigated the influence of sulfhydryl compounds on the activity of the enzyme, after preliminary studies with coenzyme A (containing glutathione) indicated an influence of thiol groups on the allosteric behaviour of pyruvate kinase. MATERIALS AND METHODS
Pyruvate kinase type L was isolated from rat liver according to the isolation procedure described earlier 4, except that during this procedure I mM mercaptoethanol was omitted and the final preparation was dissolved in 0.25 M Tris-HC1 pH 8.0. Pyruvate kinase activity was assayed by following the decrease in absorbance at 34 ° n m in a coupled reaction with lactate dehydrogenase at 23 °C according to Valentine and Tanaka TM. The triethanol-HC1 buffer (0.4 M, pH 7.5) was replaced by Tris-HC1 buffer (0.25 M, pH 8.0). Oxidized mercaptoethanol was prepared by bubbling 02 through a ioo-mM solution of reduced mercaptoethanol for 24 h. Oxidized L-type pyruvate kinase was prepared by incubating the enzyme for 6 h at 5 °C with I mM oxidized mercaptoethanol or 2.5 mM oxidized glutathione. ADP, PEP, NADH, Fru-I,6-P 2, Glc-I,6-P 2 and oxidized glutathione were obtained from Boehringer (Mannheim, Germany). Glc-I,6-P 2 was free of Fru-I,6-P 2, when analysed by the method of Bficher and Hohorst 13. Reduced glutathione was obtained from Sigma. All other reagents were of analytical grade purity. RESULTS
Fig. I shows the v v s [PEPJ plot at EADP] = 0.5 mM for the fleshly prepared pyruvate kinase (L-type) and the oxidized enzyme (preincubated with oxidized mercaptoethanol; compare Materials and Methods). In the presence of Fru-I,6-P 2
~A/rnin 0.21
0-
~
~
[PEP]raM Fig. I. T h e v v s [ P E P ] plot of t h e f r e s h l y isolated a n d oxidized L - t y p e p y r u v a t e kinase at EADP] = 0. 5 m M m e a s u r e d in t h e presence a n d a b s e n c e of F r u - I , 6 - P 2 (0. 5 mM). J - - I , t h e a c t i v i t y of t h e f r e s h l y isolated L - t y p e p y r u v a t e k i n a s e ; V - - T , t h e a c t i v i t y of t h e freshly isolated L - t y p e p y r u v a t e k i n a s e m e a s u r e d in t h e presence of F r u - I , 6 - P , ; A--~k, t h e a c t i v i t y of t h e oxidized L - t y p e p y r u v a t e kinase (by oxidized m e r c a p t o e t h a n o l ) ; O---O, t h e a c t i v i t y of t h e oxidized L - t y p e p y r u v a t e kinase (by oxidized m e r c a p t o e t h a n o l ) m e a s u r e d in t h e presence of F r u - I , 6 - P 2.
TH. J. c. VAN BERKEL et al.
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the freshly p r e p a r e d e n z y m e gives an a p p a r e n t Km value of o.I mM for P E P , whereas in the absence of F r u - i , 6 - P 2 a K0, ~ value of 2. 5 mM was obtained. O x i d a t i o n of the e n z y m e leads to a m a r k e d change in these a c t i v i t y curves. I n the presence of Fru1,6-P 2 the Km value for P E P is increased to o. 7 mM and the Ko, 5 value in the absence of F r u - I , 6 - P 2 was not measurable. This means t h a t at least u n d e r our test conditions the oxidized e n z y m e cannot reach its m a x i m a l a c t i v i t y in the absence of Fru-I,6-P2. The a d d i t i o n of F r u - I , 6 - P 2 s t i m u l a t e s the e n z y m a t i c a c t i v i t y to the same value as o b t a i n e d with the freshly p r e p a r e d enzyme, i n d i c a t i n g t h a t no loss of m a x i m a l activ i t y occurred after o x i d a t i o n of the enzyme. ~A/rnin
Q2
0:1-
iPEP] mM Fig. 2. The v v s [PEP] plot of the oxidized L-type pyruvate kinase (by oxidized glutathione) and the oxidized L-type pyruvate kinase after incubation for I h at IO °C with I mM mercaptoethanol (reduced enzyme) measured in the presence and absence of Fru-i,6-P~; A - - A , the activity of the oxidized L-type pyruvate kinase (by oxidized glutathione); O---Q, tile activity of the oxidized I.-type pyruvate kinase (by oxidized glutathione) measured in the presence of Fru-I,6-P.a; I - - R , the activity of the reduced L-type pyruvate kinase; Y - - V , the activity of the reduced Ltype pyruvate kinase measured in the presence of Fru-I,6-P v
Fig. 2 shows t h a t the L - t y p e p y r u v a t e kinase can also be oxidized b y incub a t i o n with oxidized g l u t a t h i o n e I n the absence a n d presence of F r u - I , 6 - P 2 the same p a t t e r n was o b t a i n e d as in Fig. I. Fig. 2 also shows t h a t o x i d a t i o n of the e n z y m e is a reversible process. B y i n c u b a t i n g the oxidized e n z y m e with I mM r e d u c e d m e r c a p t o e t h a n o l for I h at IO °C (reduced enzyme) the same kinetic d a t a were o b t a i n e d as with the freshly isolated enzyme. The Ko, ~ value in the absence of F r u - I , 6 - P 2 becomes again 2 mM a n d the a p p a r e n t Km for P E P in the presence of F r u - I , 6 - P 2 is again lowered to o . i mM. The same curves were o b t a i n e d when the enzyme was oxidized b y using oxidized m e r c a p t o e t h a n o l a n d i n c u b a t e d again with r e d u c e d m e r c a p t o e t h a n o l . W h e n the oxidized e n z y m e was i n c u b a t e d with r e d u c e d g l u t a t h i o n e (5 raM), interm e d i a t e curves were o b t a i n e d (Fig. 3). F r o m Figs I, 2 a n d 3 we can conclude t h a t there exist at least two forms of p y r u v a t e kinase with different kinetic properties, which can be i n t e r c o n v e r t e d , p r o b a b l y b y o x i d a t i o n a n d r e d u c t i o n of the - S H groups of the enzyme. The increase in Km value for P E P in the presence of Frn-I,6-P~, o b t a i n e d with the oxidized e n z y m e as c o m p a r e d with the r e d u c e d enzyme, m i g h t be of physiological i m p o r t a n c e . Therefore the e n z y m a t i c a c t i v i t y of the oxidized a n d r e d u c e d
INTERCONVERTIBLE FORMS OF L-TYPE PYRUVATE KINASE
I2I
~A/min
0.1
0
-[PEP] rnM
Fig. 3. The v vs [PEP] plot of the oxidized L-type pyruvate kinase after incubation for I h at Io °C with 5 mM reduced glutathione measured in the presence and absence of Fru-I,6-P v • - - I L the activity of the oxidized L-type (by oxidized mercaptoethanol) incubated with reduced glutathione; O - - O , the activity of the oxidized L-type (by oxidized mercaptoethanol) which has been incubated with reduced glutathione measured in the presence of Fru-I,6-P~; , - - A , the activity of the oxidized L-type (by oxidized glutathione) incubated with reduced glutathione; y - - y , the activity of the oxidized L-type (by oxidized glutathione) incubated with reduced glutathione measured in the presence of Fru-I,6-P 2. ,,Almin 0.3 w
•
(12-
0.1-
0
o
Fig. 4. The
o~o2 v vs
~o~
606
olo8
o'.lo
[Fru-I,6-P2] plot of the oxidized L - t y p e p y r u v a t e kinase and the oxidized L - t y p e
after incubation for i h at io °C with 5 mM reduced glutathione or I mM mercaptoethanol at [PEP] = i mM. I~--tP, oxidized L-type p y r u v a t e kinase (by oxidized mercaptoethanol) ; A - - A , oxidized L-type pyruvate kinase (by oxidized glutathione); , - - . , oxidized L-type pyruvate kinase (by oxidized glutathione) after incubation with reduced glutathione; V - - V , oxidized L-type pyruvate kinase (by oxidized mercaptoethanol) after incubation with reduced mercaptoethanol. e n z y m e s as a f u n c t i o n o f t h e [ F r u - I , 6 - P 2 ] w a s s t u d i e d . Fig. 4 s h o w s t h a t t h e r e d u c e d e n z y m e is a l r e a d y f u l l y a c t i v a t e d a t 5 # M F r u - I , 6 - P 2 , w h i l e t h e o x i d i z e d e n z y m e r e a c h e s t h i s m a x i m a l a c t i v i t y a t a c o n c e n t r a t i o n o f a b o u t 3o # M F r u - I , 6 - P 2. Fig. 4 also s h o w s t h e d i f f e r e n c e in m a x i m a l a c t i v i t i e s o f t h e r e d u c e d a n d o x i d i z e d e n z y m e s a t [ P E P ] = I m M a n d [ A D P J = o.5 m M . W h e n t h e o x i d i z e d e n z y m e is i n c u b a t e d w i t h r e d u c e d m e r c a p t o e t h a n o l , t h e s a m e c u r v e is o b t a i n e d as w i t h t h e f r e s h l y p r e pared enzyme. Incubation of the oxidized enzyme with reduced glutathione gives
I22
TH. J. c. VAN BERKEL
et
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0.2
03
0
0.1
o.2
o3
;~
~5
Fig. 5. The v v s EGlc-I,6-P~ plot of the oxidized L-type p y r u v a t e kinase and the oxidized L-type after incubation for I h at io °C with 5 mM reduced glutathione or I mM mereaptoethanol at [PEPI ~ i mM. Q - - Q , oxidized L-type p y r u v a t e kinase (by oxidized mercaptoethanol); A - - & , oxidized L-type p y r u v a t e kinase (by oxidized glutathione); II--II, oxidized L-type p y r u v a t e kinase (by oxidized glutathione) after incubation with reduced glutathione; V - - Y , oxidized Ltype p y r u v a t e kinase (by oxidized mercaptoethanol) after incubation with reduced mercaptoethanol.
an intermediate curve; when this enzyme is further incubated with reduced mereaptoethanol, the same curve is obtained as with the freshly prepared enzyme. Fig. 5 shows that the oxidized enzyme is rather insensitive to Glc-I,6-P2 stimulation. When the oxidized enzyme was incubated with reduced glutathione for i h, a more marked stimulation by Glc-I,6-P 2 is found. Incubation with (reduced) mercaptoethanol for I h gives the same stimulation with GIc-I,6-P2 as it is found with the freshly isolated enzyme. From Figs 4 and 5 the conclusion can be drawn that the oxidized enzyme is rather insensitive to Glc-I,6-P2, whereas Fru-I,6-P 2 is still able to stimulate the enzyme, although the concentration necessary for maximal stimulation has increased from 5/~M with the reduced enzyme to 3o/~M for the oxidized enzyme. DISCUSSION
From the data presented it is clear that the L-type pyruvate kinase can exist in two forms with different kinetic properties. The two forms can be interconverted by incubation with oxidized or reduced mercaptoethanol or glutathione. Since these compounds affect the - S H groups of proteins, it seems likely that thiol groups are involved in the interconversion. When glutathione (5 mM) was used to reduce the oxidized enzyme, the results obtained were not identical with those obtained with mercaptoethanol (i mM) as a reducing agent. This might be due to incomplete reduction of the enzyme with glutathione, suggesting at least two different types of - S H groups in the enzyme, involved in the interconversion. For pyruvate formiate lyase from Escherichia coli 14,15 and also for xanthine oxidase (EC 1.2.3.2) from rat liver 1~-18 an enzyme regulation by the oxidation and reduction of thiol groups has been shown and it is also likely that these interconversions are enzyme-catalysed. Also the fructose-I,6-diphosphatase (EC 3.1.3.11) activity from rabbit liver seems to be influenced by SH reagents ~9-21. For plants the re-
INTERCONVERTIBLE FORMS OF L - T Y P E PYRUVATE KINASE
123
dox state of the SH groups of a number of enzymes is a general kind of control 2~. The existence of such an enzyme-catalysed interconversion for rat liver pyruvate kinase cannot be concluded from the presented data. The in vitro oxidation is a rather slow process (for full oxidation 6 h are required, data not shown) and subsequent reduction takes I h. These data are obtained with the partially purified enzyme. By analogy with other enzymes, it is tempting to speculate the existence of an in vivo enzyme-catalysed interconversion. One condition for physiological regulation is fullfilled in that the interconversion is reversible. The difference in kinetic properties between the oxidized and reduced enzymes makes regulation of the enzymatic activity possible. In the oxidized enzyme the interaction of the P E P binding sites is raised (Figs I and 2) and Fru-I,6-P2 is still able to stimulate the reaction. However the [Fru-I,6-P2] needed for full activity is approximately 6 times higher than for the reduced enzyme and exceeds the liver [Fru-I,6P21 (ref. 23). This decrease in affinity for P E P and Fru-i,6-P 2 can be important during fasting, when P E P is needed for gluconeogenesis. In this view the markedly decreased influence of Glc-I,6-P 2 (of which the concentration in the liver remains constant during fasting and feeding, at least in the mouse 24) on the oxidized enzyme is of great importance. These properties can make the interconversion of the two forms of pyruvate kinase (L-type) of physiological importance, if a system exists which can catalyse these interconversions more rapidly than under our in vitro conditions.
ACKNOWLEDGEMENTS
We thank Mr K. K r u y t for expert technical assistance. This study was partially supported by the Foundation for Fundamental Medical Research (FUNGO). REFERENCES I T. T a n a k a , Y. H a r a n o , H. M o r i m u r a a n d R. Mori, Biochem. Biophys. Res. Commun., 21 (1965) 55. 2 T. T a n a k a , Y. H a r a n o , F. Sue a n d H. M o r i m u r a , J. Biochem. Tokyo, 62 (1967) 71. 3 T. T a n a k a , F. Sue a n d H. M o r i m u r a , Biochem. Biophys. Res. Commun., 258 (1967) 444. 4 J. F. Noster, R. G. Slee, G. E. J. Staal a n d Th. J. C. v a n Berkel, Biochim. Biophys. Acta, 258 (1972 ) 763. 5 J. F. N o s t e r a n d W. C. H f l l s m a n n , Arch. Biochem. Biophys., 14i (197 o) 98. 6 P. Llorente, R. Marco a n d A. Sols, Eur. J. Biochem., 13 (197 ° ) 45. 7 Th. J. c. v a n Berkel, J. F. K o s t e r a n d W, C. Ht~lsmann, Biochim. Biophys..4cta, 276 (1972) 425 • 8 C. S t a r k a n d E. A. N e w s h o l m e , Biochem. J., lO 7 (1968) 411. 9 Th. Bticher, K. Krejei, W. R i i s s m a n n , H. S c h n i t g e r a n d W. W e s s e m a n n , in B. Chance, R. H. E i s e n h a r d t , Q. H. Gibson a n d K. K. L o n b e r g h o l u m , Rapid Mixing and Sampling Techniques in Biochemistry, A c a d e m i c Press, New York, 1964, p. 255. Io G. Weber, M. A. L e a a n d N. B. S t a m m , Adv. Enzyme Reg., 6 (1968) i o i . i i D. H. Williamson, O. Lopes-Vieira a n d B. W a l k e r , l?iochem. J., lO 4 (1967) 497. 12 w . N. V a l e n t i n e a n d K. R. T a n a k a , in S. P. Colowick a n d N. O. K a p l a n , Methods in Enzymology, A c a d e m i c Press, New York, 1966, p. 468. 13 T. Biicher a n d H. J. H o h o r s t , in H. U. B e r g m e y e r , Methods in Enzymatic Analysis, Verlag Chemic a n d A c a d e m i c Press, N e w York, 1963, p. 246. 14 J. K n a p p e , E. B o h n e r t a n d W. B r ~ m m e r , Biochim. Biophys. Acta, lO 7 (1965) 6o3. 15 J. K n a p p e , J. Schacht, W. M6ckel, Th. H 6 p n e r , H. Vetter, J r a n d R. E d e n h a r d e r , Eur. J. Biochem., i i (1969) 316. 16 E. Della Corte a n d F. Stirpe, Biochem. J., lO8 (1968) 349.
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F. Stirpe a n d 2. Della Corte, J. Biol. Chem., 244 (1969) 3855 . F. Stirpe a n d 2. Della Corte, Bioehim. Biophys. Acta, 212 (197 o) 195. K. N a k a s h i m a , S. P o n t r e m o l i a n d 13. L. Horecker, Proc. Natl. Acad. Sci. U.S., 64 (1969) 947. K. N a k a s h i m a , B. L. H o r e c k e r a n d S. P o n t r e m o l i , Arch. Biochem. Biophys., 141 (197 o) 579. K. N a k a s h i m a , B. L. Horecker, S. Traniello a n d S. P o n t r e m o l i , Arch. Biochem. Biophys., 139 /I97O) 19o. 22 J. Preiss a n d T. K o s u g e , A n ~ u . Rev. Plant Physiol., 21 (197 o) 433. 23 J. R. \ ¥ i U i a m s o n , A. J ~ k o b a n d R. Scholz, Metabolism, 2o (I97 I) 13. 24 J. V. P a s s o n n e a u , O. H. Lowry, D. W. Schulz a n d J. G. Brown, J. Biol. Chem., 244 (1969) 902. 17 18 19 20 21