THE JOURNAL OF VITAMINOLOGY 14, 59-67 (1968)

MECHANISM F FORMATION OF OXALOACETATE AND PHOSPHOENOL PYRUVATE FROM PYRUVATE1

HARLAND G. WOOD

Department of Biochemistry, Case Western Reserve University Cleveland, Ohio 44, 106, U.S.A.

Pyruvate, oxaloacetate and P-enolpyruvate have been known to be key compounds in metabolism for many years; pyruvate since 1911 when Neuberg found that yeast decarboxylates pyruvate to acetaldehyde, oxaloacetate since 1937 when Krebs pro posed the citric acid cycle, and P-enolpyruvate since 1934-1936 when Lohmann, Meyerhof and Kiessling discovered that 3-phosphoglycerate is converted to P enolpyruvate by dialyzed yeast or muscle extracts. These three compounds are the hub of numerous reactions leading to both synthesis and breakdown of carbo hydrates, fats and nitrogenous compounds (See the recent review by Utter (1) ). Furthermore, they are interlocked within themselves by a series of reactions re sulting in the synthesis of each fromn the other with accompanying breakdown of the companion compound. There has been a long debate concerning the mechanism of formation of P enolpyruvate from pyruvate. The conversion of P-enolpyruvate to pyruvate in glycolysis by pyruvate kinase was shown by Lardy and Ziegler (2) in 1945 to be reversible, but the equilibrium lies far to the right and the physiological signi ficance of the reversibility is questionable. The possibility that P-enolpyruvate might be formed by a pathway other than via pyruvate kinase was first raised by Kalckar (3) who found that kidney preparations could form P-enolpyruvate from malate and fumarate. At that time Wood and Werkman had discovered fixation of CO2 by heterotrophs and had postulated that the fixation occurs by combination of CO2 with pyruvate to give oxaloacetate. When Solomon et al. (4) in 1940 found that a considerable amount of 14CO2 is incorporated into liver glycogen by rats during its synthesis from lactate, it provided support to the hypothesis that lactate or pyruvate passes through oxaloacetate and a symmetrical 4-carbon dicarboxylic acid during the conversion to P-enolpyruvate and thus to glycogen. Furthermore, using various types of labeled lactate (5) it was calculated from the degree of randomization of label in the glycogen that a major portion of the carbon probably had passed through a symmetrical dicarboxylic acid and the Krebs cycle on its path to glycogen. However, a satisfactory explanation at the enzymatic level for the formation of P-enolpyruvate from oxaloacetate was not found until 1954, at which time Utter and Kurahashi (6) discovered the enzyme, P-enolpyruvate carboxykinase, in chicken liver. It catalyzes Reaction C. It was then postulated by Utter and 1 Assisted by grants from the Atomic Energy Commission under Contract AT-(30-1)-1,320 and from the Nationl Institutes of Health (GM 1,397-01).

59 60 WOOD 1968

Kurahashi (7) and Krebs (8) that P-enolpyruvate is synthesized from pyruvate as follows: Pyruvate+CO2+TPNH+H+_??_Malate+TPN+ (A) Malate+DPN+_??_Oxaloacetate+DPNH+H+ (B) Oxaloacetate+GTP_??_P-enolpyruvate+CO2+GDP (C) Sum: Pyruvate+GTP+TPNH+H++DPN+_??_P-enolpyruvate+GDP+TPN++DPNH+H+ (D) This mechanism involved linking the malate enzyme (Reaction A), malate dehydrogenase (Reaction B) and P-enolpyruvate carboxykinase (Reaction C). How ever, this hypothesis did not stand the test of time. Utter (9) found that mito chondria from chicken liver contained only trace amounts of pyruvate kinase and malate enzyme, yet they could still form significant amounts of P-enolpyruvate from pyruvate (10). This fact prompted Utter to re-examine the liver mitochondria re sulting in the discovery of a new CO2 fixing enzyme, pyruvate carboxylase (11), which catalyzes Reaction E. It then became clear that P-enolpyruvate is probably synthesized from pyruvate by the following linked reactions: Pyruvate+CO2+ATP_??_Oxaloacetate+ADP+Pi (E) Oxaloacetate+GTP_??_P-enolpyruvate+GDP+CO2 (C) Sum: Pyruvate+ATP+GTP_??_ P-enolpyruvate+ADP+GDP+Pi (F) The energetics of Reaction F are very favorable for synthesis of P-enolpyruvate, since it requires the participation of two molecules of nucleotide triphosphate. All available evidence is consistent with the view that formation of P-enolpyruvate from pyruvate occurs by this mechanism in animals and yeast (See the reviews by Utter (12,1)). It is noted that a symmetrical dicarboxylic acid is not involved in Reactions F and C. It is of interest that earlier tracer results obtained with intact animals, in which the isotope pattern in liver glycogen was used as an indicator of meta bolism, are in accord with this mechanism. For example, with 2 labeled lactate (5) or pyruvate (13) the isotope was only partly randomized during its conversion to glycogen, carbons 2 and 5 of the glucose containing about 40 per cent more tracer than 1 and 6 (5). These results were interpreted as evidence that in part there was direct conversion of pyruvate to P-enolpyruvate by the pyruvate kinase reaction and thus to glucose. We now know that this probably was not the case, rather it is likely the oxaloacetate was not completely equilibrated with a sym metrical dicarboxylic acid and thus the tracer was not completely randomized. In contrast, when similar experiments were done with 2-or 3-labeled propionate, the tracer concentration in carbons 2 and 5 always equalled that in 1 and 6, indicating that the metabolism of propionate involved a symmetrical compound as an essential step in the conversion (14). We now know from the enzyme studies of Ochoa and collaborators that propionate is metabolized by formation of methylmalonyl-CoA, then succipyl-CoA, succinate, fumarate, malate, oxaloacetate and P-enolpyruvate. Thus a symmetrical compound is an essential part of the pathway and isotope randomization is complete as was observed. The dicarboxylic acid pathway for forming P-enolpyruvate from pyruvate is not universal; certain bacteria possess another mechanism for forming P-enolpyruvate. Vol. 14 OXALOACETATE, PHOSPHOENOL PYRUVATE FORMATION 61

The pathway has been studied in propionic acid bacteria, E. coli and other bacteria. When Siu and Wood (15) discovered that the propionic acid bacteria contain an enzyme, P-enolpyruvate carboxytransphosphorylase, which catalyzes Reaction G, it seemed possible that these bacteria might use this reaction in conjunction with the pyruvate carboxylase reaction to form P-enolpyruvate from pyruvate much as in animals and as illustrated below:

Pyruvate+ATP+CO2_??_Oxaloacetate+ADP+Pi (E) Oxaloacetate+PPi_??_P-enolpyruvate+Pi+CO2 (G) Sum: Pyruvate+ATP+PPi P-enolpyruvate+ADP+2Pi (H)

Carboxytransphosphorylase has the interesting property of forming P-enolpyru vate from oxaloacetate, but in contrast to carboxykinase (Reaction C) it uses in organic pyrophosphate (PPi) instead of GTP. PPi is formed as a byproduct of the many synthetic reactions of growth and it seemed possible that these bacteria utilize the energy of PPi instead of wasting it by hydrolysis to inorganic Pi. This view was attractive because the propionic acid bacteria are known to possess an unusually high growth efficiency (16) and Bauchop and Elsden (16) have proposed these bacteria must possess some unique feature for formation (or conservation) of ATP. A test of linked Reactions E and G using P-enolpyruvate carboxytransphos phorylase in combination with pyruvate carboxylase (from chicken liver) showed that synthesis of P-enolpyruvate from pyruvate, PPi and ATP could be obtained and furthermore the energetics of Reaction H are quite favorable (17). Therefore, a search for pyruvate carboxylase in propionibacteria was undertaken in our labora tories by Herbert J. Evans. He found that extracts from these bacteria do catalyze the formation of oxaloacetate from pyruvate, CO2 and ATP as would be expected for pyruvate carboxylase, Reaction E. However, there was a requirement for Pi which was puzzling. The extracts were known to contain carboxytransphosphorylase and pyruvate kinase and it seemed possible the oxaloacetate was being synthesized as follows: Pyruvate+ATP_??_P-enolpyruvate+ADP (I) P-enolpyruvate+CO2+Pi_??_Oxaloacetate+PPi (G) Sum: Pyruvate+ATP+CO2+Pi_??_Oxaloacetate+PPi+ADP (J)

However, it was shown that this was not the explanation, since it was found that pyruvate kinase and carboxytransphosphorylase formed little or no oxaloacetate from pyruvate, ATP, CO2 and Pi. At this time a publication by Cooper and Kornberg (18) appeared which provided the answer to the mystery. They described the discovery of a new enzyme, P-enolpyruvate synthetase, in E. coli which cata lyzes Reaction K. It was obvious that this enzyme in combination with carboxy transphosphorylase might be responsible for the observed synthesis of oxaloacetate as illustrated below:

Pyruvate+ATP_??_P-enolpyruvate+AMP+Pi (K) P-enolpyruvate+CO2+Pi_??_Oxaloacetate+PPi (G) Sum: Pyruvate+ATP+CO2_??_Oxaloacetate+AMP+PPi (L) 62 WOOD 1968

A search for pyruvate synthetase was therefore undertaken and indeed it was found to be present in propionibacteria. Evans has now purified the enzyme some 300-fold. It is of interest that linked Reactions K and G provide the mechanism by which the Wood and Werkman reaction occurs in propionic acid bacteria. These bacteria do not contain pyruvate carboxylase and thus do not catalyze a direct Wood and Werkman reaction. It also is noteworthy that carboxykinase in animals and car boxytransphosphorylase in the propionibacteria which catalyze rather similar reac tions (C and G) function quite differently. Carboxykinase serves as a decarboxylase in Reaction F, whereas carboxytransphosphorylase functions as a carboxylase in Reaction L. The reaction catalyzed by P-enolpyruvate synthetase utilizes both high energy bonds of ATP (Reaction K) and thus makes direct phosphorylation of pyruvate energetically feasible. The mechanism of this reaction is of considerable interest. Cooper and Kornberg (19) have recently proposed the following mechanism :

Enzyme+ATP_??_Enzyme-P-O-P+AMP (M) Enzyme-P-O-P+H2O_??_Enzyme-P+Pi (N) Enzyme-P+Pyruvate_??_Enzyme+P-enolpyruvate (O)

In support of this scheme they observed the following: (ƒ¿) AMP exchanges into ATP (Reaction M) and pyruvate is not required but a high concentration of inorganic phosphate (0.1 M) is required. ADP does not influence the rate of the reaction. (b) Pi exchanges into ATP in the presence of AMP and in the absence of pyruvate. The exchange does not occur in the absence of AMP (Reactions M and N). (c) Pyruvate exchanges into P-enolpyruvate in the absence of nucleotides or phosphate (Reaction O). They also state that [ƒÁ-32P•n-ATP yields exclusively 32Pi, whereas [ƒÀ-32P•n-ATP gives rise to 32P-enolpyruvate. In addition, P-enolpyruvate, Pi and AMP can be converted to pyruvate and ATP, while in the presence of arsenate

ADP is formed instead of ATP.

This is a rather surprising development. It seemed likely that the reaction

might occur so mewhat as follows

Enzyme+ATP_??_Enzyme-P-O-P+AMP (M) Enzyme-P-O-P+Pyruvate_??_Enzyme-P-O-P-enolpyruvate (P) Enzyme-P-O-P-enolpyruvate_??_Enzyme+Pi+P-enolpyruvate (Q)

The cleavage of the pyrophosphate bond in Reaction Q presumably would give an equilibrium far to the right, thus accounting for net synthesis of P-enolpyruvate in contrast to the pyruvate kinase reaction. In Reactions M, N, O it would appear that the thermodynamic advantage gained from the formation of enzyme-P-O-P would largely be lost through cleavage of the pyrophosphate bond in Reaction N. The equilibrium of Reaction N would no doubt be far toward enzyme-P. Neverthe less, if the free energy of Reaction O were unfavorable, little P-enolpyruvate would be formed. The free energy of hydrolysis of P-enolpyruvate is much greater than that of any of the other known phosphate esters. Comparison of ATP, phospho creatine and acetyl phosphate discloses that acetyl phosphate has the highest free energy of hydrolysis, but it is about 2.5 kilocalories less negative than that of Vol. 14 OXALOACETATE, PHOSPHOENOL PYRUVATE FORMATION 63

P-enolpyruvate. Therefore, one asks what type of bond is present in the enzyme-P which permits net formation of P-enolpyruvate in Reaction O ? Perhaps there is a conformational change in the protein during Reaction N which retains part of the energy released in the cleavage of the enzyme-P-O-P. Clearly the mechanism of the synthetase reaction warrants further study. One is led to consider the pos sibility that the enzyme lacks specificity and can catalyze exchange Reactions N and O, but these are not part of the normal sequence of the net synthesis of P- enolpyruvate. We have seen that metabolic pathways have different solutions in different cells. Thus E. coli, the propionic acid bacteria, Acetobacter xylinium (20) and Cholorbium thiosulfatophum (21) phosphorylate pyruvate with ATP directly, whereas in animals and yeast this phosphorylation is accomplished indirectly via oxaloacetate (Reaction F). We have also seen that animals and yeast form oxaloacetate directly from pyruvate by carboxylation, whereas the propionic acid bacteria convert the pyruvate to P-enolpyruvate and then to oxaloacetate, Reaction L. E. coli accom plishes this same synthesis using a different fixation reaction (22) catalyzed by P-enolpyruvate carboxylase (Reaction R) which was first observed in plants by Bandurski. The synthesis occurs as follows:

Pyruvate+ATP_??_P-enolpyruvate+AMP+Pi (K) P-enolpyruvate+CO2_??_Oxaloacetate+Pi (R) Sum: Pyruvate+ATP+CO2_??_Oxaloacetate+AMP+2Pi (S) In addition to the problem of different mechanisms in different cells there is the problem of control mechanisms in a given cell. Pyruvate, oxaloacetate and P -enolpyruvate are all interlocked by a series of reactions in animal cells as illustrated in Fig. 1. What "turns off" pyruvate kinase during gluconeogenesis to prevent it from converting the P-enolpyruvate back to pyruvate after it has been formed

FIG. 1 Pathways Which Interlink Pyruvate, P-enolpyruvate and Oxaloacetate in Animals The reactions are catalyzed by the following enzymes: (a) malate enzyme (Reaction A); (b) malate dehydrogenase (Reaction B); (c) P-enolpyruvate carboxykinase (Reaction C); (d) pyruvokinase (Reaction I); (e) pyruvate carboxylase (Reaction E) and (f) fumarase. Fumarase catalyzes formation of a symmetrical dicarboxylic acid and thus cause randomization of 14C in tracer experiments. The reaction refer to those in the text. 64 WOOD 1968 from pyruvate by way of oxaloacetate ? Likewise during glycolysis and fatty acid synthesis what "turns on" pyruvate kinase and "turns off" carboxykinase and pyruvate carboxylase ? We have only limited understanding of the factors which control the flux of metabolites through the pathways but the intracellular location of the enzymes certainly is of importance in these considerations. Pyruvate kinase is in the cytosol of cells. On the other hand, both pyruvate carboxylase and P- enolpyruvate carboxykinase are in the mitochondria of cells from livers of chickens, beef and rabbits (1). In rats, however, the pyruvate carboxylase is in both the mitochondria and the cytosol (23) and practically all the P-enolpyruvate carboxy kinase is in the cytosol (24). In liver cells of guinea pigs the P-enolpyruvate carboxykinase is about equally distributed in the cytosol and mitochondria (24). The amount of these enzymes can be increased or decreased by subjecting the rats to dietary or hormonal regimes, but it is only the soluble form of both pyruvate carboxylase (23) and of P-enolpyruvate carboxykinase (24) in the cytosol which undergoes the change. These changes occur in a matter of hours and therefore appear to be too slow to account for the immediate regulatory mechanisms which are required. The interaction and control of these enzymes undoubtedly is dependent on the location of the enzymes in the cells. In chicken liver where both pyruvate car boxylase and P-enolpyruvate carboxykinase are mitochondrial, the situation may be represented as shown in Fig. 2. The P-enolpyruvate is formed in the mitochondria and passes through the membrane of the mitochondria into the cytosol. This view is based on the observations of Mendicino and Utter (10). They coupled the for mation of P-enolpyruvate produced from malate by mitochondria of chicken liver to the synthesis of carbohydrates by added glycolytic enzymes. For this synthesis to occur the P-enolpyruvate which was formed inside the mitochondria must be transported to the glycolytic enzymes on the outside. Within the mitochondria pyruvate is converted to acetyl-CoA. The latter is required for the activation of pyruvate carboxylase and this provides a very attractive mechanism for control

FIG. 2 Synthesis of P-enolpyruvate from Pyruvate when Both the Pyruvate Carboxylase and P-enolpyruvate Carboxykinase are in the Mitochondria Acetyl-CoA activates the pyruvate carboxylase. P-enolpyruvate is trans ported through the mitochondrial membrane, See Utter (1). Vol. 14 OXALOACETATE, PHOSPHOENOL PYRUVATE FORMATION 65 of the synthesis of P-enolpyruvate. Utter and coworkers have shown that there is an absolute requirement of acetyl-CoA for activation of the pyruvate carboxylase from liver. The activation occurs in the range of 10-5 to 10-4 M and the rela tionship between acetyl-CoA concentration and reaction velocity is sigmoidal (25). Thus acetyl-CoA has all the properties of an effective control factor. There is indirect evidence from several laboratories that gluconeogenesis is increased in liver preparations by the presence of fatty acids. These results have been inter preted to be due to the increase in acetyl-CoA concentration due to oxidation of fatty acids which then activate the pyruvate carboxylase. The problem of deter mining with certainty that acetyl-CoA has such a regulating effect on gluconeo genesis presents many problems, however, and probably cannot be answered de finitely until methods have been devised for the determination of the concentration of acetyl-CoA in the mitochondria of intact cells. The situation is quite different when the two enzymes occur in different compartments. For example, P-enolpyruvate carboxykinase is in the cytosol and pyruvate carboxylase in the mitochondria in some species. This requires that ox aloacetate formed in the mitochondria to pass through the mitochondrial membrane. Lardy and coworkers have proposed that oxaloacetate as such is not transported but it is converted to other metabolites for transport. The scheme shown in Fig. 3 reflects the observations of Shrago and Lardy (26). They found that rat liver mitochondria formed aspartate, malate and citrate from pyruvate and bicarbonate. If P-enolpyruvate carboxykinase, glutamate and glutamate-oxaloacetate transaminase were added in addition, there was a very substantial formation of P-enolpyruvate. If glutamate or transaminase were omitted, the yield was decreased. The gluta mate presumably aids in the conversion of oxaloacetate to aspartate in the

FIG. 3 Synthesis of P-enolpyruvate from Pyruvate when Pyruvate Carboxylase is in the Mitochondria and P-enolpyruvate Carboxykinase is in the Cytosol The oxaloacetate is converted to aspartate by transamination or to malate and transported from the mitochondria. These compounds are again con verted to oxaloacetate in the cytosol and then to P-enolpyruvate, See Utter (1). 66 WOOD 1968

TABLE 1 Occurrance of Enzymes Involved in the Synthesis of Oxaloacetate and P-enolpyruvate from Pyruvate

mitochondria and after transport it is again reconverted to oxaloacetate by action

of the transaminase using the ƒ¿-ketoglutarate which has been transferred to the

cytosol.

Clearly the situation illustrated in Fig. 3 presents very different conditions for

control than those illustrated in Fig. 2. Malate formation from oxaloacetate and

aspartate formation from oxaloacetate would be influenced respectively by the NAD/

reduced NADP ratio and glutamate concentration. Furthermore, the pathways

competing for oxaloacetate, i.e. the tricarboxylic acid cycle and the formation of

P-enolpyruvate are in separate compartments in the situation of Fig. 3 as contrasted

to that of Fig. 2. The two processes therefore are subject to control by the rate

of transport of the oxaloacetate derivatives between the two compartments in the

case of Fig. 3.

Yeast presents still a different situation. Both pyruvate carboxylase and the

P-enolpyruvate carboxykinase are in the cytosol in this case. Thus a third set of

circumstances are presented for control which could be considered. Furthermore,

the Pyruvate carboxylase of yeast has quite different properties than that of liver.

The activation by acetyl-CoA is quite non-specific (1). For example, benzoyl-CoA

stimulates the yeast enzyme but is quite inert with the chicken liver enzyme.

It is to be noted that the synthesis of carbohydrate occurs in the cytosol and pyruvate kinase is present in this compartment. It is difficult to see why the P- enolpyruvate remains available for gluconeogenesis when the kinase present in this

compartment would convert P-enolpyruvate to pyruvate. It seems probable that

there is a strong negative control on pyruvate kinase which prevents its activity during gluconeogenesis. However, such control has not yet been reported in the literature.

SUMMARY

The pathways by which pyruvate, oxaloacetate and P-enolpyruvate are interlinked have been reviewed. The enzymes involved (see Table 1) and pathways differ markedly in different cells. Animals and yeast possessing pyruvate carboxylase are able to convert pyruvate to oxaloacetate directly by CO2 fixation, but the propionic acid bacteria and E. toll use an indirect mechanism. These bacteria lack pyruvate carboxylase but possess an enzyme, P-enolpyruvate synthetase, which phosphorylates pyruvate directly with ATP forming P-enolpyruvate, AMP and Pi. They then con vert the P-enolpyruvate to oxaloacetate; E, coil by the enzyme, P-enolpyruvate Vol. 14 OXALOACETATE, PHOSPHOENOL PYRUVATE FORMATION 67 carboxylase, and the propionic acid bacteria by P-enolpyruvate carboxytransphos phorylase. Animals lack P-enolpyruvate synthetase and are not able with pyruvate kinase to catalyze the direct synthesis of P-enolpyruvate effectively from pyruvate. They make P-enolpyruvate from oxaloacetate by catalysis with P-en olpyruvate carboxykinase. Not only do the animal cells have different enzymes and use different mechanisms than bacteria, the distribution of the enzymes in the cytosol and mitochondria of cells differ in the different species of animals. This variation in distribution presents a possibility for control of metabolism which varies from species to species. The mechanisms of these controls are still subject to much speculation, but have been considered briefly.

REFERENCES

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