Volume 24, number 2
MOLECULAR ,~' CELLULARBIOCHEMISTRY
March 19, 1979
KINETIC PROPERTIES OF PYRUVATE KINASE OF RABBIT BRAIN* Makepeace U. TSAO
University of California School of Medicine, Davis, California (Received October 5, 1978)
Summ~ The kinetic properties of rabbit brain pyruvate kinase have been studied to determine its role in the regulation of glycolysis. One of the substrates of the enzyme, phosphoenolpyruvate, exhibits homotropic cooperativity (Hill coeff, of 1.45); thus, it is a moderate activator of the enzyme. The other substrate, ADP, shows normal Michaelis-Menton kinetics. Fructose-6phosphate and glucose-6-phosphate activate the enzyme only slightly at the l m u level and inhibit slightly at higher levels, and hence have no metabolic influence on the enzyme activity. Fructose-l, 6-diphosphate also has a slight activation up to 0.5 mM but no inhibition at higher level; therefore, it has no influence either. ATP, 2-phosphoglycerate, and phenylalanine are inhibitors of the enzyme. ATP, being the energy reservoir derived from glycolysis as well as a product of the reaction catalyzed by the enzyme, is a significant feedback inhibitor of the enzyme. These kinetic properties suggest a key role for pyruvate kinase in the regulation of glycolysis. Phenylalanine inhibition of the enzyme has been reported to be a possible mechanism of damage to the developing brain in phenylketonuria. The inhibition by phenylalanine at 10 mu in the assay mixture is reversed by alanine, cysteine, or serine at 0.2 mM level. Furthermore, the effect of these amino acids in reversing the * A preliminary report has been presented at the American Society of Biological Chemists Meeting at Atlanta, Georgia, June 1978.
phenylalanine inhibition are mutually enhancing. Consequently phenylalanine cannot have a significant inhibition on the activity of pyruvate kinase in brain.
Introduction The inhibition of human brain pyruvate kinase by L-phenylalanine has been proposed as a possible mechanism of damage to developing brain in phenylketonuria. 1 Because of the similarity of the kinetic properties of the adult and fetal human enzymes the enzyme activity which is very low in the fetal brain was suggested to be the vulnerable point of phenylalanine inhibition. Muscle pyruvate kinase has also been shown to be inhibited by phenylalanine but the inhibition can be reversed by alanine, cysteine and serine. 2'3 In this study, first the role of pyruvate kinase in the regulation of glycolysis in rabbit brain is demonstrated by its kinetic properties. Then the phenylalanine inhibition of this enzyme and its reversal by other amino acids are investigated.
Methods Reagents All biochemicals were obtained from Sigma Chemical Company. Inorganic chemicals were reagent grade obtained from J. T. Baker Chemical Company or Mallinekrodt Chemical Works. Rabbit brain. Acetone powder of unstripped brains was purchased from Pel-Freeze Biologicals, Inc. The
Dr. W. Junk b.v. Publishers - The Hague, The Netherlands
75
brains were from mixed breed, mixed sex and mixed age rabbits.
Extraction and isolation of pyruvate kinase. The following procedure is carried out at 0-4 ° . Ten g of rabbit brain acetone powder is suspended in 100 ml of cold phosphate buffer (0.1 M, pH 7.0; and 10 mr~ with respect to mercaptoethanol)~ The mixture is homogenized at maximum power (Omnimix) intermittently in two minute intervals for a total of 20 minutes. The homogenate is centrifuged at 40,000 g for 90 min. The supernatant is separated with a syringe. To the 90 ml of supernatant is added, with stirring, 28.17 g of ammonium sulfate to make a 50% saturated solution. The pH is maintained at 6.8 by addition of 4 N ammonium hydroxide. The precipitate is gathered by centrifugation at 20,000 g for 30 minutes (further addition of ammonium sulfate to the supernatant yields a precipitate from 70% saturation with ammonium sulfate. This fraction contains lower specific activity than the precipitate from 50% saturation with ammonium sulfate). The precipitate is taken up in 10 ml of phosphate buffer and the milky suspension is centrifuged at 30,000 g for 30 minutes. The opalescent supernatant is transferred to a dialysis bag. The contents are dialyzed against 150 ml of phosphate buffer with gentle agitation for six hours. This process is repeated twice with fresh buffer. The dialyzed material is then centrifuged at 40,000 g for three hours. The opalescent supernatant is collected and kept frozen at - 8 0 ° until use.
strate, cofactor, or buffer pH vary from the standard conditions. The desired amounts of substrate, cofactor, or effector are then added and the final volume of 1 ml is made up with calculated amounts of water. Duplicate analyses are made on each reaction. Velocity measurement and the mean are used for the graphs. The range of duplicate determinations is less than the dimensions of points shown in the graphs.
Experimental Isolation of pyruvate kinase The supernatant of the homogenate of 10 g of rabbit brain acetone powder contained 9.4 mg/ml of protein with a specific activity of 3.7 units/mg of protein. From this preparation a final dialyzed preparation with 1.1 mg/ml of protein and a specific activity of 73 units/mg of protein was obtained in a yield of 53%.
pH-activity curve Using triethanolamine hydrochloride as buffer with pH adjusted to various values by the addition of 1 N HC1 or NaOH, the pyruvate kinase activity is determined. When the readings of activity are plotted against the measured pH of the assay mixture after the activity determination a symmetrical bellshape curve is obtained with optimal activity at pH 7.0. This characteristic permits the study of the kinetics of the enzyme at a cellular hydrogen ion concentration.
Metal ions as cofactors Method for assay of pyruvate kinase activity. The conditions used in the assay are a modification of the coupled assay procedure described by BUCHERand PFLEIDERER. 4 The rate of oxidation of NADH is followed by the decrease in absorbance at 340 mM at 25 °, using a Gilford Model 2400-S recording spectrophotometer. The assay mixture contains the following components in a total volume of 1 ml; NADH, 0.08 mM; ADP, 1.0 mM; phosphoenolpyruvate, 0.2 raM; MgCI2, 10 mM, KC1, 100 mM; lactate dehydrogenase, 6 units (3.5 mg/1); triethanolamine hydrochloride buffer (pH 6.80), 100 mM. The assay mixture is prepared in bulk for each experiment and the same amount is measured into each cuvette, except where sub76
The requirement for both a monovalent and divalent metal ion for pyruvate kinase activity is shown in Figure 1. Potassium ion at 100 mM and Mg2÷ at 10 mM are used for assay of pyruvate kinase activity to maintain a saturated concentration under any circumstance.
Substrate concentration and pyruvate kinase activity (K,~) Both phosphoenolypyruvate and ADP seem to exhibit normal Michaelis-Menton kinetics under the standard assay condition. However, when Hill coefficients for the relationship between the substrate concentration and the reaction velocity are calculated the coefficient for phosphoenolpyruvate is found to be 1.45. Thus, a slight
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Fig. 1. Requirement o[ K ÷ and Mg2÷ for rabbit brain
pyruvate kinase activity. Assay mixture of 1 ml contains: NADH, 0.08 mM; ADP, 1.0 mu; phosphoenolpyruvate, 0.2 mu; MgCI2, 10 ram; KC1, 100 mM; lactate dehydrogenase, 6 units (3.5 mg/L); triethanolamine hydrochloride buffer (pH 6.80), 100 mM. The pyruvate kinase is diluted with 1% bovine serum albumin so that 20 ul in the cuvette will cause an absorbance change approximately 0.1 per min. The cation concentrations are varied by adding calculated amounts of 1M KC1 and 0.1M MgCI2. cooperative binding of this substrate to the enzyme occurs at the substrate concentration range from 0.02 to 0.10 mM. From a Lineweaver-Burk plot the K,, for A D P is 0.42 mu.
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Fructose-6-phosphate and glucose-6-phosphate activate the rabbit brain pyruvate kinase slightly at the concentration range of 0 to 1.0 mM. At higher concentration these metabolites mildly inhibit the enzyme. In the absence of ATP, fructose-l, 6-diphosphate activates pyruvate kinase maximally but only moderately at 0.5 mM. (see Figure 2) In effect, no activator with metabolic consequence, other than the substrates of this enzyme has been found.
Negative effectors of pyruvate kinase Three metabolically significant inhibitors of pyruvate kinase are: 2-phosphoglycerate, ATP, and phenylalanine. The inhibition by A T P increases linearly with A T P concentration up to 5 mM of A T P which caused a 50% inhibition.-
, i i 2 3 4 PEP mM
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Positive effectors of pyruvate kinase
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I I
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Fig. 2. Reversal of ATP inhibition of rabbit brain pyruvate kinase. (a) by ADP: (b) by phosphoenolpyruvate; (c) by fructose-l, 6-diphosphate. The assay conditions are similar to those for Figure 1 except that ATP is added to give the concentration indicated in the graph. Fructose-l, 6-diphosphate is added to give the indicated concentrations in (c).
(Figure 3) A M P up to 5 mM has no effect on the pyruvate kinase activity. The inhibition by A T P is reduced by increasing the substrate concentration. (Figure 2) The Moderate activation of the enzyme by fructose-I, 6-diphosphate is also eliminated by ATP. (Figure 2) The inhibition of pyruvate kinase by 2-phosphoglycerate, due to its competition with the substrate phosphoenolypyruvate for the same binding site on 77
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the enzyme, is examined by a reciprocal plot of substrate concentration versus reaction velocity with 2-phosphoglycerate at 0, 2.5, and 5 mM to determine the intersects at the ordinate. (Figure
4) NADH, in the concentration range from 0.048 to 1.16 mM, does not inhibit pyruvate kinase.
Phenylalanine inhibition of pyruvate kinase The amino acids Ala, Asp, Cys, GABA, Glu, Gin, Gly, His, Leu, Lys, Met, Phe, Pro, Set, Thr, Val were studied for their effect on the rabbit brain pyruvate kinase at 5 and 10 mM concentrations. Only Phe has a marked inhibitory effect on the activity of the enzyme. Lys has a slight inhibitory effect and it also augments the Phe inhibition. None of the other amino acids tested inhibits the enzyme or enhances the Phe inhibition. However, several amino acids either reduces or totally reverses the Phe inhibition. This will be discussed later in the section dealing with the reversing of the Phe inhibition. At the phosphoenolpyruvate concentration of 5 m i Phe has only a 35% inhibition even at Phe concentration of 20 raM. From a Dixon plot of the same data the Ki is calculated to be 78
5
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I I I I0 15 [ P E P ] BM -I
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Fig. 4. Effect of 2-phosphoglycerate on rabbit brain pyruvate kinase. The assay conditions are similar to those in Figure 1, except that fresh 2-phosphoglycerate solution (0.1 M) is prepared from sodium salt and its pH is adjusted to 6.80. Calculated amounts of 2-phosphoglycerate is added to the assay mixture to give the concentrations indicated in the graph.
1.2 m i . The interaction of Phe with the substrates is illustrated by the double reciprocal plots shown in Figures 5 and 6. The effect of fructose-I, 6-diphosphate on the Phe inhibition of pyruvate kinase is shown in Figure 7. In contrast to its lack of effect on ATP inhibition of pyruvate kinase, fructose-I, 6-diphosphate causes a reduction of inhibition due to phenylalanine. The inhibition of pyruvate kinase by Phe can be reduced or completely reversed by several amino acids, even though these amino acids do
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Fig. 5. Effect of phenylalanin¢ on [phosphoenolpyruvate] vs. reaction velocity of rabbit brain pyruvate kinase. A Lineweaver-Burk plot. The assay conditions are similar to those for Figure 1, except that the substrate concentration is set at 0.04, 0.05, 0.06, 0.1, and 0.2 mM. The levels of added phenylalanine are: none (0), 2.5 mM (A), and 5 mM (0).
not have a direct effect on the enzyme. The effect of the amino acids that have been found to reduce the Phe inhibition are summarized in Table I. The relationship between the concentration of the amino acid and the reduction of the Phe inhibition of pyruvate kinase is demonstrated with Ala in Figure 8. The combined effect of Cys and Thr, and Ala and Ser on the Phe inhibition are investigated at 0.02-0.20 mM concentration range. The effect of these two pairs of amino acids on the reduction of Phe inhibition of pyruvate kinase when Phe is maintained at 10 mM level are mutually enhancing.
TABLE
I 0
raM-'
Fig. 6. Effect of phenylalanine on [ADP] vs. reaction velocity of rabbit brain pyruvate kinase. A LineweaverBurk plot. The assay conditions are similar to those for Figure 5, except that the substrate concentration is set at 0.1, 0.25, 0.5, 0.75, and 1.0 mM. The levels of added phenylalanine are: none (©), and 10 mM (Q). Discussion
WEBER1 has presented evidence to show that L-phenylalanine can inhibit glycolysis through its effect on the pyruvate kinase of the fetal human brain to cause damage in phenylketonuria. In addition he also presented a similar effect of phenylpyruvate on hexokinase. However, at the time of this discovery the effect of several amino acids on the reversal of the inhibition of pyruvate kinase of muscle by phenylalanine had not yet been reported. Shortly thereafter ROZENGURT 2 and CARMINATI 3 reported the reduction of phenylalanine inhibition of pyruvate kinase from muscle by the
I
Effect of 1 mM of selected amino acids on the i n h i b i t i o n o f r a b b i t brain pyruvate kinase caused by I0 mM of phenylalanine
Control
Phe
Phe+ Cys
100%
44
102
Phe+ Ser
99
Phe+ Ala
89
Phe+ Thr
75
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56
Phe+ GABA
54
Phe+ Val
51
Phe+ Ala
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Phe+ Pro
44 79
"i 0.10 7, Co
IOmM~)
0.050
I
I
I
I
I
2
4
6
8
I0
[FDP] mM Fig. 7. Effect of fructose-l, 6-diphosphate on the phenylalanine inhibition of rabbit brain pyruvate kinase. The assay conditions are similar to those for Figure 1 except that fructose-l, 6-diphosphate is added to give the indicated concentrations and the levels of added phenylalanine are: none (O), and 10 mM (0).
presence of alanine. It appeared probable that the inhibition of the brain enzyme by phenylalanine might also be reversed by amino acids. The results contained herein show that alanine, cysteine, serine and threonine at 0.2 mM level can indeed reverse the inhibition. Furthermore, the reversal of phenylalanine inhibition by these amino acids is mutually enhancing in their effect. The joint effect of these amino acids at their physiological concentrations is sui~cient to cancel out the inhibitory effect of as much as 10 mM of phenylalanine. However, a possible species difference between human and rabbit brain pyruvate kinase remains to be investigated.
O m M ~:>
-~ °tsI
~ 0.10 I 0.25
I 0.5
I 0.75
I 1,00
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ALANINE mM
Fig. 8. Reversal of phenylalanine inhibition of rabbit brain pyruvate kinase by alanine. The assay conditions are similar to those of Figure 7, except that the phenylalanine concentration is maintained at 10 mM and the alanine concentration is varied as indicated in ihe graph.
80
Since the usually powerful activator of pyruvate kinases, fructose-I, 6-diphosphate, has little effect on the enzyme from rabbit brain, the inhibitors appear to be the significant effectors regulating the activity of this enzyme. The inhibition by ATP at its physiological levels is of particular importance. Among the inhibitors, 2-phosphoglycerate is of interest also because its molecular configuration is similar to that of phosphoenolpyruvate. The reciprocal plot of substrate concentration versus reaction velocity where the intercepts on the ordinate converge (Figure 4b) indicates that 2-phosphoglycerate is in competition with phosphoenolpyruvate for the same site on this enzyme. Phenylalanine, on the other hand, is a mixed type of inhibitor with respect to ADP. (Figure 6) The pyruvate kinase of rabbit brain is similar to that of the rabbit muscle in the inhibition by phenylalanine as well as the inhibition by 2-phosphoglycerate. 2'3 The inhibition by pyruvate kinase by alanine has been observed in the enzyme from rat adipose tissue 5, rat kidney cortex types I and I I 6, r a t liver type L 7, pig liver s, leucocytes9, rabbit liver TM and bovine muscle types K, L, and M 11. From these observations it is surprising to find that alanine, instead of inhibiting the rabbit brain enzyme, has the capacity to reverse the inhibition by phenylalanine with the latter at a concentration fifty times greater. ATP inhibition of pyruvate kinase has been reported on enzyme isolated from rat liver type L but not type M7, rat kidney cortex type II but not type 16, rabbit liver which is also inhibited by GTP TM, and pig liver 8. Thus the inhibition of brain pyrnvate kinase by ATP places the enzyme in a class with the liver enzymes. With respect to the allosteric activation by phosphoenolypyruvate, the rabbit brain enzyme also shares this property with the enzyme from pig liver s, rat liver 12, rabbit liver TM, and enzyme type I from rat kidney cortex 6. The lack of significant activation of the brain enzyme by fructose-l, 6-diphosphate, on the other hand, places the enzyme in the same group as rat kidney cortex enzyme t y p e / 6 and rat liver enzyme type M 7 rather than with the enzyme from pig liver s, rabbit liver TM, rat liver enzyme type L t2, and human erythrocyte enzyme ~3. These comparative kinetic properties of rabbit brain pyruvate kinase with reported characteristics of other mammalian enzymes indicate that
the rabbit brain enzyme is distinct from either the muscle or the liver types. Indeed, a similar conclusion was drawn for the guinea pig brain enzyme on the basis of its kinetic properties TM. The existence of isoenzymes of pyruvate kinase in other tissues suggests their occurrence in the brain as well. On the basis of the foregoing and on the assumption of comparable levels of tissue metabolites from data on guinea pig brain 15, one might deduce the key factors regulating the activity of pyruvate kinase. ATP, the enzyme inhibitor, is important in determining the activity. The ATP store, being the ultimate reservoir of chemical energy deriving from glycolysis, would be an effective feed-back regulator of glycolysis through its inhibition of pyruvate kinase. The homotropic cooperativity of phosphoenolpyruvate to the enzyme makes the substrate a moderate activator. Since the reaction catalyzed by pyruvate kinase is not the only reaction generating ATP the regulation of glycolysis in rabbit brain through the activity of pyruvate kinase is thus not merely a net result of opposing effects of substrate activation and product inhibition. Weber has suggested the causing of hyperglycemia and its consequent increase of metabolic intermediates, specifically phosphoenolpyruvate, as a means to overcome the inhibition of pyruvate kinase by phenylalanine. This would be a reasonable approach under circumstances where the inhibition is due to either an excessively high level of phenylalanine or unusually low levels of alanine, cysteine, serine, and threonine. Since the ATP level would be low as a result of phenylalanine inhibition of pyruvate kinase, only activation by augmented phosphoenolpyruvate level could restore the reaction rate. A more direct way to relieve phenylalanine inhibition would be to increase the concentration of any or all of the amino acids, alanine, cysteine, serine, and threonine.
References 1. Weber, G., 1969. Proc. Natl. Acad. Sci. 63, 1365-1369. 2. Rozengurt, E. et al. 1970. FEBS Letters 11,284-286. 3. Carminati, H. et al. 1971. J. Biol. Chem. 246, 72847288. 4. Bucher, T. and Pfleiderer, G., 1955. In Methods in Enzymology (Kaplan, N. and Colowick, S. eds.) Vol. 1, pp. 435. Academic Press, New York. 5. Marco, R. et al. 1971. Biochem., Biophy. Res. Commun. 43, 126-132. 6. Costa, L. et al. 1972. Biochim., Biophy. Acta 289, 128-136. 7. Van Berkel, T. J. C. et al. 1972. Biochim. Biophys. Acta 276,425-429. 8. Kutzbach, C. et al. 1973. Hoppe-Seyler's Z. Physiol. Chem. 354, 1473-1489. 9. Van Berkel, T. J. C. and Koster, J. F. 1973. Biochim. Biophys. Acta 293, 134-139. 10. Irving, M. G. and Williams, J. F. 1973. Biochem. J. 131, 287-301. 11. Cardenas, J. M. et al. 1975. Biochemistry 14, 40414045. 12. Van Berkel, T. J. C. et al. 1973. Biochim. Biophys. Acta 321, 171-180. 13. Black, J. A. and Henderson, M. H. 1972. Biochim. Biophys. Acta 284, 115-127. 14. Nicholas, P. C. and Bachelard, H. S. 1974. Biochem J. 141, 165-171. 15. Rolleston, F. S. and Newsholme, E. A. 1967. Biochem. J. 104, 524-533.
Acknowledgement The author thanks PROFESSORRICHARDA. FREEDLANDfor his discussions in the course of this study and PROFESSORPIERRE DREYFUSfor his advice in the preparation of the manuscript. 81 2
MOLECULAR ,~' CELLULARBIOCHEMISTRY
March 19, 1979
KINETIC PROPERTIES OF PYRUVATE KINASE OF RABBIT BRAIN* Makepeace U. TSAO
University of California School of Medicine, Davis, California (Received October 5, 1978)
Summ~ The kinetic properties of rabbit brain pyruvate kinase have been studied to determine its role in the regulation of glycolysis. One of the substrates of the enzyme, phosphoenolpyruvate, exhibits homotropic cooperativity (Hill coeff, of 1.45); thus, it is a moderate activator of the enzyme. The other substrate, ADP, shows normal Michaelis-Menton kinetics. Fructose-6phosphate and glucose-6-phosphate activate the enzyme only slightly at the l m u level and inhibit slightly at higher levels, and hence have no metabolic influence on the enzyme activity. Fructose-l, 6-diphosphate also has a slight activation up to 0.5 mM but no inhibition at higher level; therefore, it has no influence either. ATP, 2-phosphoglycerate, and phenylalanine are inhibitors of the enzyme. ATP, being the energy reservoir derived from glycolysis as well as a product of the reaction catalyzed by the enzyme, is a significant feedback inhibitor of the enzyme. These kinetic properties suggest a key role for pyruvate kinase in the regulation of glycolysis. Phenylalanine inhibition of the enzyme has been reported to be a possible mechanism of damage to the developing brain in phenylketonuria. The inhibition by phenylalanine at 10 mu in the assay mixture is reversed by alanine, cysteine, or serine at 0.2 mM level. Furthermore, the effect of these amino acids in reversing the * A preliminary report has been presented at the American Society of Biological Chemists Meeting at Atlanta, Georgia, June 1978.
phenylalanine inhibition are mutually enhancing. Consequently phenylalanine cannot have a significant inhibition on the activity of pyruvate kinase in brain.
Introduction The inhibition of human brain pyruvate kinase by L-phenylalanine has been proposed as a possible mechanism of damage to developing brain in phenylketonuria. 1 Because of the similarity of the kinetic properties of the adult and fetal human enzymes the enzyme activity which is very low in the fetal brain was suggested to be the vulnerable point of phenylalanine inhibition. Muscle pyruvate kinase has also been shown to be inhibited by phenylalanine but the inhibition can be reversed by alanine, cysteine and serine. 2'3 In this study, first the role of pyruvate kinase in the regulation of glycolysis in rabbit brain is demonstrated by its kinetic properties. Then the phenylalanine inhibition of this enzyme and its reversal by other amino acids are investigated.
Methods Reagents All biochemicals were obtained from Sigma Chemical Company. Inorganic chemicals were reagent grade obtained from J. T. Baker Chemical Company or Mallinekrodt Chemical Works. Rabbit brain. Acetone powder of unstripped brains was purchased from Pel-Freeze Biologicals, Inc. The
Dr. W. Junk b.v. Publishers - The Hague, The Netherlands
75
brains were from mixed breed, mixed sex and mixed age rabbits.
Extraction and isolation of pyruvate kinase. The following procedure is carried out at 0-4 ° . Ten g of rabbit brain acetone powder is suspended in 100 ml of cold phosphate buffer (0.1 M, pH 7.0; and 10 mr~ with respect to mercaptoethanol)~ The mixture is homogenized at maximum power (Omnimix) intermittently in two minute intervals for a total of 20 minutes. The homogenate is centrifuged at 40,000 g for 90 min. The supernatant is separated with a syringe. To the 90 ml of supernatant is added, with stirring, 28.17 g of ammonium sulfate to make a 50% saturated solution. The pH is maintained at 6.8 by addition of 4 N ammonium hydroxide. The precipitate is gathered by centrifugation at 20,000 g for 30 minutes (further addition of ammonium sulfate to the supernatant yields a precipitate from 70% saturation with ammonium sulfate. This fraction contains lower specific activity than the precipitate from 50% saturation with ammonium sulfate). The precipitate is taken up in 10 ml of phosphate buffer and the milky suspension is centrifuged at 30,000 g for 30 minutes. The opalescent supernatant is transferred to a dialysis bag. The contents are dialyzed against 150 ml of phosphate buffer with gentle agitation for six hours. This process is repeated twice with fresh buffer. The dialyzed material is then centrifuged at 40,000 g for three hours. The opalescent supernatant is collected and kept frozen at - 8 0 ° until use.
strate, cofactor, or buffer pH vary from the standard conditions. The desired amounts of substrate, cofactor, or effector are then added and the final volume of 1 ml is made up with calculated amounts of water. Duplicate analyses are made on each reaction. Velocity measurement and the mean are used for the graphs. The range of duplicate determinations is less than the dimensions of points shown in the graphs.
Experimental Isolation of pyruvate kinase The supernatant of the homogenate of 10 g of rabbit brain acetone powder contained 9.4 mg/ml of protein with a specific activity of 3.7 units/mg of protein. From this preparation a final dialyzed preparation with 1.1 mg/ml of protein and a specific activity of 73 units/mg of protein was obtained in a yield of 53%.
pH-activity curve Using triethanolamine hydrochloride as buffer with pH adjusted to various values by the addition of 1 N HC1 or NaOH, the pyruvate kinase activity is determined. When the readings of activity are plotted against the measured pH of the assay mixture after the activity determination a symmetrical bellshape curve is obtained with optimal activity at pH 7.0. This characteristic permits the study of the kinetics of the enzyme at a cellular hydrogen ion concentration.
Metal ions as cofactors Method for assay of pyruvate kinase activity. The conditions used in the assay are a modification of the coupled assay procedure described by BUCHERand PFLEIDERER. 4 The rate of oxidation of NADH is followed by the decrease in absorbance at 340 mM at 25 °, using a Gilford Model 2400-S recording spectrophotometer. The assay mixture contains the following components in a total volume of 1 ml; NADH, 0.08 mM; ADP, 1.0 mM; phosphoenolpyruvate, 0.2 raM; MgCI2, 10 mM, KC1, 100 mM; lactate dehydrogenase, 6 units (3.5 mg/1); triethanolamine hydrochloride buffer (pH 6.80), 100 mM. The assay mixture is prepared in bulk for each experiment and the same amount is measured into each cuvette, except where sub76
The requirement for both a monovalent and divalent metal ion for pyruvate kinase activity is shown in Figure 1. Potassium ion at 100 mM and Mg2÷ at 10 mM are used for assay of pyruvate kinase activity to maintain a saturated concentration under any circumstance.
Substrate concentration and pyruvate kinase activity (K,~) Both phosphoenolypyruvate and ADP seem to exhibit normal Michaelis-Menton kinetics under the standard assay condition. However, when Hill coefficients for the relationship between the substrate concentration and the reaction velocity are calculated the coefficient for phosphoenolpyruvate is found to be 1.45. Thus, a slight
0.2
0.10
@ATP--O
-
Jj SrnMATP M9 =* at I0 mM
0.1 r-
0.05
-
I I00
,
I 150
i
I 200
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I
,
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'
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2 3 4 ADP mM
'
5
=
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I I0
Mgz+ m M
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Fig. 1. Requirement o[ K ÷ and Mg2÷ for rabbit brain
pyruvate kinase activity. Assay mixture of 1 ml contains: NADH, 0.08 mM; ADP, 1.0 mu; phosphoenolpyruvate, 0.2 mu; MgCI2, 10 ram; KC1, 100 mM; lactate dehydrogenase, 6 units (3.5 mg/L); triethanolamine hydrochloride buffer (pH 6.80), 100 mM. The pyruvate kinase is diluted with 1% bovine serum albumin so that 20 ul in the cuvette will cause an absorbance change approximately 0.1 per min. The cation concentrations are varied by adding calculated amounts of 1M KC1 and 0.1M MgCI2. cooperative binding of this substrate to the enzyme occurs at the substrate concentration range from 0.02 to 0.10 mM. From a Lineweaver-Burk plot the K,, for A D P is 0.42 mu.
I--O
0
o
Fructose-6-phosphate and glucose-6-phosphate activate the rabbit brain pyruvate kinase slightly at the concentration range of 0 to 1.0 mM. At higher concentration these metabolites mildly inhibit the enzyme. In the absence of ATP, fructose-l, 6-diphosphate activates pyruvate kinase maximally but only moderately at 0.5 mM. (see Figure 2) In effect, no activator with metabolic consequence, other than the substrates of this enzyme has been found.
Negative effectors of pyruvate kinase Three metabolically significant inhibitors of pyruvate kinase are: 2-phosphoglycerate, ATP, and phenylalanine. The inhibition by A T P increases linearly with A T P concentration up to 5 mM of A T P which caused a 50% inhibition.-
, i i 2 3 4 PEP mM
i 5
[ ~
k&
0.05 F
/ 0
Positive effectors of pyruvate kinase
I I
mMATP
•
=
i
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I .5
I I
FDP
I 1.5
~.--/0 l 2
mM
Fig. 2. Reversal of ATP inhibition of rabbit brain pyruvate kinase. (a) by ADP: (b) by phosphoenolpyruvate; (c) by fructose-l, 6-diphosphate. The assay conditions are similar to those for Figure 1 except that ATP is added to give the concentration indicated in the graph. Fructose-l, 6-diphosphate is added to give the indicated concentrations in (c).
(Figure 3) A M P up to 5 mM has no effect on the pyruvate kinase activity. The inhibition by A T P is reduced by increasing the substrate concentration. (Figure 2) The Moderate activation of the enzyme by fructose-I, 6-diphosphate is also eliminated by ATP. (Figure 2) The inhibition of pyruvate kinase by 2-phosphoglycerate, due to its competition with the substrate phosphoenolypyruvate for the same binding site on 77
0.20
B
A.
0.15 ¢-
L
= 0.15
2.5ram
I0
OmU
the enzyme, is examined by a reciprocal plot of substrate concentration versus reaction velocity with 2-phosphoglycerate at 0, 2.5, and 5 mM to determine the intersects at the ordinate. (Figure
4) NADH, in the concentration range from 0.048 to 1.16 mM, does not inhibit pyruvate kinase.
Phenylalanine inhibition of pyruvate kinase The amino acids Ala, Asp, Cys, GABA, Glu, Gin, Gly, His, Leu, Lys, Met, Phe, Pro, Set, Thr, Val were studied for their effect on the rabbit brain pyruvate kinase at 5 and 10 mM concentrations. Only Phe has a marked inhibitory effect on the activity of the enzyme. Lys has a slight inhibitory effect and it also augments the Phe inhibition. None of the other amino acids tested inhibits the enzyme or enhances the Phe inhibition. However, several amino acids either reduces or totally reverses the Phe inhibition. This will be discussed later in the section dealing with the reversing of the Phe inhibition. At the phosphoenolpyruvate concentration of 5 m i Phe has only a 35% inhibition even at Phe concentration of 20 raM. From a Dixon plot of the same data the Ki is calculated to be 78
5
I
0
I
5
i
I I I I0 15 [ P E P ] BM -I
,
I 20
Fig. 4. Effect of 2-phosphoglycerate on rabbit brain pyruvate kinase. The assay conditions are similar to those in Figure 1, except that fresh 2-phosphoglycerate solution (0.1 M) is prepared from sodium salt and its pH is adjusted to 6.80. Calculated amounts of 2-phosphoglycerate is added to the assay mixture to give the concentrations indicated in the graph.
1.2 m i . The interaction of Phe with the substrates is illustrated by the double reciprocal plots shown in Figures 5 and 6. The effect of fructose-I, 6-diphosphate on the Phe inhibition of pyruvate kinase is shown in Figure 7. In contrast to its lack of effect on ATP inhibition of pyruvate kinase, fructose-I, 6-diphosphate causes a reduction of inhibition due to phenylalanine. The inhibition of pyruvate kinase by Phe can be reduced or completely reversed by several amino acids, even though these amino acids do
60-
•
° I ", 2o3O
40
/
o
I/V 30
7
"
20
15
I0
5
I 5
o
I I0
I/[pEp]
I 15
I 20
I
t
I
I
]
2
4
6
8
I0
t/i'AOP]
I 25
mM -I
Fig. 5. Effect of phenylalanin¢ on [phosphoenolpyruvate] vs. reaction velocity of rabbit brain pyruvate kinase. A Lineweaver-Burk plot. The assay conditions are similar to those for Figure 1, except that the substrate concentration is set at 0.04, 0.05, 0.06, 0.1, and 0.2 mM. The levels of added phenylalanine are: none (0), 2.5 mM (A), and 5 mM (0).
not have a direct effect on the enzyme. The effect of the amino acids that have been found to reduce the Phe inhibition are summarized in Table I. The relationship between the concentration of the amino acid and the reduction of the Phe inhibition of pyruvate kinase is demonstrated with Ala in Figure 8. The combined effect of Cys and Thr, and Ala and Ser on the Phe inhibition are investigated at 0.02-0.20 mM concentration range. The effect of these two pairs of amino acids on the reduction of Phe inhibition of pyruvate kinase when Phe is maintained at 10 mM level are mutually enhancing.
TABLE
I 0
raM-'
Fig. 6. Effect of phenylalanine on [ADP] vs. reaction velocity of rabbit brain pyruvate kinase. A LineweaverBurk plot. The assay conditions are similar to those for Figure 5, except that the substrate concentration is set at 0.1, 0.25, 0.5, 0.75, and 1.0 mM. The levels of added phenylalanine are: none (©), and 10 mM (Q). Discussion
WEBER1 has presented evidence to show that L-phenylalanine can inhibit glycolysis through its effect on the pyruvate kinase of the fetal human brain to cause damage in phenylketonuria. In addition he also presented a similar effect of phenylpyruvate on hexokinase. However, at the time of this discovery the effect of several amino acids on the reversal of the inhibition of pyruvate kinase of muscle by phenylalanine had not yet been reported. Shortly thereafter ROZENGURT 2 and CARMINATI 3 reported the reduction of phenylalanine inhibition of pyruvate kinase from muscle by the
I
Effect of 1 mM of selected amino acids on the i n h i b i t i o n o f r a b b i t brain pyruvate kinase caused by I0 mM of phenylalanine
Control
Phe
Phe+ Cys
100%
44
102
Phe+ Ser
99
Phe+ Ala
89
Phe+ Thr
75
Phe+ Gly
56
Phe+ GABA
54
Phe+ Val
51
Phe+ Ala
50
Phe+ Pro
44 79
"i 0.10 7, Co
IOmM~)
0.050
I
I
I
I
I
2
4
6
8
I0
[FDP] mM Fig. 7. Effect of fructose-l, 6-diphosphate on the phenylalanine inhibition of rabbit brain pyruvate kinase. The assay conditions are similar to those for Figure 1 except that fructose-l, 6-diphosphate is added to give the indicated concentrations and the levels of added phenylalanine are: none (O), and 10 mM (0).
presence of alanine. It appeared probable that the inhibition of the brain enzyme by phenylalanine might also be reversed by amino acids. The results contained herein show that alanine, cysteine, serine and threonine at 0.2 mM level can indeed reverse the inhibition. Furthermore, the reversal of phenylalanine inhibition by these amino acids is mutually enhancing in their effect. The joint effect of these amino acids at their physiological concentrations is sui~cient to cancel out the inhibitory effect of as much as 10 mM of phenylalanine. However, a possible species difference between human and rabbit brain pyruvate kinase remains to be investigated.
O m M ~:>
-~ °tsI
~ 0.10 I 0.25
I 0.5
I 0.75
I 1,00
I I.z5
I 1.50
ALANINE mM
Fig. 8. Reversal of phenylalanine inhibition of rabbit brain pyruvate kinase by alanine. The assay conditions are similar to those of Figure 7, except that the phenylalanine concentration is maintained at 10 mM and the alanine concentration is varied as indicated in ihe graph.
80
Since the usually powerful activator of pyruvate kinases, fructose-I, 6-diphosphate, has little effect on the enzyme from rabbit brain, the inhibitors appear to be the significant effectors regulating the activity of this enzyme. The inhibition by ATP at its physiological levels is of particular importance. Among the inhibitors, 2-phosphoglycerate is of interest also because its molecular configuration is similar to that of phosphoenolpyruvate. The reciprocal plot of substrate concentration versus reaction velocity where the intercepts on the ordinate converge (Figure 4b) indicates that 2-phosphoglycerate is in competition with phosphoenolpyruvate for the same site on this enzyme. Phenylalanine, on the other hand, is a mixed type of inhibitor with respect to ADP. (Figure 6) The pyruvate kinase of rabbit brain is similar to that of the rabbit muscle in the inhibition by phenylalanine as well as the inhibition by 2-phosphoglycerate. 2'3 The inhibition by pyruvate kinase by alanine has been observed in the enzyme from rat adipose tissue 5, rat kidney cortex types I and I I 6, r a t liver type L 7, pig liver s, leucocytes9, rabbit liver TM and bovine muscle types K, L, and M 11. From these observations it is surprising to find that alanine, instead of inhibiting the rabbit brain enzyme, has the capacity to reverse the inhibition by phenylalanine with the latter at a concentration fifty times greater. ATP inhibition of pyruvate kinase has been reported on enzyme isolated from rat liver type L but not type M7, rat kidney cortex type II but not type 16, rabbit liver which is also inhibited by GTP TM, and pig liver 8. Thus the inhibition of brain pyrnvate kinase by ATP places the enzyme in a class with the liver enzymes. With respect to the allosteric activation by phosphoenolypyruvate, the rabbit brain enzyme also shares this property with the enzyme from pig liver s, rat liver 12, rabbit liver TM, and enzyme type I from rat kidney cortex 6. The lack of significant activation of the brain enzyme by fructose-l, 6-diphosphate, on the other hand, places the enzyme in the same group as rat kidney cortex enzyme t y p e / 6 and rat liver enzyme type M 7 rather than with the enzyme from pig liver s, rabbit liver TM, rat liver enzyme type L t2, and human erythrocyte enzyme ~3. These comparative kinetic properties of rabbit brain pyruvate kinase with reported characteristics of other mammalian enzymes indicate that
the rabbit brain enzyme is distinct from either the muscle or the liver types. Indeed, a similar conclusion was drawn for the guinea pig brain enzyme on the basis of its kinetic properties TM. The existence of isoenzymes of pyruvate kinase in other tissues suggests their occurrence in the brain as well. On the basis of the foregoing and on the assumption of comparable levels of tissue metabolites from data on guinea pig brain 15, one might deduce the key factors regulating the activity of pyruvate kinase. ATP, the enzyme inhibitor, is important in determining the activity. The ATP store, being the ultimate reservoir of chemical energy deriving from glycolysis, would be an effective feed-back regulator of glycolysis through its inhibition of pyruvate kinase. The homotropic cooperativity of phosphoenolpyruvate to the enzyme makes the substrate a moderate activator. Since the reaction catalyzed by pyruvate kinase is not the only reaction generating ATP the regulation of glycolysis in rabbit brain through the activity of pyruvate kinase is thus not merely a net result of opposing effects of substrate activation and product inhibition. Weber has suggested the causing of hyperglycemia and its consequent increase of metabolic intermediates, specifically phosphoenolpyruvate, as a means to overcome the inhibition of pyruvate kinase by phenylalanine. This would be a reasonable approach under circumstances where the inhibition is due to either an excessively high level of phenylalanine or unusually low levels of alanine, cysteine, serine, and threonine. Since the ATP level would be low as a result of phenylalanine inhibition of pyruvate kinase, only activation by augmented phosphoenolpyruvate level could restore the reaction rate. A more direct way to relieve phenylalanine inhibition would be to increase the concentration of any or all of the amino acids, alanine, cysteine, serine, and threonine.
References 1. Weber, G., 1969. Proc. Natl. Acad. Sci. 63, 1365-1369. 2. Rozengurt, E. et al. 1970. FEBS Letters 11,284-286. 3. Carminati, H. et al. 1971. J. Biol. Chem. 246, 72847288. 4. Bucher, T. and Pfleiderer, G., 1955. In Methods in Enzymology (Kaplan, N. and Colowick, S. eds.) Vol. 1, pp. 435. Academic Press, New York. 5. Marco, R. et al. 1971. Biochem., Biophy. Res. Commun. 43, 126-132. 6. Costa, L. et al. 1972. Biochim., Biophy. Acta 289, 128-136. 7. Van Berkel, T. J. C. et al. 1972. Biochim. Biophys. Acta 276,425-429. 8. Kutzbach, C. et al. 1973. Hoppe-Seyler's Z. Physiol. Chem. 354, 1473-1489. 9. Van Berkel, T. J. C. and Koster, J. F. 1973. Biochim. Biophys. Acta 293, 134-139. 10. Irving, M. G. and Williams, J. F. 1973. Biochem. J. 131, 287-301. 11. Cardenas, J. M. et al. 1975. Biochemistry 14, 40414045. 12. Van Berkel, T. J. C. et al. 1973. Biochim. Biophys. Acta 321, 171-180. 13. Black, J. A. and Henderson, M. H. 1972. Biochim. Biophys. Acta 284, 115-127. 14. Nicholas, P. C. and Bachelard, H. S. 1974. Biochem J. 141, 165-171. 15. Rolleston, F. S. and Newsholme, E. A. 1967. Biochem. J. 104, 524-533.
Acknowledgement The author thanks PROFESSORRICHARDA. FREEDLANDfor his discussions in the course of this study and PROFESSORPIERRE DREYFUSfor his advice in the preparation of the manuscript. 81 2
