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