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Kinetic properties of pig pyruvate kinases type A from kidney and type M from muscle
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 195, No. 2, July, pp. 347-361, 1979
Kinetic
Properties
of Pig Pyruvate Kinases Type A from Kidney and Type M from Muscle’
LARS BERGLUND* Institute
of Medical
ELISABET
AND
HUMBLE
and Physiological Chemistry, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden Received August 28, 1978; revised January
University
of Uppsala,
29, 1979
Kinetic properties of homogeneous preparations of pig kidney and pig muscle pyruvate kinases (EC 2.7.1.40) were studied. Both isozymes showed a hyperbolic relationship to ADP with an apparent K, of 0.3 mM. K+ and MgZ+ were necessary for the activity of both isozymes, and their dependences on these cations were similar. The muscle isozyme expressed Michaelis-Menten type of kinetics with respect to phosphoenolpyruvate, and the apparent K,,, was the same (0.03 mM) from pH 5.5 to pH 8.0. In contrast, the dependence on phosphoenolpyruvate changed with pH for the kidney isozyme. It showed similar properties to the muscle isozyme at pH 5.5-7.0 (apparent K,,, of 0.08 mM), while two apparent K,, values for this substrate were present at pH 7.5-8.0, one low (0.1 mM) and one high (0.3-0.6 mM). At pH 7.5, fructose 1,6-bisphosphate converted the kidney isozyme to a kinetical form where only the lower apparent K,,? for phosphoenolpyruvate was detected. On the other hand, in the presence of alanine or phenylalanine the kidney pyruvate kinase showed only the higher K, for this substrate. At low phosphoenolpyruvate levels both isozymes were inhibited by phenylalanine, and half-maximal inhibition was found at 0.3 and 2.2 mM for the kidney and muscle isozymes, respectively. At a 5 mM concentration of the substrate only the kidney isozyme was inhibited, the apparent K, being the same. Alanine inhibited the kidney isozyme (apparent K, at 0.3 mM, irrespective of substrate concentration). No effect was seen on the muscle isozyme. Fructose 1,6-bisphosphate was an activator of the kidney isozyme at phosphoenolpyruvate concentrations below 1.~ ..__,. It also counteracted the inhibition by alanine or phenylalanine of this isozyme. A’,‘P inhibited both isozymes, and this inhibition was not counteracted by fructose 1,6-bisphosphate. The kidney isozyme showed both a high and a low apparent K,,z for phosphoenolpyruvate in the presence of ATP. The influence of the effecters on the activity of both isozymes varied markedly with pH, except for the action of ATP. At low substrate concentrations, however, the inhibitor action of ATP on the muscle enzyme was diminished around pH 7.5, in contrast to higher or lower pH values. Alanine or phenylalanine were more effective as inhibitors at higher pH values, and fructose 1,6-bisphosphate stimulated the kidney isozyme only at pH levels above pH 6.5. The influence of activators and inhibitors on the regulation of the kidney and muscle pyruvate kinases is discussed.
The distribution of different isozymes of mammalian pyruvate kinase (ATP:pyruvate phosphotransferase; EC 2.7.1.40) in various tissues is well documented (l-3). The most widely distributed isozyme is the A type 1 This investigation was supported by the Swedish Medical Research Council (Project 13X-50). 2 Present address: Division of Metabolic Disease, Department of Medicine, School of Medicine, M-013, University of California, San Diego, La Jolla, California 92093.
(or M, type), which is found in most tissues hitherto investigated (3). The M-type isozyme is present in skeletal muscle and brain tissue, whereas the L-type isozyme is found in hepatocytes, in the small intestine and as the minor pyruvate kinase isozyme in renal cortex (l-7). The A-type pyruvate kinase from pig kidney cortex was recently purified and studied in our laboratory (8), and it was found that the amino acid composition of the kidney enzyme was very similar to that of pig muscle pyruvate kinase. In this 347
0003.9861/79/080347-15$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reprorluction in any form reserved.
348
BERGLUND
AND HUMBLE
respect they differed considerably from the L-type pyruvate kinase from pig liver (9). Furthermore, concerning pig pyruvate kinases, it has been shown that the activity of the L-type isozyme, in contrast to the M and A types of isozymes, is regulated by a cyclic AMP-dependent phosphorylation reaction (8, 10). In immunological experiments on enzymes from rat tissues, the similarity between the M and A types of pyruvate kinases has also been documented, as well as their difference from the L-type isozyme (11). Because of the structural similarities noted between the M and A types of pyruvate kinases (8), it was of interest to compare the kinetic properties of these isozymes. The pig pyruvate kinases have not been examined in this respect earlier, and the kinetic properties of a homogeneous mammalian A-type pyruvate kinase have not so far been investigated. The regulation of this type of pyruvate kinase isozyme should be of considerable interest in view of its widespread occurrence. It is known that the assay conditions are of great importance when determining the activity of pyruvate kinase (9, 12-14), which indicates that comparisons made between the same type of pyruvate kinase isozyme from different species assayed under various conditions cannot be evaluated without reservation. The pig liver pyruvate kinase has recently been the subject of a kinetic study in this laboratory (14). In the investigation reported below on pig kidney and pig muscle pyruvate kinase, the same assay conditions as for the pig liver pyruvate kinase were chosen so as to allow a direct comparison of the kinetic properties between these three pyruvate kinase isozymes. EXPERIMENTAL
PROCEDURE
Materials Deionized, glass-distilled water was used in all solutions in the kinetic experiments. P-enolpyruvate,’ Fru-1,6-P,, ADP, ATP, L-phenylalanine, L-alanine, and bovine serum albumin were products of Sigma Chemical Co., St. Louis, Missouri. Lactate dehydro3 Abbreviations used: P-enolpyruvate, phosphoenolpyruvate; Fru-1,6-P,, fructose 1,6-bisphosphate.
genase (EC 1.1.1.27) from rabbit muscle and NADH were obtained from Boehringer Mannheim, GmbH, Germany. In order to remove the ammonium sulfate from the lactate dehydrogenase, prior to use the enzyme suspension was dialyzed at 4°C for 24 h against several changes of 20 mM imidazoleiHC1 buffer, pH 7.5, containing 0.1 mM dithiothreitol. This was found necessary since sulfate ions influence the kinetic properties of the pyruvate kinases, as earlier shown for the pig liver enzyme (14). CarboxymethylSephadex C-50 and Sepharose 6B were products of Pharmacia Fine Chemicals, Uppsala, Sweden, and were treated according to the instructions supplied. All other chemicals were of analytical grade and were used without further purification.
Methods Purification of pig kidney pyruvate kinuse. Pig kidney pyruvate kinase was purified as described earlier (8). The specific activity of the enzyme used was 500 units/mg of protein, assuming that A$$ was 1.0 cm-‘. Pur$cation of pig muscle pyruvate kinase. Pig muscle pyruvate kinase was prepared essentially as described by Cardenas et al. for the bovine muscle pyruvate kinase (15). The initial purification steps, including the chromatography of the enzyme on carboxymethyl-Sephadex, were carried out in all essential details according to this procedure. The pig muscle enzyme was further purified by chromatography on Sepharose 6B in the presence of 100 mM potassium phosphate buffer, pH 7.0, containing 10 mM mercaptoethanol and 30% (v/v) glycerol. When this purification step was omitted, the final product was found to contain some contaminating proteins of lower apparent molecular weights. Hence, to get an optimal degree of purification, it was necessary to include the chromatography on Sepharose 6B. The result of a typical chromatography is shown in Fig. 1A. The purified pig muscle pyruvate kinase was homogeneous as judged from the Sepharose 6B chromatography. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate only one band was seen (Fig. 1B). The specific activity of the purified enzyme was about 340 units/mg of protein, using an absorbance coefficient (A P&90)of 0.55 cm-‘, as reported for the purified bovine muscle pyruvate kinase (15). The yield in a typical preparation of the pig muscle enzyme was about 280 mg from 800 g of pig muscle tissue. This was a considerably higher recovery than obtained for the kidney or liver enzymes (8, 10). Both the kidney and the muscle pyruvate kinases were found to be stable for several months when kept at -18°C in the respective buffers used in the Sepharose 6B chromatography. Assay of pymvate kinase activity. During the purification procedures the enzyme activity was
KINETICS
OF PIG PYRUVATE
I
KINASES
A AND M
349
i
90 FRACTION
NUMBER
FIG. 1. (A) Sepharose 6B chromatography of pig muscle pyruvate kinase. About 70,000 units in a volume of 5 ml were loaded on the column (1.8 x 95 cm), which was equilibrated and eluted with 100 mM potassium phosphate buffer, pH 7.0, containing 10 mM mercaptoethanol and 30% (v/v) glycerol. (0) = pyruvate kinase activity; (0) = absorbance at 280 nm. (B) Polyacrylamide gel electrophoresis in detergent of purified pig muscle pyruvate kinase. The amounts of protein loaded on the gels were (i) 14, (ii) ‘7, and (iii) 2 pg. The position of the band corresponded to a molecular weight of 59,000, using reference proteins as described earlier (23). The same molecular weight, under these conditions, has been found previously for pig kidney pyruvate kinase type A (8).
assayed by a modification of the calorimetric procedure described by Kimberg and Yielding (16, 1’7). One unit of enzyme activity is the amount of enzyme that converts 1 pmol of substrate per minute under the conditions used. In the kinetic experiments, the activity of pyruvate kinase at 30°C was determined in a coupled assay with lactate dehydrogenase (18). Before use, the pyruvate kinase concentration was adjusted to about 1 (kidney enzyme) and 1.2 pgiml (muscle enzyme). The dilution was performed in buffer, pH 7.5, containing 30% 50 mM imidazole/HCl (v/v) glycerol, 100 mM potassium chloride, and However, since this represented 0.1 mM dithiothreitol. a large dilution of the enzymes resulting in a low protein concentration, it was found necessary to add bovine serum albumin (final concentration, 2 mg/ml) in order to stabilize the kidney enzyme. No addition was required for the stability of the muscle enzyme. The presence of the bovine serum albumin did not change any of the kinetic properties of the kidney enzyme. The diluted enzymes were found to be stable for at least 8 h at +4”C. The standard incubation mixture contained 50 pmol of imidazole/HCl buffer, pH 7.5, 100 pmol of potassium chloride, 5 +mol of magnesium chloride, 0.2 Fmol of NADH, 1 /*mol of ADP, 0.1 pmol of dithiothreitol, and 1.5 units of lactate dehydrogenase in a volume of 0.9 ml. After preincubation at 30°C for 3 min, 25 ~1 of the enzyme solution was added, and the preincubation was
continued for a further 3 min. The reaction was then started by the addition of P-enolpyruvate, giving a total incubation volume of 1,025 ml. It was ascertained that the amount of lactate dehydrogenase was not a rate-limiting factor at any pH, and that the pyruvate kinase activity was linear with time and enzyme concentration. In the experiments in which the effects of various pH values on the reaction velocity were tested, all additives were adjusted to the respective pH values. Polyacrglamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was carried out essentially according to Dunker and Reuckert (19, 20). Calculations. Calculations of apparent K,, values were based on Lineweaver-Burk plots as well as on data from Michaelis-Menten tabulations. The apparent K, values were given as the respective effector concentrations corresponding to 50% of the decrease in enzyme activity. Each kinetic experiment was performed in duplicate or triplicate with a very small variation (~10%). The day-to-day variation was somewhat larger primarily at low P-enolpyruvate concentrations. However, each experiment was repeated several times, in typical cases three to four times, with enzymes representing different purification batches, and similar results were obtained. Thus, the variation in the experiments did consequently not influence the result to any measurable degree.
350
BERGLUND
AND HUMRIJC
CONCENTRATION
OF P-ENOLPYWVATE
ImM)
8
05 CONCENTRATION
OF PmENOLPYRUVATE
ImMI
KINETICS
OF PIG PYRUVATE
RESULTS
Rate Dependence on Substrate Concentration
The influence of the substrates P-enolpyruvate and ADP on the reaction rate of kidney and muscle pyruvate kinases was investigated. As seen in Fig. 2 the muscle enzyme displayed a hyperbolic saturation curve with respect to P-enolpyruvate. The apparent K, at pH 7.5 for this substrate was found to be 0.02 mM. In contrast, the kidney enzyme showed more complex kinetic properties with this substrate. The P-enolpyruvate concentration giving half-maximal velocity at pH 7.5 for the kidney enzyme was found to be 0.25 mM. However, despite the different numerical values obtained for the two enzymes, both apparent K, figures are within the reported physiological P-enolpyruvate concentration range (21,22). Neither of the two enzymes showed sigmoidal dependence on this substrate. A straight line was achieved in a double reciprocal plot for the muscle enzyme, but this was not the case for the kidney isozyme. The Hill constant was calculated to 0.8 for this isozyme and to 1.0 for the muscle enzyme. This phenomenon was investigated further, and two apparent K, values for P-enolpyruvate were found for the kidney isozyme. Since this was dependent on the pH of the reaction mixture, these experiments were presented in connection with the pH studies below. In the further studies only two concentrations of P-enolpyruvate were used, representing saturating and unsaturating conditions. For both isozymes 5 mM P-enolpyruvate was found to be a saturating concentration (Fig. 2). Since the apparent K,n values for this substrate varied considerably between the two enzymes, the concentrations of P-enolpyruvate chosen to represent unsaturating conditions were selected to give approximately the same enzyme activity and to have a similar close relation to the apparent K,. This latter
KINASES
351
A AND M
concentration range was considered important in relation to the physiological importance of the various effecters. The activity of the two enzymes as a function of the second substrate, ADP, is outlined in Fig. 3. Both enzymes expressed hyperbolic relationships to this substrate, with the apparent K, values for ADP of 0.3 mM at saturating and 0.2 mM at unsaturating P-enolpyruvate concentrations. The Hill constant was calculated to be 1.0 under all conditions tested. These results are in agreement with those obtained for the pig liver pyruvate kinase (14). The dependence on ADP, in contrast to P-enolpyruvate, seerned thus to be similar for the three pig pyruvate kinases. InJluence of Mono- and Divalent
Cations
Both the pig muscle and kidney pyruvate kinases were found to be inactive in the absence of potassium or magnesium ions. The same findings have been reported for other pyruvate kinases (23). The two isozymes revealed a similar dependence on the potassium ion concentration. No qualitative differences were detected when saturating or unsaturating P-enolpyruvate concentrations were tested. Maximal activity was obtained at 50 mM potassium chloride, and the activity remained unchanged up to 100 mM, which was the highest potassium concentration tested. In each case a hyperbolic relationship to the potassium ion concentration was seen, and half-maximal concentration was found to be 8-10 mM potassium chloride. The dependence on magnesium ions, however, differed between the two isozymes when assayed in the presence of unsaturating P-enolpyruvate concentrations. All figures given for the magnesium concentration in the following represent free Mg2+, calculated as described by van Berkel(24). The muscle enzyme showed an activity maximum at 2-4 mM Mg’+, and concentrations above
FIG. 2. Dependence of pyruvate kinase activity on the concentration of P-enolpyruvate. The figure shows the activity in the absence of effecters (O), and in the presence of 5 pM Fru-1,6-P, (O), 5 mM alanine (A), 5 mM ATP (A), and phenylalanine (Cl). The concentrations of phenylalanine were 0.5 and 5.0 mM in the experiments with the kidney and muscle pyruvate kinases, respectively. (A) and (C) Muscle pyruvate kinase. (B) and (D) Kidney pyruvate kinase.
352
BERGLUND
AND HUMBLE
20
10
FIG. 3. Effect of ADP on the pyruvate kinase activity. (0) Pig kidney pyruvate kinase activity, (0) pig muscle pyruvate kinase activity. (A) 5 mM P-enolpyruvate and (B) low P-enolpyruvate concentrations, 0.1 mM for the kidney isozyme and 0.01 mM for the muscle isozyme.
4 mM were inhibitory. Half-maximal inhibition, given as 50% of the maximal activity, was seen at 20 mM Me+. The kidney enzyme on the other hand displayed a broad activity maximum (5-15 mM Mg2+), and 35 lllM magnesium chloride was found to give half-maximal inhibition. At 5 mM P-enolpyruvate the dependence on Mg2+ was similar for the isozymes. Low magnesium concentrations were optimal (2-8 mM Mg2+) with a relative inhibition seen at higher Mg2+ concentrations. The activity at the highest Mg2+ concentration tested (45 mM) was found to be 66 and 61% of the maximal activity for the muscle and kidney enzymes, respectively.
saturating and unsaturating P-enolpyruvate concentrations (Fig. 4). The degree of inhibition was almost the same for the two enzymes at the lower substrate level with an apparent Ki of l-l.5 mM ATP. At saturating P-enolpyruvate concentrations this value was higher for the kidney enzyme (3.5 mM) than for the muscle enzyme (1.8 ITIM). Thus, under these conditions ATP was a more efficient inhibitor of the muscle isozyme. The effect of ATP as a function of the P-enolpyruvate concentration was also studied. As shown in Fig. 2, the inhibition by 5 mM ATP on the activity of the muscle enzyme was present throughout the substrate concentration range, giving a decrease in the apparent V. Not even very high P-enolpyruvate concentrations could Effect of ATP overcome this inhibition. The ratio between The influence of ATP has been studied the apparent V values in the presence vs for other pyruvate kinases (13, 23, 25-29). absence of ATP was 0.7. Similar findings Conflicting results have, however, been were seen for the kidney isozyme, and the reported especially regarding the influence apparent K,,, values for P-enolpyruvate of ATP on the activity of A-type pyruvate in the presence of ATP are given in Table I. kinases (28, 29). In view of these results As is evident in Fig. 2, two apparent it was of interest to compare the effect of K, values for P-enolpyruvate were still ATP on the pig enzymes. In order to keep found for the kidney isozyme under these the Mg2+ concentration constant, which conditions. as described above is of great importance, In order to investigate whether Fru-1,6-P,, ATP was added as Mg-ATP. which is a known positive effector of other ATP was found to inhibit the activity of pyruvate kinases (23), could influence the muscle and kidney pyruvate kinase at both inhibition by ATP, the sugar was added in
KINETICS
OF PIG PYRUVATE
I
I
1
,
I
I
/
/
1
I
2
3
4
5
6
7
8
9
10
I 7
I 8
/ 9
1 10
I 1
I 2
I 3
I 4
FIG.
353
(lllM1
I 5
I MgATP I
activity
A AND M
1
[MgATPI
I
KINASES
I 6 (mM1
4. Effect of ATP on the activity of muscle (A) and kidney (B) pyruvate kinase. The in the presence of P-enolpyruvate in concentrations of 5.0 (O), 0.5 (a), and 0.02 ItIM (0) is shown.
a final concentration of 0.1 mM together with increased in the presence of Fru-1,6-P,. No ATP. No effect was found on the ATP- change in basal activity or degree of dependent inhibition of the muscle enzyme. inhibition by ATP was found at 5 mM The activity of the kidney isozyme was, P-enolpyruvate. Since the inhibitory effect however, influenced at the lower P-enol- of ATP on the activity of both isozymes pyruvate concentration. In the presence of thus remained unchanged in the presence ATP and Fru-1,6-P,, the activity of this of Fru-1,6-P,, the results demonstrate the enzyme was the same at 0.5 and 5 mM inability of the sugar to counteract the P-enolpyruvate. This increase in the activity inhibition by ATP on either enzyme. of the kidney enzyme at the low substrate concentration was due to the stimulatory Influence of Alanine and Phenylalanine effect of Fru-1,6-P, as shown below. Neither Several amino acids have been shown to the degree of inhibition by ATP nor the apparent Ki values were, however, affected be inhibitory to other pyruvate kinases even if the basal enzyme activity was (23). Phenylalanine has been found to
354
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AND HUMBLE
TABLE I COMPARISONOFAPPARENTK, VALUES FOR P-ENOLPYRUVATEFORTHREE PIG PYRUVATE KINASE ISOZYMESO Apparent K, for P-enolpyruvate (mM)
Conditions Control ATP Alanine Fru-1,6-P, Phenylalanine
Muscle pyruvate kinase 0.02 0.04 0.02 0.02* 0.06d
Kidney pyruvate kinase
Liver pyruvate kinase
0.07 and 0.25 0.07 and 0.6 0.6 0.07” 0.6'
0.3 0.6 0.7 0.03’ n.d.’
fl The apparent K, values for P-enolpyruvate were investigated at pH 7.5 for pyruvate kinases from pig liver, kidney, and muscle. All enzymes were assayed in the absence or presence of various effecters in 50 mM imidazole/HCl buffer, pH 7.5, containing 100 kmol of potassium chloride, 5 pmol of magnesium chloride, 0.2 pmol of NADH, 1 pmol of ADP, 0.1 pmol of dithiothreitol, and 1.5 units of lactate dehydrogenase in a final volume of 0.9 ml. The results given for the liver isozyme are those of Ref. (14). The concentrations of alanine or ATP were 5 mM for the muscle and kidney isozyme and 2 mM for the liver isozyme. * 5 FM Fru-1,6-P,. r 20 PM Fru-1,6-P,. d 0.05 mM phenylalanine. ’ 0.5 mM phenylalanine. ’ n.d. = not determined.
Phenylalanine was found to be an inhibitor of both isozymes (Fig. 5). The muscle enzyme expressed a sigmoidal dependence on P-enolpyruvate at pH 7.5 in the presence of phenylalanine, and the Hill constant increased from 1.0 to 1.4. As seen in Fig. 2A the inhibitory effect of phenylalanine was highly dependent on the P-enolpyruvate concentration, and virtually no inhibition was found at concentrations above 0.2 lTlM P-enolpyruvate. At low substrate concentrations,. however, the muscle enzyme was inhibited by phenylalanine, the apparent Ki at 0.02 mM P-enolpyruvate being 2.2 11IM. Fru-1,6-P, had no influence on the inhibition of the muscle enzyme by phenylalanine, whereas 5 mM alanine totally counteracted the effect of phenylalanine, giving an uninhibited muscle isozyme. The kidney enzyme differed considerably from the muscle enzyme regarding the effect of phenylalanine. The enzyme was inhibited as seen in Fig. 5. The apparent Ki values were found to be the same (0.3-0.4 InM) at both 0.5 and 5 InM P-enolpyruvate. No signs of sigmoidal dependence on P-enolpyruvate could be detected for the kidney enzyme in the presence of phenylalanine (Fig. 2B). The phenylalanine inhibition of this isozyme was almost totally abolished by 5 PM Fru-1,6-P,. Addition of alanine (5 mM) did not completely remove the phenylalanine inhibition of the A-type isozyme, although the effect of the latter inhibitor was far less pronounced. Both alanine and phenylalanine when added separately influenced the dependence on P-enolpyruvate for the kidney isozyme. As seen in Fig. 2D, only one apparent K, value for this substrate was obtained under these conditions. This value was somewhat higher than the higher K, value found for the control enzyme (Table I). These amino acids seemed thus to convert the kidney isozyme kinetically to a “high K, form.”
inhibit the activity of rabbit muscle pyruvate kinase (type M) (30, 31), and this inhibition was counteracted by alanine (25, 32). Whether this is the case also for A-type pyruvate kinase has not so far been investigated. Alanine had no effect on the activity of the pig muscle pyruvate kinase, the highest effector concentration tested being 10 mM. The kidney enzyme was in contrast inhibited by addition of the amino acid. The apparent Ki for alanine was found at a concentration of 0.3 mM in the presence of either 0.5 or 5 mu P-enolpyruvate. In the presence of 2-10 mM alanine the activity of the kidney pyruvate kinase was 60 or 80% of the activity in the absence of the amino acid Effect of Fm-1,6-P, at low or high P-enolpyruvate concentration, respectively. In contrast, no inhibition by The enhancing effect of Fru-1,6-P, on the alanine of the kidney enzyme was seen in activity of several pyruvate kinases is well the presence of 5 PM Fru-1,6-P,. documented (23). Most investigators have
KINETICS
OF PIG PYRUVATE
5 CONCENTRATION
OF PHENYLALANINE
CONCENTRATION
355
KINASES A AND M
5 OF PHENYLALANINE
10 I mM 1
10 I mMI
FIG. 5. Effect of phenylalanine on the activity of muscle (A) and kidney (B) pyruvate kinase. In some experiments Fru-1,6-P, or alanine was added at final concentrations of 5 pM and 5 mM, respectively, together with phenylalanine. The activities at 5.0 mM P-enolpyruvate (0) and in the presence of Fru-1,6-P, (0) or alanine (Cl) are shown. The corresponding symbols at the lower P-enolpyruvate concentrations (0.5 mM for the kidney enzyme and 0.02 mM for the muscle enzyme) are (A), (A), and (W).
not been able to detect a stimulation of the M-type isozyme (23), but at least in one case Fru-1,6-P, was found to effect the activity of this isozyme (32). Regarding the A-type isozyme several authors describe a positive effect of Fru-1,6-P, on the enzyme activity (6, 13, 23), whereas in some cases no apparent effect of the sugar was found (32). In this characterization of the kinetic properties of the pig pyruvate
kinases type A and M, it was therefore obvious that the role of Fru-1,6-P, was of interest. As shown above even low concentrations of this effector were able to relieve the inhibition by amino acids on the kidney enzyme, whereas such an effect was not seen for the muscle enzyme. The lack of influence of Fru-1,6-P, on the muscle enzyme activity explained these findings. No effect was seen with 0.1-100 PM con-
356
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centrations of the sugar, irrespective of the P-enolpyruvate concentration. The influence of Fru-1,6-P, on the activity of the kidney pyruvate kinase was found to be dependent on the P-enolpyruvate concentration. The effector stimulated the enzyme activity at low P-enolpyruvate levels decreasing the apparent K, for this substrate at 5 j.,&M Fru-1,6-P, to 0.06 mM (Fig. 2B). This value was the same as the low K, for P-enolpyruvate expressed by the control enzyme (Table I). Fru-1,6-P, seemed thus to convert the kidney isozyme kinetically to a “low K, form.” At saturating substrate concentrations, however, no effect was detected. In the presence of 0.5 mM P-enolpyruvate the maximal activity of the kidney enzyme was attained at 5 j.,&M Fru-1,6-P, with an apparent K, of less than 0.1 PM. The effect of this effector on the kidney enzyme activity was, however, counteracted by some negative effecters, as discussed above. ATP (3 InM) and phenylalanine (0.5 IrIM) were therefore
added together with Fru-1,6-P,. These levels were chosen in order to approach physiological effector concentrations (22). Under these conditions a considerably higher concentration of Fru-1,6-P, (1.5 PM) was needed to obtain half-maximal activation of the kidney enzyme, indicating the importance of a combined effector action. Dependence on pH
The activity of both muscle and kidney pyruvate kinase varied considerably with pH, both at unsaturating and saturating P-enolpyruvate concentrations (Fig. 6). At both substrate levels the pH optimum for the muscle enzyme was found to be at pH 7.5. The kidney enzyme had, however, a more acidic pH optimum (pH 6.5) at the lower substrate concentration, but showed a pH profile very similar to the muscle enzyme at 5 mM P-enolpyruvate. At pH levels below pH 7.0 the kidney enzyme appeared to follow Michaelis-Menten-
FIG. 6. Activity of pyruvate kinase as a function of pH. (A) Muscle enzyme at 0.02 mM P-enolpyruvate, (B) kidney enzyme at 0.5 mM P-enolpyruvate, (C) muscle enzyme at 5.0 mM P-enolpyruvate, and (D) kidney enzyme at 5.0 mM P-enolpyruvate. In these experiments the concentration of each enzyme was 200% of that in the standard assay. Symbols as in Fig. 2. The concentrations of the added effecters were: 5 pM Fru-1,6-P,, 3 mM ATP, and 5 mM alanine. Phenylalanine was added at a concentration of 5.0 mM except at the low concentration of P-enolpyruvate in the case of the kidney enzyme, where the phenylalanine concentration was 0.5 mM. All additions were corrected to the respective pH values.
KINETICS
OF PIG PYRUVATE
type of enzyme kinetics with respect to Penolpyruvate. However, at pH 7.5 and 8.0 this was not the case, and a straight line was not obtained in a double reciprocal plot. The value for the Hill constant reflected this phenomenon, being 1.0 between pH 5.5-7.0 and 0.8 at the higher pH levels. At these higher pH values the kidney enzyme was represented by two straight lines in a Lineweaver-Burk plot (Fig. 7), and consequently two apparent K, values for P-enolpyruvate were found, as compared to the findings at lower pH levels. The lower K, value remained essentially the same over the pH range and was similar to the apparent K, for P-enolpyruvate for the muscle enzyme (Table I). As seen in Table II, the higher K, value increased slightly from pH 7.5 to pH 8.0.
FIG.
7. Activity Lineweaver-Burk
KINASES
357
A AND M
In order to further investigate the influence of effecters on the pyruvate kinase activity at different pH values, ATP, alanine, phenylalanine, or Fru-1,6-P, were added to both isozymes over the pH range studied. The enzyme activity was thereafter assayed at saturating or unsaturating P-enolpyruvate concentrations (Fig. 6). ATP inhibited the muscle isozyme under both conditions. However, at a low P-enolpyruvate level the inhibition by ATP was found to be considerably less around pH 7.5 than at other pH values tested. This result was very reproducible and occurred with all enzyme batches tested. In order to further investigate this phenomenon a substrate concentration of 0.1 InM, which approaches a saturating level, was used. Under these conditions the inhibitory effect
CONCENTRATION
OF P-ENOLPYRUVATE
ImMl
CONCENTRATION
OF P-ENOLPYRUVATE
fmM1
of pig kidney pyruvate kinase as a function of the concentration plots inserted. (A) pH 5.5, (B) pH 7.5.
of P-enolpyruvate.
358
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AND HUMBLE
between pyruvate kinases from pig kidney
TABLE II
(A-type), pig muscle (M-type), and pig APPARENT K, FORP-ENOLPYRUVATEATDIFFERENT liver (L-type) (14). The muscle and kidney pH VALUESFORMUSCLEANDKIDNEY PYRUVATE KINASE Apparent K, (mM) for P-enolpyruvate
PH
Muscle pyruvate kinase
Kidney pyruvate kinase
5.5 6.0 6.5 7.0 7.5 8.0
0.03 0.04 0.03 0.03 0.02 0.03
0.11 0.08 0.06 0.11 0.07 and 0.25 0.10 and 0.59
of ATP was still relatively small at pH 7.5, and at more basic or acidic pH values ATP was a more effective inhibitor. The difference was, however, not as pronounced as at the lower P-enolpyruvate concentration. The inhibition by phenylalanine of the muscle enzyme occurred above pH 6.5. Alanine or FIX-1,6-P, were without effect on the muscle enzyme at all pH levels tested. Fru-1,6-P, activated the kidney enzyme primarily at neutral or alkaline pH values and hardly any effect was seen at pH 5.5-6.5. The influence of the negative effecters was similar at pH 5.5-6.5 in the presence of a low substrate concentration. Above pH 6.5, phenylalanine was the most efficient inhibitor. As seen in Fig. 6D, the inhibitory effect of the amino acids on the kidney enzyme activity increased with increasing pH at 5 mM P-enolpyruvate. The ATP inhibition was fairly similar over the pH range. DISCUSSION
So far the A-type pyruvate kinase has been purified to homogeneity from only a few sources (8, 11, 33), and the kinetic properties of the homogeneous enzyme are to a large extent unexplored. In the present paper the kinetic properties of pyruvate kinases type A from kidney and type M from muscle were studied. Our choice of assay conditions also allowed a direct comparison
isozymes were found to have similar properties with respect to the dependence of enzyme activity on the concentrations of ADP, potassium ions, and magnesium ions. These properties also closely resembled those of the liver isozyme (14), which indicates that the activity pattern as a function of these cations or ADP may be a basic, common property of the three types of pig pyruvate kinase isozymes. The dependence on the substrate, P-enolpyruvate, differed to a large extent for the various isozymes. The pig liver isozyme expresses sigmoidal kinetic properties for this substrate (14), while the muscle isozyme showed Michaelis-Menten type of enzyme kinetics with a low apparent K, for P-enolpyruvate. A more complex picture was seen for the kidney isozyme. The two apparent K, values found for P-enolpywvate for the latter enzyme is consistent with the finding that this type of isozyme may exist in two kinetically different forms (34, 35). The low K, for P-enolpyruvate was similar to that obtained for the muscle enzyme, whereas the high K, value approached that given for the liver isozyme (14). The presence of these two kinetical forms of the kidney isozyme was, however, dependent on pH. It was only at pH 7.5 and 8.0 that two K, values were found. At lower pH values the kidney enzyme showed only one apparent K, value for P-enolpyruvate. This value corresponded to the low K, value for this substrate found at pH 7.5. It is also noteworthy that the apparent K, values increased from pH 7.5 to pH 8.0. Thus, at a given P-enolpyruvate concentration the activity of the kidney enzyme was successively more inhibited with increasing pH. The increase in P-enolpyruvate concentration necessary for half-maximal activity with pH for this isozyme indicates that the high K, form of the enzyme dominates at high pH, while the low K, form is found at low pH levels. The occurrence of the kidney pyruvate kinase in these two kinetical, or K, forms, was further dependent on the presence of various effecters. Interestingly, only the low apparent K, for P-enol-
KINETICS
OF PIG PYRUVATE
pyruvate was expressed in the presence of Fru-1,6-P, even at higher pH values. In contrast, the amino acids alanine or phenylalanine converted the kidney isozyme to the high K, form. These changes are summarized as a hypothetical model in Fig. 8. The changes obtained suggest the possibility of a conformational R-T pair, according to the model of Monod et al. (36). The changes in the kinetical forms could be of large importance in the regulation of the kidney pyruvate kinase activity, since the physiological P-enolpyruvate concentration is low (21, 22). A relatively small change in K,, could thus result in a large change of enzyme activity. The suggested kinetical changes for the kidney isozyme are to some extent analogous to the factors influencing the activity of the liver isozyme. In the latter case, the effect of phosphorylation and allosteric effecters are also dependent on pH (14). In contrast, the muscle isozyme showed kinetical properties resembling the A-state of the model in Fig. 8. The negative allosteric effecters also influenced the action of each other. Alanine did not effect the activity of the muscle enzyme but eliminated the inhibition by phenylalanine, which might indicate competition for the same binding site (or sites) (3’7). However, two separate binding sites may also be involved with a resultant conformation change of the enzyme. It is noteworthy that the combined action of alanine and phenylalanine inhibited the
FIG. 8. Hypothetical model for the various kinetical forms of pig kidney pyruvate kinase. The low K, form is represented by A, whereas C is the high K, form. The enzyme form present at pH 7.5 with two is illusapparent K,,, values for P-enolpyruvate trated by B.
KINASES
A AND M
359
kidney enzyme less than each amino acid when added separately. The sigmoidal inhibition curve found for the latter enzyme in the presence of both amino acids also showed the complex type of interaction present in this case. The results do not, however, rule out the possibility of a common binding site. The effect of ATP was different from that of the amino acids and pH. Two K,, values for P-enolpyruvate were again found at higher pH values for the kidney isozyme. Another interesting finding was that the ATP inhibition, in contrast to that of pH or amino acids, was not counteracted by Fru-1,6-P,. These results indicated that the effect of ATP on the activity of the pyruvate kinase isozymes may be the result of a different mechanism than indicated in Fig. 8. This is also supported by the similar action of ATP on both the muscle and kidney pyruvate kinase over the pH range studied. One very striking difference in the ATP action was, however, consistently found between the two isozymes. At 0.02 mM P-enolpyruvate the muscle isozyme showed a relatively small sensitivity to the inhibitory action of ATP around pH 7.5, in contrast to higher or lower pH values. This effect decreased with increasing P-enolpyruvate concentrations and was not present at 5 mM P-enolpyruvate. No such phenomenon could be detected for the kidney enzyme. It is important to point out that the relatively small inhibition by ATP of the muscle enzyme occurred in the physiologically important pH range 7.07.5-8.0. A small change in pH in this pH area could thus considerably increase the inhibitory effect of ATP. This finding and the fact that the effect only was present at low, physiological P-enolpyruvate concentrations indicates that it may be of significance for the regulation of the muscle isozyme activity. In contrast, the concentration of phenylalanine necessary to appreciably influence the activity in vitro of the muscle isozyme was high, and the importance of this type of inhibition in vivo therefore seems doubtful. The concentrations necessary of all effecters tested in this study to influence the activity of the kidney isozyme were
360
BERGLUND
AND HUMBLE
low and approached the respective physiological concentrations (22). A physiological regulation of this isozyme by a combined effector action therefore seems conceivable. It is interesting in this context to mention the increase in apparent K, for Fru-1,6-P, found for the kidney isozyme in the presence of ATP and phenylalanine, demonstrating the importance of a concerted effector influence. Engstrijm and collaborators showed that in addition to allosteric effecters the activity of the pig liver pyruvate kinase is affected by a cyclic AMP-dependent phosphorylation of the enzyme (10, 14). There is apparently no such regulation for the pig kidney and muscle pyruvate kinases (8, 38, 39), and in this respect these isozymes seem to be less complex regulated than the liver isozyme. On the other hand, the M-type isozyme is found in glycolytic organs and the A-type isozyme is widely distributed among various organs, while the L-type pyruvate kinase has been found in organs capable of gluconeogenesis where the demand for pyruvate kinase regulation is large. ACKNOWLEDGMENTS The valuable discussions with Professor L. Engstrom and Dr. S. M%rdh at this institute are gratefully acknowledged. REFERENCES 1. TANAKA, T., HARANO, Y., MORIMURA, H., AND MORI, R. (1965) Biochem. Biophys. Res. Commun. 21, 55-60. 2. SUSOR, W. A., AND RUTTER, W. J. (1968) Biochem. Biophys. Res. Commun. 30, 14-20. 3. IMAMURA, K., ANDTANAKA, T. (1972)J. Biochem. fToky0)
71, 1043-1051.
4. CRISP, D. M., AND POGSON, C. I. (1972) Biochem. J. 126, 1009-1023. 5. VAN BERKEL, T. J. C., KOSTER, J. F., AND HULSMANN, W. C. (1972) Biochim. Biophys.
Acta 276, 425-429. 6. JIMENI?Z DE ASIA, L., ROZENGURT, E., AND CARMINATTI, H. (1971) FEBS Letl. 14,22-24. 7. OSTERMAN, J., AND FRITZ, P. J. (1974) Biochemistry 13, 1731-1736. 8. BERGLUND, L., LJUNGSTR~M, O., AND ENGSTROM, L. (1977) J. Biol. Chem. 252, 6108-6111. 9. KUTZBACH, C., BISCHOFBERGER, H., HESS, B., AND ZIMMERMAN-TELSCHOW, H. (1973)
Hoppe-Seyler’s
2. Physiol.
Chem. 354, 1473-
1489. 10. ENGSTR~M, L., BERGLUND, L., BERGSTROM, G., HJELMQUIST, G., AND LJUNGSTR~M, 0. (1974)
in Lipmann Symposium: Energy, Biosynthesis and Regulation in Molecular Biology (Richter, D., ed.), pp. 192-204, Walter de Gruyter Inc., Berlin-New York. 11. IMAMURA, K., TANIUCHI, K., AND TANAKA, T. (1972) J. B&hem. (Tokyo) 72, 1001-1015. 12. LLORENTE, P., MARCO, R., AND SOLS, A. (1970) Eur. J. Biochem. 13, 45-54. 13. VAN BERKEL, T. J. C., KOSTER, J. F., AND HOLSMANN, W. C. (1973) Biochim. Biophys. Acta 321, 171-180. 14. LJUNGSTR~M, O., BERGLUND, L., AND ENGSTROM, L. (1976) Eur. J. Biochem. 68, 497-506. 15. CARDENAS, J. M., DYSON, R. D., AND STRANDHOLM, J. J. (1973) J. Biol. Chem. 248,
6931-6937. 16. KIMBERG, D. V., AND YIELDING, K. L. (1962) J. Biol. Chem. 237, 3233-3239. 17. LJUNGSTR~M, O., HJELMQUIST, G., AND ENGSTR~M, L. (1974) Biochim. Biophys. Acta 358, 289-298. 18. BOCHER, T., AND PFLEIDERER, G. (1955) in
Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 435-440, Academic Press, New York. 19. DUNKER, A. K., AND REUCKERT, R. R. (1969) J. Biol. Chem. 244, 5074-5080. 20. LJUNGSTR~M, O., BERGLUND, L., HJELMQUIST, G., HUMBLE, E., AND ENGSTROM, L. (1974) Upsala J. Med. Sci. 79, 129-137. 21. BARWELL, C., AND HESS, B. (1972) HoppeSeyler’s 2. Physiol. Chem. 353, 1178-1184. 22. FLORY, W., PECZON, B. D., KOEPPE, R. E., AND SPIVEY, H. 0. (1974)Biochem. J. 141,127-131. 23. KAYNE, F. J. (1973) in The Enzymes (Boyer, P.,
ed.), 3rd Ed., Vol. 8, pp. 353-382, Academic Press, New York-London. 24. VAN BERKEL, T. J. C. (1974) Biochim. Biophys. Acta 370, 140-152. 25. IBSEN, K. H., AND TRIPPET, P. (1973) Arch. Biochem. Biophys. 156, 730-744. 26. SEUBERT, W., AND SCHONER, W. (1971) in
Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds.), Vol. 3, pp. 237-267, Academic Press, New YorkLondon. 27. TANAKA, T., HARANO, Y., SUE, F., AND MORIMURA, H. (1967) J. Biochem. (Tokyo) 62,
71-91. 28. VAN BERKEL, T. J. C., DE JONGE, H. R., KOSTER, J. F., AND HOLSMANN, W. C. (1974) Biochem. Biophys. Res. Commun. 60, 398-405. 29. COSTA, L., JIM~NEZ DE ASIA, L., ROZENGURT,
KINETICS
30.
31.
32.
33.
OF PIG PYRUVATE
E., BADE, E., AND CARMINATTI, H. (1972) Biochim. Biophys. Acta 289, 128-136. VIJAYVARGIYA, R., SCHWARK, W. S., AND SINGHAL, R. L. (1969) Canad. J. Biochem. 47, 895-898. CARMINATTI, H., JIM~NEZ DE ASIA, L., LEIDERMAN, B., AND ROZENGURT, E. (1971) J. Biol. Chem. 246, 7284-7288. JIM~NEZ DE ASIA, L., ROZENGURT, E., DEVALLE, J. J., AND CARMINATTI, H. (1971) Biochim. Biophys. Acta 235, 326-334. CORCORAN, E., PHELAN, J. J., AND FOTTRELL, P. F. (1976) Biochim. Biophys. Acta 446, 96-104.
KINASES
361
A AND M
34. POGSON, C. I. (1968) Biochem.
J. 110, 67-77.
35. NISHIMURA, J., HAMASAKI, N., AND MINAKAMI, S. (1978) Biochim. Biophys. Acta 522,104-112. 36. MONOD, J., WYMAN, J., AND CHANGEUX, J.-P. (1965) J. Mol. Biol. 12, 88-118. 37. KAYNE, F. J., AND PRICE, N. C. (1973) Arch. Biochem. Biophys. 159, 292-296. 38. COHEN, P., WATSON, D. C., AND DIXON, G. H. (1975) Eur. J. Biochem. 51, 79-92. 39. HUMBLE, E., BERGLUND, L., TITANJI, V., LJUNGSTR~M, O., EDLUND, B., ZETTERQVIST, ii., AND ENGSTROM. L. (1975) Biochem. Biophys. Res. Comma. 66, 614-621.
Kinetic
Properties
of Pig Pyruvate Kinases Type A from Kidney and Type M from Muscle’
LARS BERGLUND* Institute
of Medical
ELISABET
AND
HUMBLE
and Physiological Chemistry, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden Received August 28, 1978; revised January
University
of Uppsala,
29, 1979
Kinetic properties of homogeneous preparations of pig kidney and pig muscle pyruvate kinases (EC 2.7.1.40) were studied. Both isozymes showed a hyperbolic relationship to ADP with an apparent K, of 0.3 mM. K+ and MgZ+ were necessary for the activity of both isozymes, and their dependences on these cations were similar. The muscle isozyme expressed Michaelis-Menten type of kinetics with respect to phosphoenolpyruvate, and the apparent K,,, was the same (0.03 mM) from pH 5.5 to pH 8.0. In contrast, the dependence on phosphoenolpyruvate changed with pH for the kidney isozyme. It showed similar properties to the muscle isozyme at pH 5.5-7.0 (apparent K,,, of 0.08 mM), while two apparent K,, values for this substrate were present at pH 7.5-8.0, one low (0.1 mM) and one high (0.3-0.6 mM). At pH 7.5, fructose 1,6-bisphosphate converted the kidney isozyme to a kinetical form where only the lower apparent K,,? for phosphoenolpyruvate was detected. On the other hand, in the presence of alanine or phenylalanine the kidney pyruvate kinase showed only the higher K, for this substrate. At low phosphoenolpyruvate levels both isozymes were inhibited by phenylalanine, and half-maximal inhibition was found at 0.3 and 2.2 mM for the kidney and muscle isozymes, respectively. At a 5 mM concentration of the substrate only the kidney isozyme was inhibited, the apparent K, being the same. Alanine inhibited the kidney isozyme (apparent K, at 0.3 mM, irrespective of substrate concentration). No effect was seen on the muscle isozyme. Fructose 1,6-bisphosphate was an activator of the kidney isozyme at phosphoenolpyruvate concentrations below 1.~ ..__,. It also counteracted the inhibition by alanine or phenylalanine of this isozyme. A’,‘P inhibited both isozymes, and this inhibition was not counteracted by fructose 1,6-bisphosphate. The kidney isozyme showed both a high and a low apparent K,,z for phosphoenolpyruvate in the presence of ATP. The influence of the effecters on the activity of both isozymes varied markedly with pH, except for the action of ATP. At low substrate concentrations, however, the inhibitor action of ATP on the muscle enzyme was diminished around pH 7.5, in contrast to higher or lower pH values. Alanine or phenylalanine were more effective as inhibitors at higher pH values, and fructose 1,6-bisphosphate stimulated the kidney isozyme only at pH levels above pH 6.5. The influence of activators and inhibitors on the regulation of the kidney and muscle pyruvate kinases is discussed.
The distribution of different isozymes of mammalian pyruvate kinase (ATP:pyruvate phosphotransferase; EC 2.7.1.40) in various tissues is well documented (l-3). The most widely distributed isozyme is the A type 1 This investigation was supported by the Swedish Medical Research Council (Project 13X-50). 2 Present address: Division of Metabolic Disease, Department of Medicine, School of Medicine, M-013, University of California, San Diego, La Jolla, California 92093.
(or M, type), which is found in most tissues hitherto investigated (3). The M-type isozyme is present in skeletal muscle and brain tissue, whereas the L-type isozyme is found in hepatocytes, in the small intestine and as the minor pyruvate kinase isozyme in renal cortex (l-7). The A-type pyruvate kinase from pig kidney cortex was recently purified and studied in our laboratory (8), and it was found that the amino acid composition of the kidney enzyme was very similar to that of pig muscle pyruvate kinase. In this 347
0003.9861/79/080347-15$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reprorluction in any form reserved.
348
BERGLUND
AND HUMBLE
respect they differed considerably from the L-type pyruvate kinase from pig liver (9). Furthermore, concerning pig pyruvate kinases, it has been shown that the activity of the L-type isozyme, in contrast to the M and A types of isozymes, is regulated by a cyclic AMP-dependent phosphorylation reaction (8, 10). In immunological experiments on enzymes from rat tissues, the similarity between the M and A types of pyruvate kinases has also been documented, as well as their difference from the L-type isozyme (11). Because of the structural similarities noted between the M and A types of pyruvate kinases (8), it was of interest to compare the kinetic properties of these isozymes. The pig pyruvate kinases have not been examined in this respect earlier, and the kinetic properties of a homogeneous mammalian A-type pyruvate kinase have not so far been investigated. The regulation of this type of pyruvate kinase isozyme should be of considerable interest in view of its widespread occurrence. It is known that the assay conditions are of great importance when determining the activity of pyruvate kinase (9, 12-14), which indicates that comparisons made between the same type of pyruvate kinase isozyme from different species assayed under various conditions cannot be evaluated without reservation. The pig liver pyruvate kinase has recently been the subject of a kinetic study in this laboratory (14). In the investigation reported below on pig kidney and pig muscle pyruvate kinase, the same assay conditions as for the pig liver pyruvate kinase were chosen so as to allow a direct comparison of the kinetic properties between these three pyruvate kinase isozymes. EXPERIMENTAL
PROCEDURE
Materials Deionized, glass-distilled water was used in all solutions in the kinetic experiments. P-enolpyruvate,’ Fru-1,6-P,, ADP, ATP, L-phenylalanine, L-alanine, and bovine serum albumin were products of Sigma Chemical Co., St. Louis, Missouri. Lactate dehydro3 Abbreviations used: P-enolpyruvate, phosphoenolpyruvate; Fru-1,6-P,, fructose 1,6-bisphosphate.
genase (EC 1.1.1.27) from rabbit muscle and NADH were obtained from Boehringer Mannheim, GmbH, Germany. In order to remove the ammonium sulfate from the lactate dehydrogenase, prior to use the enzyme suspension was dialyzed at 4°C for 24 h against several changes of 20 mM imidazoleiHC1 buffer, pH 7.5, containing 0.1 mM dithiothreitol. This was found necessary since sulfate ions influence the kinetic properties of the pyruvate kinases, as earlier shown for the pig liver enzyme (14). CarboxymethylSephadex C-50 and Sepharose 6B were products of Pharmacia Fine Chemicals, Uppsala, Sweden, and were treated according to the instructions supplied. All other chemicals were of analytical grade and were used without further purification.
Methods Purification of pig kidney pyruvate kinuse. Pig kidney pyruvate kinase was purified as described earlier (8). The specific activity of the enzyme used was 500 units/mg of protein, assuming that A$$ was 1.0 cm-‘. Pur$cation of pig muscle pyruvate kinase. Pig muscle pyruvate kinase was prepared essentially as described by Cardenas et al. for the bovine muscle pyruvate kinase (15). The initial purification steps, including the chromatography of the enzyme on carboxymethyl-Sephadex, were carried out in all essential details according to this procedure. The pig muscle enzyme was further purified by chromatography on Sepharose 6B in the presence of 100 mM potassium phosphate buffer, pH 7.0, containing 10 mM mercaptoethanol and 30% (v/v) glycerol. When this purification step was omitted, the final product was found to contain some contaminating proteins of lower apparent molecular weights. Hence, to get an optimal degree of purification, it was necessary to include the chromatography on Sepharose 6B. The result of a typical chromatography is shown in Fig. 1A. The purified pig muscle pyruvate kinase was homogeneous as judged from the Sepharose 6B chromatography. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate only one band was seen (Fig. 1B). The specific activity of the purified enzyme was about 340 units/mg of protein, using an absorbance coefficient (A P&90)of 0.55 cm-‘, as reported for the purified bovine muscle pyruvate kinase (15). The yield in a typical preparation of the pig muscle enzyme was about 280 mg from 800 g of pig muscle tissue. This was a considerably higher recovery than obtained for the kidney or liver enzymes (8, 10). Both the kidney and the muscle pyruvate kinases were found to be stable for several months when kept at -18°C in the respective buffers used in the Sepharose 6B chromatography. Assay of pymvate kinase activity. During the purification procedures the enzyme activity was
KINETICS
OF PIG PYRUVATE
I
KINASES
A AND M
349
i
90 FRACTION
NUMBER
FIG. 1. (A) Sepharose 6B chromatography of pig muscle pyruvate kinase. About 70,000 units in a volume of 5 ml were loaded on the column (1.8 x 95 cm), which was equilibrated and eluted with 100 mM potassium phosphate buffer, pH 7.0, containing 10 mM mercaptoethanol and 30% (v/v) glycerol. (0) = pyruvate kinase activity; (0) = absorbance at 280 nm. (B) Polyacrylamide gel electrophoresis in detergent of purified pig muscle pyruvate kinase. The amounts of protein loaded on the gels were (i) 14, (ii) ‘7, and (iii) 2 pg. The position of the band corresponded to a molecular weight of 59,000, using reference proteins as described earlier (23). The same molecular weight, under these conditions, has been found previously for pig kidney pyruvate kinase type A (8).
assayed by a modification of the calorimetric procedure described by Kimberg and Yielding (16, 1’7). One unit of enzyme activity is the amount of enzyme that converts 1 pmol of substrate per minute under the conditions used. In the kinetic experiments, the activity of pyruvate kinase at 30°C was determined in a coupled assay with lactate dehydrogenase (18). Before use, the pyruvate kinase concentration was adjusted to about 1 (kidney enzyme) and 1.2 pgiml (muscle enzyme). The dilution was performed in buffer, pH 7.5, containing 30% 50 mM imidazole/HCl (v/v) glycerol, 100 mM potassium chloride, and However, since this represented 0.1 mM dithiothreitol. a large dilution of the enzymes resulting in a low protein concentration, it was found necessary to add bovine serum albumin (final concentration, 2 mg/ml) in order to stabilize the kidney enzyme. No addition was required for the stability of the muscle enzyme. The presence of the bovine serum albumin did not change any of the kinetic properties of the kidney enzyme. The diluted enzymes were found to be stable for at least 8 h at +4”C. The standard incubation mixture contained 50 pmol of imidazole/HCl buffer, pH 7.5, 100 pmol of potassium chloride, 5 +mol of magnesium chloride, 0.2 Fmol of NADH, 1 /*mol of ADP, 0.1 pmol of dithiothreitol, and 1.5 units of lactate dehydrogenase in a volume of 0.9 ml. After preincubation at 30°C for 3 min, 25 ~1 of the enzyme solution was added, and the preincubation was
continued for a further 3 min. The reaction was then started by the addition of P-enolpyruvate, giving a total incubation volume of 1,025 ml. It was ascertained that the amount of lactate dehydrogenase was not a rate-limiting factor at any pH, and that the pyruvate kinase activity was linear with time and enzyme concentration. In the experiments in which the effects of various pH values on the reaction velocity were tested, all additives were adjusted to the respective pH values. Polyacrglamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate was carried out essentially according to Dunker and Reuckert (19, 20). Calculations. Calculations of apparent K,, values were based on Lineweaver-Burk plots as well as on data from Michaelis-Menten tabulations. The apparent K, values were given as the respective effector concentrations corresponding to 50% of the decrease in enzyme activity. Each kinetic experiment was performed in duplicate or triplicate with a very small variation (~10%). The day-to-day variation was somewhat larger primarily at low P-enolpyruvate concentrations. However, each experiment was repeated several times, in typical cases three to four times, with enzymes representing different purification batches, and similar results were obtained. Thus, the variation in the experiments did consequently not influence the result to any measurable degree.
350
BERGLUND
AND HUMRIJC
CONCENTRATION
OF P-ENOLPYWVATE
ImM)
8
05 CONCENTRATION
OF PmENOLPYRUVATE
ImMI
KINETICS
OF PIG PYRUVATE
RESULTS
Rate Dependence on Substrate Concentration
The influence of the substrates P-enolpyruvate and ADP on the reaction rate of kidney and muscle pyruvate kinases was investigated. As seen in Fig. 2 the muscle enzyme displayed a hyperbolic saturation curve with respect to P-enolpyruvate. The apparent K, at pH 7.5 for this substrate was found to be 0.02 mM. In contrast, the kidney enzyme showed more complex kinetic properties with this substrate. The P-enolpyruvate concentration giving half-maximal velocity at pH 7.5 for the kidney enzyme was found to be 0.25 mM. However, despite the different numerical values obtained for the two enzymes, both apparent K, figures are within the reported physiological P-enolpyruvate concentration range (21,22). Neither of the two enzymes showed sigmoidal dependence on this substrate. A straight line was achieved in a double reciprocal plot for the muscle enzyme, but this was not the case for the kidney isozyme. The Hill constant was calculated to 0.8 for this isozyme and to 1.0 for the muscle enzyme. This phenomenon was investigated further, and two apparent K, values for P-enolpyruvate were found for the kidney isozyme. Since this was dependent on the pH of the reaction mixture, these experiments were presented in connection with the pH studies below. In the further studies only two concentrations of P-enolpyruvate were used, representing saturating and unsaturating conditions. For both isozymes 5 mM P-enolpyruvate was found to be a saturating concentration (Fig. 2). Since the apparent K,n values for this substrate varied considerably between the two enzymes, the concentrations of P-enolpyruvate chosen to represent unsaturating conditions were selected to give approximately the same enzyme activity and to have a similar close relation to the apparent K,. This latter
KINASES
351
A AND M
concentration range was considered important in relation to the physiological importance of the various effecters. The activity of the two enzymes as a function of the second substrate, ADP, is outlined in Fig. 3. Both enzymes expressed hyperbolic relationships to this substrate, with the apparent K, values for ADP of 0.3 mM at saturating and 0.2 mM at unsaturating P-enolpyruvate concentrations. The Hill constant was calculated to be 1.0 under all conditions tested. These results are in agreement with those obtained for the pig liver pyruvate kinase (14). The dependence on ADP, in contrast to P-enolpyruvate, seerned thus to be similar for the three pig pyruvate kinases. InJluence of Mono- and Divalent
Cations
Both the pig muscle and kidney pyruvate kinases were found to be inactive in the absence of potassium or magnesium ions. The same findings have been reported for other pyruvate kinases (23). The two isozymes revealed a similar dependence on the potassium ion concentration. No qualitative differences were detected when saturating or unsaturating P-enolpyruvate concentrations were tested. Maximal activity was obtained at 50 mM potassium chloride, and the activity remained unchanged up to 100 mM, which was the highest potassium concentration tested. In each case a hyperbolic relationship to the potassium ion concentration was seen, and half-maximal concentration was found to be 8-10 mM potassium chloride. The dependence on magnesium ions, however, differed between the two isozymes when assayed in the presence of unsaturating P-enolpyruvate concentrations. All figures given for the magnesium concentration in the following represent free Mg2+, calculated as described by van Berkel(24). The muscle enzyme showed an activity maximum at 2-4 mM Mg’+, and concentrations above
FIG. 2. Dependence of pyruvate kinase activity on the concentration of P-enolpyruvate. The figure shows the activity in the absence of effecters (O), and in the presence of 5 pM Fru-1,6-P, (O), 5 mM alanine (A), 5 mM ATP (A), and phenylalanine (Cl). The concentrations of phenylalanine were 0.5 and 5.0 mM in the experiments with the kidney and muscle pyruvate kinases, respectively. (A) and (C) Muscle pyruvate kinase. (B) and (D) Kidney pyruvate kinase.
352
BERGLUND
AND HUMBLE
20
10
FIG. 3. Effect of ADP on the pyruvate kinase activity. (0) Pig kidney pyruvate kinase activity, (0) pig muscle pyruvate kinase activity. (A) 5 mM P-enolpyruvate and (B) low P-enolpyruvate concentrations, 0.1 mM for the kidney isozyme and 0.01 mM for the muscle isozyme.
4 mM were inhibitory. Half-maximal inhibition, given as 50% of the maximal activity, was seen at 20 mM Me+. The kidney enzyme on the other hand displayed a broad activity maximum (5-15 mM Mg2+), and 35 lllM magnesium chloride was found to give half-maximal inhibition. At 5 mM P-enolpyruvate the dependence on Mg2+ was similar for the isozymes. Low magnesium concentrations were optimal (2-8 mM Mg2+) with a relative inhibition seen at higher Mg2+ concentrations. The activity at the highest Mg2+ concentration tested (45 mM) was found to be 66 and 61% of the maximal activity for the muscle and kidney enzymes, respectively.
saturating and unsaturating P-enolpyruvate concentrations (Fig. 4). The degree of inhibition was almost the same for the two enzymes at the lower substrate level with an apparent Ki of l-l.5 mM ATP. At saturating P-enolpyruvate concentrations this value was higher for the kidney enzyme (3.5 mM) than for the muscle enzyme (1.8 ITIM). Thus, under these conditions ATP was a more efficient inhibitor of the muscle isozyme. The effect of ATP as a function of the P-enolpyruvate concentration was also studied. As shown in Fig. 2, the inhibition by 5 mM ATP on the activity of the muscle enzyme was present throughout the substrate concentration range, giving a decrease in the apparent V. Not even very high P-enolpyruvate concentrations could Effect of ATP overcome this inhibition. The ratio between The influence of ATP has been studied the apparent V values in the presence vs for other pyruvate kinases (13, 23, 25-29). absence of ATP was 0.7. Similar findings Conflicting results have, however, been were seen for the kidney isozyme, and the reported especially regarding the influence apparent K,,, values for P-enolpyruvate of ATP on the activity of A-type pyruvate in the presence of ATP are given in Table I. kinases (28, 29). In view of these results As is evident in Fig. 2, two apparent it was of interest to compare the effect of K, values for P-enolpyruvate were still ATP on the pig enzymes. In order to keep found for the kidney isozyme under these the Mg2+ concentration constant, which conditions. as described above is of great importance, In order to investigate whether Fru-1,6-P,, ATP was added as Mg-ATP. which is a known positive effector of other ATP was found to inhibit the activity of pyruvate kinases (23), could influence the muscle and kidney pyruvate kinase at both inhibition by ATP, the sugar was added in
KINETICS
OF PIG PYRUVATE
I
I
1
,
I
I
/
/
1
I
2
3
4
5
6
7
8
9
10
I 7
I 8
/ 9
1 10
I 1
I 2
I 3
I 4
FIG.
353
(lllM1
I 5
I MgATP I
activity
A AND M
1
[MgATPI
I
KINASES
I 6 (mM1
4. Effect of ATP on the activity of muscle (A) and kidney (B) pyruvate kinase. The in the presence of P-enolpyruvate in concentrations of 5.0 (O), 0.5 (a), and 0.02 ItIM (0) is shown.
a final concentration of 0.1 mM together with increased in the presence of Fru-1,6-P,. No ATP. No effect was found on the ATP- change in basal activity or degree of dependent inhibition of the muscle enzyme. inhibition by ATP was found at 5 mM The activity of the kidney isozyme was, P-enolpyruvate. Since the inhibitory effect however, influenced at the lower P-enol- of ATP on the activity of both isozymes pyruvate concentration. In the presence of thus remained unchanged in the presence ATP and Fru-1,6-P,, the activity of this of Fru-1,6-P,, the results demonstrate the enzyme was the same at 0.5 and 5 mM inability of the sugar to counteract the P-enolpyruvate. This increase in the activity inhibition by ATP on either enzyme. of the kidney enzyme at the low substrate concentration was due to the stimulatory Influence of Alanine and Phenylalanine effect of Fru-1,6-P, as shown below. Neither Several amino acids have been shown to the degree of inhibition by ATP nor the apparent Ki values were, however, affected be inhibitory to other pyruvate kinases even if the basal enzyme activity was (23). Phenylalanine has been found to
354
BERGLUND
AND HUMBLE
TABLE I COMPARISONOFAPPARENTK, VALUES FOR P-ENOLPYRUVATEFORTHREE PIG PYRUVATE KINASE ISOZYMESO Apparent K, for P-enolpyruvate (mM)
Conditions Control ATP Alanine Fru-1,6-P, Phenylalanine
Muscle pyruvate kinase 0.02 0.04 0.02 0.02* 0.06d
Kidney pyruvate kinase
Liver pyruvate kinase
0.07 and 0.25 0.07 and 0.6 0.6 0.07” 0.6'
0.3 0.6 0.7 0.03’ n.d.’
fl The apparent K, values for P-enolpyruvate were investigated at pH 7.5 for pyruvate kinases from pig liver, kidney, and muscle. All enzymes were assayed in the absence or presence of various effecters in 50 mM imidazole/HCl buffer, pH 7.5, containing 100 kmol of potassium chloride, 5 pmol of magnesium chloride, 0.2 pmol of NADH, 1 pmol of ADP, 0.1 pmol of dithiothreitol, and 1.5 units of lactate dehydrogenase in a final volume of 0.9 ml. The results given for the liver isozyme are those of Ref. (14). The concentrations of alanine or ATP were 5 mM for the muscle and kidney isozyme and 2 mM for the liver isozyme. * 5 FM Fru-1,6-P,. r 20 PM Fru-1,6-P,. d 0.05 mM phenylalanine. ’ 0.5 mM phenylalanine. ’ n.d. = not determined.
Phenylalanine was found to be an inhibitor of both isozymes (Fig. 5). The muscle enzyme expressed a sigmoidal dependence on P-enolpyruvate at pH 7.5 in the presence of phenylalanine, and the Hill constant increased from 1.0 to 1.4. As seen in Fig. 2A the inhibitory effect of phenylalanine was highly dependent on the P-enolpyruvate concentration, and virtually no inhibition was found at concentrations above 0.2 lTlM P-enolpyruvate. At low substrate concentrations,. however, the muscle enzyme was inhibited by phenylalanine, the apparent Ki at 0.02 mM P-enolpyruvate being 2.2 11IM. Fru-1,6-P, had no influence on the inhibition of the muscle enzyme by phenylalanine, whereas 5 mM alanine totally counteracted the effect of phenylalanine, giving an uninhibited muscle isozyme. The kidney enzyme differed considerably from the muscle enzyme regarding the effect of phenylalanine. The enzyme was inhibited as seen in Fig. 5. The apparent Ki values were found to be the same (0.3-0.4 InM) at both 0.5 and 5 InM P-enolpyruvate. No signs of sigmoidal dependence on P-enolpyruvate could be detected for the kidney enzyme in the presence of phenylalanine (Fig. 2B). The phenylalanine inhibition of this isozyme was almost totally abolished by 5 PM Fru-1,6-P,. Addition of alanine (5 mM) did not completely remove the phenylalanine inhibition of the A-type isozyme, although the effect of the latter inhibitor was far less pronounced. Both alanine and phenylalanine when added separately influenced the dependence on P-enolpyruvate for the kidney isozyme. As seen in Fig. 2D, only one apparent K, value for this substrate was obtained under these conditions. This value was somewhat higher than the higher K, value found for the control enzyme (Table I). These amino acids seemed thus to convert the kidney isozyme kinetically to a “high K, form.”
inhibit the activity of rabbit muscle pyruvate kinase (type M) (30, 31), and this inhibition was counteracted by alanine (25, 32). Whether this is the case also for A-type pyruvate kinase has not so far been investigated. Alanine had no effect on the activity of the pig muscle pyruvate kinase, the highest effector concentration tested being 10 mM. The kidney enzyme was in contrast inhibited by addition of the amino acid. The apparent Ki for alanine was found at a concentration of 0.3 mM in the presence of either 0.5 or 5 mu P-enolpyruvate. In the presence of 2-10 mM alanine the activity of the kidney pyruvate kinase was 60 or 80% of the activity in the absence of the amino acid Effect of Fm-1,6-P, at low or high P-enolpyruvate concentration, respectively. In contrast, no inhibition by The enhancing effect of Fru-1,6-P, on the alanine of the kidney enzyme was seen in activity of several pyruvate kinases is well the presence of 5 PM Fru-1,6-P,. documented (23). Most investigators have
KINETICS
OF PIG PYRUVATE
5 CONCENTRATION
OF PHENYLALANINE
CONCENTRATION
355
KINASES A AND M
5 OF PHENYLALANINE
10 I mM 1
10 I mMI
FIG. 5. Effect of phenylalanine on the activity of muscle (A) and kidney (B) pyruvate kinase. In some experiments Fru-1,6-P, or alanine was added at final concentrations of 5 pM and 5 mM, respectively, together with phenylalanine. The activities at 5.0 mM P-enolpyruvate (0) and in the presence of Fru-1,6-P, (0) or alanine (Cl) are shown. The corresponding symbols at the lower P-enolpyruvate concentrations (0.5 mM for the kidney enzyme and 0.02 mM for the muscle enzyme) are (A), (A), and (W).
not been able to detect a stimulation of the M-type isozyme (23), but at least in one case Fru-1,6-P, was found to effect the activity of this isozyme (32). Regarding the A-type isozyme several authors describe a positive effect of Fru-1,6-P, on the enzyme activity (6, 13, 23), whereas in some cases no apparent effect of the sugar was found (32). In this characterization of the kinetic properties of the pig pyruvate
kinases type A and M, it was therefore obvious that the role of Fru-1,6-P, was of interest. As shown above even low concentrations of this effector were able to relieve the inhibition by amino acids on the kidney enzyme, whereas such an effect was not seen for the muscle enzyme. The lack of influence of Fru-1,6-P, on the muscle enzyme activity explained these findings. No effect was seen with 0.1-100 PM con-
356
BERGLUND
AND HUMBLE
centrations of the sugar, irrespective of the P-enolpyruvate concentration. The influence of Fru-1,6-P, on the activity of the kidney pyruvate kinase was found to be dependent on the P-enolpyruvate concentration. The effector stimulated the enzyme activity at low P-enolpyruvate levels decreasing the apparent K, for this substrate at 5 j.,&M Fru-1,6-P, to 0.06 mM (Fig. 2B). This value was the same as the low K, for P-enolpyruvate expressed by the control enzyme (Table I). Fru-1,6-P, seemed thus to convert the kidney isozyme kinetically to a “low K, form.” At saturating substrate concentrations, however, no effect was detected. In the presence of 0.5 mM P-enolpyruvate the maximal activity of the kidney enzyme was attained at 5 j.,&M Fru-1,6-P, with an apparent K, of less than 0.1 PM. The effect of this effector on the kidney enzyme activity was, however, counteracted by some negative effecters, as discussed above. ATP (3 InM) and phenylalanine (0.5 IrIM) were therefore
added together with Fru-1,6-P,. These levels were chosen in order to approach physiological effector concentrations (22). Under these conditions a considerably higher concentration of Fru-1,6-P, (1.5 PM) was needed to obtain half-maximal activation of the kidney enzyme, indicating the importance of a combined effector action. Dependence on pH
The activity of both muscle and kidney pyruvate kinase varied considerably with pH, both at unsaturating and saturating P-enolpyruvate concentrations (Fig. 6). At both substrate levels the pH optimum for the muscle enzyme was found to be at pH 7.5. The kidney enzyme had, however, a more acidic pH optimum (pH 6.5) at the lower substrate concentration, but showed a pH profile very similar to the muscle enzyme at 5 mM P-enolpyruvate. At pH levels below pH 7.0 the kidney enzyme appeared to follow Michaelis-Menten-
FIG. 6. Activity of pyruvate kinase as a function of pH. (A) Muscle enzyme at 0.02 mM P-enolpyruvate, (B) kidney enzyme at 0.5 mM P-enolpyruvate, (C) muscle enzyme at 5.0 mM P-enolpyruvate, and (D) kidney enzyme at 5.0 mM P-enolpyruvate. In these experiments the concentration of each enzyme was 200% of that in the standard assay. Symbols as in Fig. 2. The concentrations of the added effecters were: 5 pM Fru-1,6-P,, 3 mM ATP, and 5 mM alanine. Phenylalanine was added at a concentration of 5.0 mM except at the low concentration of P-enolpyruvate in the case of the kidney enzyme, where the phenylalanine concentration was 0.5 mM. All additions were corrected to the respective pH values.
KINETICS
OF PIG PYRUVATE
type of enzyme kinetics with respect to Penolpyruvate. However, at pH 7.5 and 8.0 this was not the case, and a straight line was not obtained in a double reciprocal plot. The value for the Hill constant reflected this phenomenon, being 1.0 between pH 5.5-7.0 and 0.8 at the higher pH levels. At these higher pH values the kidney enzyme was represented by two straight lines in a Lineweaver-Burk plot (Fig. 7), and consequently two apparent K, values for P-enolpyruvate were found, as compared to the findings at lower pH levels. The lower K, value remained essentially the same over the pH range and was similar to the apparent K, for P-enolpyruvate for the muscle enzyme (Table I). As seen in Table II, the higher K, value increased slightly from pH 7.5 to pH 8.0.
FIG.
7. Activity Lineweaver-Burk
KINASES
357
A AND M
In order to further investigate the influence of effecters on the pyruvate kinase activity at different pH values, ATP, alanine, phenylalanine, or Fru-1,6-P, were added to both isozymes over the pH range studied. The enzyme activity was thereafter assayed at saturating or unsaturating P-enolpyruvate concentrations (Fig. 6). ATP inhibited the muscle isozyme under both conditions. However, at a low P-enolpyruvate level the inhibition by ATP was found to be considerably less around pH 7.5 than at other pH values tested. This result was very reproducible and occurred with all enzyme batches tested. In order to further investigate this phenomenon a substrate concentration of 0.1 InM, which approaches a saturating level, was used. Under these conditions the inhibitory effect
CONCENTRATION
OF P-ENOLPYRUVATE
ImMl
CONCENTRATION
OF P-ENOLPYRUVATE
fmM1
of pig kidney pyruvate kinase as a function of the concentration plots inserted. (A) pH 5.5, (B) pH 7.5.
of P-enolpyruvate.
358
BERGLUND
AND HUMBLE
between pyruvate kinases from pig kidney
TABLE II
(A-type), pig muscle (M-type), and pig APPARENT K, FORP-ENOLPYRUVATEATDIFFERENT liver (L-type) (14). The muscle and kidney pH VALUESFORMUSCLEANDKIDNEY PYRUVATE KINASE Apparent K, (mM) for P-enolpyruvate
PH
Muscle pyruvate kinase
Kidney pyruvate kinase
5.5 6.0 6.5 7.0 7.5 8.0
0.03 0.04 0.03 0.03 0.02 0.03
0.11 0.08 0.06 0.11 0.07 and 0.25 0.10 and 0.59
of ATP was still relatively small at pH 7.5, and at more basic or acidic pH values ATP was a more effective inhibitor. The difference was, however, not as pronounced as at the lower P-enolpyruvate concentration. The inhibition by phenylalanine of the muscle enzyme occurred above pH 6.5. Alanine or FIX-1,6-P, were without effect on the muscle enzyme at all pH levels tested. Fru-1,6-P, activated the kidney enzyme primarily at neutral or alkaline pH values and hardly any effect was seen at pH 5.5-6.5. The influence of the negative effecters was similar at pH 5.5-6.5 in the presence of a low substrate concentration. Above pH 6.5, phenylalanine was the most efficient inhibitor. As seen in Fig. 6D, the inhibitory effect of the amino acids on the kidney enzyme activity increased with increasing pH at 5 mM P-enolpyruvate. The ATP inhibition was fairly similar over the pH range. DISCUSSION
So far the A-type pyruvate kinase has been purified to homogeneity from only a few sources (8, 11, 33), and the kinetic properties of the homogeneous enzyme are to a large extent unexplored. In the present paper the kinetic properties of pyruvate kinases type A from kidney and type M from muscle were studied. Our choice of assay conditions also allowed a direct comparison
isozymes were found to have similar properties with respect to the dependence of enzyme activity on the concentrations of ADP, potassium ions, and magnesium ions. These properties also closely resembled those of the liver isozyme (14), which indicates that the activity pattern as a function of these cations or ADP may be a basic, common property of the three types of pig pyruvate kinase isozymes. The dependence on the substrate, P-enolpyruvate, differed to a large extent for the various isozymes. The pig liver isozyme expresses sigmoidal kinetic properties for this substrate (14), while the muscle isozyme showed Michaelis-Menten type of enzyme kinetics with a low apparent K, for P-enolpyruvate. A more complex picture was seen for the kidney isozyme. The two apparent K, values found for P-enolpywvate for the latter enzyme is consistent with the finding that this type of isozyme may exist in two kinetically different forms (34, 35). The low K, for P-enolpyruvate was similar to that obtained for the muscle enzyme, whereas the high K, value approached that given for the liver isozyme (14). The presence of these two kinetical forms of the kidney isozyme was, however, dependent on pH. It was only at pH 7.5 and 8.0 that two K, values were found. At lower pH values the kidney enzyme showed only one apparent K, value for P-enolpyruvate. This value corresponded to the low K, value for this substrate found at pH 7.5. It is also noteworthy that the apparent K, values increased from pH 7.5 to pH 8.0. Thus, at a given P-enolpyruvate concentration the activity of the kidney enzyme was successively more inhibited with increasing pH. The increase in P-enolpyruvate concentration necessary for half-maximal activity with pH for this isozyme indicates that the high K, form of the enzyme dominates at high pH, while the low K, form is found at low pH levels. The occurrence of the kidney pyruvate kinase in these two kinetical, or K, forms, was further dependent on the presence of various effecters. Interestingly, only the low apparent K, for P-enol-
KINETICS
OF PIG PYRUVATE
pyruvate was expressed in the presence of Fru-1,6-P, even at higher pH values. In contrast, the amino acids alanine or phenylalanine converted the kidney isozyme to the high K, form. These changes are summarized as a hypothetical model in Fig. 8. The changes obtained suggest the possibility of a conformational R-T pair, according to the model of Monod et al. (36). The changes in the kinetical forms could be of large importance in the regulation of the kidney pyruvate kinase activity, since the physiological P-enolpyruvate concentration is low (21, 22). A relatively small change in K,, could thus result in a large change of enzyme activity. The suggested kinetical changes for the kidney isozyme are to some extent analogous to the factors influencing the activity of the liver isozyme. In the latter case, the effect of phosphorylation and allosteric effecters are also dependent on pH (14). In contrast, the muscle isozyme showed kinetical properties resembling the A-state of the model in Fig. 8. The negative allosteric effecters also influenced the action of each other. Alanine did not effect the activity of the muscle enzyme but eliminated the inhibition by phenylalanine, which might indicate competition for the same binding site (or sites) (3’7). However, two separate binding sites may also be involved with a resultant conformation change of the enzyme. It is noteworthy that the combined action of alanine and phenylalanine inhibited the
FIG. 8. Hypothetical model for the various kinetical forms of pig kidney pyruvate kinase. The low K, form is represented by A, whereas C is the high K, form. The enzyme form present at pH 7.5 with two is illusapparent K,,, values for P-enolpyruvate trated by B.
KINASES
A AND M
359
kidney enzyme less than each amino acid when added separately. The sigmoidal inhibition curve found for the latter enzyme in the presence of both amino acids also showed the complex type of interaction present in this case. The results do not, however, rule out the possibility of a common binding site. The effect of ATP was different from that of the amino acids and pH. Two K,, values for P-enolpyruvate were again found at higher pH values for the kidney isozyme. Another interesting finding was that the ATP inhibition, in contrast to that of pH or amino acids, was not counteracted by Fru-1,6-P,. These results indicated that the effect of ATP on the activity of the pyruvate kinase isozymes may be the result of a different mechanism than indicated in Fig. 8. This is also supported by the similar action of ATP on both the muscle and kidney pyruvate kinase over the pH range studied. One very striking difference in the ATP action was, however, consistently found between the two isozymes. At 0.02 mM P-enolpyruvate the muscle isozyme showed a relatively small sensitivity to the inhibitory action of ATP around pH 7.5, in contrast to higher or lower pH values. This effect decreased with increasing P-enolpyruvate concentrations and was not present at 5 mM P-enolpyruvate. No such phenomenon could be detected for the kidney enzyme. It is important to point out that the relatively small inhibition by ATP of the muscle enzyme occurred in the physiologically important pH range 7.07.5-8.0. A small change in pH in this pH area could thus considerably increase the inhibitory effect of ATP. This finding and the fact that the effect only was present at low, physiological P-enolpyruvate concentrations indicates that it may be of significance for the regulation of the muscle isozyme activity. In contrast, the concentration of phenylalanine necessary to appreciably influence the activity in vitro of the muscle isozyme was high, and the importance of this type of inhibition in vivo therefore seems doubtful. The concentrations necessary of all effecters tested in this study to influence the activity of the kidney isozyme were
360
BERGLUND
AND HUMBLE
low and approached the respective physiological concentrations (22). A physiological regulation of this isozyme by a combined effector action therefore seems conceivable. It is interesting in this context to mention the increase in apparent K, for Fru-1,6-P, found for the kidney isozyme in the presence of ATP and phenylalanine, demonstrating the importance of a concerted effector influence. Engstrijm and collaborators showed that in addition to allosteric effecters the activity of the pig liver pyruvate kinase is affected by a cyclic AMP-dependent phosphorylation of the enzyme (10, 14). There is apparently no such regulation for the pig kidney and muscle pyruvate kinases (8, 38, 39), and in this respect these isozymes seem to be less complex regulated than the liver isozyme. On the other hand, the M-type isozyme is found in glycolytic organs and the A-type isozyme is widely distributed among various organs, while the L-type pyruvate kinase has been found in organs capable of gluconeogenesis where the demand for pyruvate kinase regulation is large. ACKNOWLEDGMENTS The valuable discussions with Professor L. Engstrom and Dr. S. M%rdh at this institute are gratefully acknowledged. REFERENCES 1. TANAKA, T., HARANO, Y., MORIMURA, H., AND MORI, R. (1965) Biochem. Biophys. Res. Commun. 21, 55-60. 2. SUSOR, W. A., AND RUTTER, W. J. (1968) Biochem. Biophys. Res. Commun. 30, 14-20. 3. IMAMURA, K., ANDTANAKA, T. (1972)J. Biochem. fToky0)
71, 1043-1051.
4. CRISP, D. M., AND POGSON, C. I. (1972) Biochem. J. 126, 1009-1023. 5. VAN BERKEL, T. J. C., KOSTER, J. F., AND HULSMANN, W. C. (1972) Biochim. Biophys.
Acta 276, 425-429. 6. JIMENI?Z DE ASIA, L., ROZENGURT, E., AND CARMINATTI, H. (1971) FEBS Letl. 14,22-24. 7. OSTERMAN, J., AND FRITZ, P. J. (1974) Biochemistry 13, 1731-1736. 8. BERGLUND, L., LJUNGSTR~M, O., AND ENGSTROM, L. (1977) J. Biol. Chem. 252, 6108-6111. 9. KUTZBACH, C., BISCHOFBERGER, H., HESS, B., AND ZIMMERMAN-TELSCHOW, H. (1973)
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in Lipmann Symposium: Energy, Biosynthesis and Regulation in Molecular Biology (Richter, D., ed.), pp. 192-204, Walter de Gruyter Inc., Berlin-New York. 11. IMAMURA, K., TANIUCHI, K., AND TANAKA, T. (1972) J. B&hem. (Tokyo) 72, 1001-1015. 12. LLORENTE, P., MARCO, R., AND SOLS, A. (1970) Eur. J. Biochem. 13, 45-54. 13. VAN BERKEL, T. J. C., KOSTER, J. F., AND HOLSMANN, W. C. (1973) Biochim. Biophys. Acta 321, 171-180. 14. LJUNGSTR~M, O., BERGLUND, L., AND ENGSTROM, L. (1976) Eur. J. Biochem. 68, 497-506. 15. CARDENAS, J. M., DYSON, R. D., AND STRANDHOLM, J. J. (1973) J. Biol. Chem. 248,
6931-6937. 16. KIMBERG, D. V., AND YIELDING, K. L. (1962) J. Biol. Chem. 237, 3233-3239. 17. LJUNGSTR~M, O., HJELMQUIST, G., AND ENGSTR~M, L. (1974) Biochim. Biophys. Acta 358, 289-298. 18. BOCHER, T., AND PFLEIDERER, G. (1955) in
Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 435-440, Academic Press, New York. 19. DUNKER, A. K., AND REUCKERT, R. R. (1969) J. Biol. Chem. 244, 5074-5080. 20. LJUNGSTR~M, O., BERGLUND, L., HJELMQUIST, G., HUMBLE, E., AND ENGSTROM, L. (1974) Upsala J. Med. Sci. 79, 129-137. 21. BARWELL, C., AND HESS, B. (1972) HoppeSeyler’s 2. Physiol. Chem. 353, 1178-1184. 22. FLORY, W., PECZON, B. D., KOEPPE, R. E., AND SPIVEY, H. 0. (1974)Biochem. J. 141,127-131. 23. KAYNE, F. J. (1973) in The Enzymes (Boyer, P.,
ed.), 3rd Ed., Vol. 8, pp. 353-382, Academic Press, New York-London. 24. VAN BERKEL, T. J. C. (1974) Biochim. Biophys. Acta 370, 140-152. 25. IBSEN, K. H., AND TRIPPET, P. (1973) Arch. Biochem. Biophys. 156, 730-744. 26. SEUBERT, W., AND SCHONER, W. (1971) in
Current Topics in Cellular Regulation (Horecker, B. L., and Stadtman, E. R., eds.), Vol. 3, pp. 237-267, Academic Press, New YorkLondon. 27. TANAKA, T., HARANO, Y., SUE, F., AND MORIMURA, H. (1967) J. Biochem. (Tokyo) 62,
71-91. 28. VAN BERKEL, T. J. C., DE JONGE, H. R., KOSTER, J. F., AND HOLSMANN, W. C. (1974) Biochem. Biophys. Res. Commun. 60, 398-405. 29. COSTA, L., JIM~NEZ DE ASIA, L., ROZENGURT,
KINETICS
30.
31.
32.
33.
OF PIG PYRUVATE
E., BADE, E., AND CARMINATTI, H. (1972) Biochim. Biophys. Acta 289, 128-136. VIJAYVARGIYA, R., SCHWARK, W. S., AND SINGHAL, R. L. (1969) Canad. J. Biochem. 47, 895-898. CARMINATTI, H., JIM~NEZ DE ASIA, L., LEIDERMAN, B., AND ROZENGURT, E. (1971) J. Biol. Chem. 246, 7284-7288. JIM~NEZ DE ASIA, L., ROZENGURT, E., DEVALLE, J. J., AND CARMINATTI, H. (1971) Biochim. Biophys. Acta 235, 326-334. CORCORAN, E., PHELAN, J. J., AND FOTTRELL, P. F. (1976) Biochim. Biophys. Acta 446, 96-104.
KINASES
361
A AND M
34. POGSON, C. I. (1968) Biochem.
J. 110, 67-77.
35. NISHIMURA, J., HAMASAKI, N., AND MINAKAMI, S. (1978) Biochim. Biophys. Acta 522,104-112. 36. MONOD, J., WYMAN, J., AND CHANGEUX, J.-P. (1965) J. Mol. Biol. 12, 88-118. 37. KAYNE, F. J., AND PRICE, N. C. (1973) Arch. Biochem. Biophys. 159, 292-296. 38. COHEN, P., WATSON, D. C., AND DIXON, G. H. (1975) Eur. J. Biochem. 51, 79-92. 39. HUMBLE, E., BERGLUND, L., TITANJI, V., LJUNGSTR~M, O., EDLUND, B., ZETTERQVIST, ii., AND ENGSTROM. L. (1975) Biochem. Biophys. Res. Comma. 66, 614-621.