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The stoichiometry of the citric acid cycle
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161
May 1983
and Dawson, R. M. C., eds), Ch. 16, Elsevier/North-Holland Biomedical Press, Amsterdam 4 0 p den Kamp, J. A. F. (1979) Annu. Rev. Biochem. 48, 47-71 5 Bloj, B. and Zilversmit, D. B. (1981)Mol. Cell. Biochem. 40, 163-192 6 Wirtz, K. W. A. (1982)inLipid-Protein Interact/ons (Jost, P. C. and Griffith, O. Hayes, eds), Vol. 1, Ch. 6, John Wiley and Sons, New York 7 Stein, O. and Stein, Y. (1971)Adv. LipidRes. 9, 1-72 8 Gould, R. M. and Dawson, R. M. C. (1976) J. Cell Biol. 68,480--496 9 Pagano, R. E., Longmuir, K. J., Martin, O. C. and Struck, D. K. (1981)J. CellBiol. 91,872-877 10 Struck, D. K. and Pagano, R. E. (1980)J. Biol. Chem. 255, 5405-5410
11 Pagano, R. E. and Weinstein, J. N. (1978)Annu. Rev. Biophys. Bioeng. 7,435---468 12 Pagano, R. E., Schroit, A. J. and Struck, D. K. in Liposomes: From Physical Structure to Therapeutic Applications (Knight, C. G., ed.), Ch. 11, Elsevier/Noah-Holland Biomedical Press, Amsterdam 13 Huang, L. in The Liposomes (Ostro, M. J., ed.), Ch. 3, Marcel Dekker, New York (in press) 14 Szoka, F., Jacobson, K., Derzko, Z. and Papahadjopoulos, D. (1980) Biochim. Biophys. Acta 600, 1-18 15 Virtanen, I., Ekblom, P. and Laurila, P. (1980) J. Cell Biol. 85,429-434 16 Johnson, L. V., Walsh, M. L., Bockus, B. J. and Chen, L. B. (1981)J. Cell Biol. 88, 526-535 17 Pagano, R. E., Longmuir, K. J. and Martin, O. C. (1983)J. Biol. Chem. 258, 2034-2040
18 Spector, A. A., Mathur, S. N., Kaduce, T. L. and Hyman B. T. (1981) Prog. Lipid Res. 19, 155-186 19 Edidin, M. (1981) in New Comprehensive Biochemistry (Finean, J. B. and Michell, R. H., eds), Vol. 1, Elsevier/North-Holland Biomedical Press, Amsterdam 20 Small, D. M. (1981) in Proceedings of the International Conference on Biological Membranes (Bloch, K., Bolis, L. and Tosteson, D. C., eds), Ch. 2, pp. 11-34, PSG Publishing Company, Boston 21 Van Golde, L. M. G. and Van den Bergh, S. G. (1977) in Lipid Metabolism in Mammals (Snyder, F., ed. ), Vol. 1, pp. 1-33, Plenum Press, New York 22 Bell, R. M. and Coleman, R. A. (1980) Annu. Rev. Biochem. 49, 459-487
Textbook Errors The stoichiometry of the citric acid cycle T. NormanPalmerandMaryC. Sugden Students are often asked to describe the pathway whereby palmitate (or related long-chain fatty acids containing an even number of carbon atoms) is converted to glucose in mammalian liver. Needless to say, this is a 'trick question'. Mammalian liver and kidney are unable to convert palmitate into glucose. This fact is related to the stoichiometry of the citric acid cycle. The entry of the two-carbon acetyl unit of acetyl CoA (the product of/3-oxidation of palmitate) into the cycle is balanced by the loss of two carbon atoms by decarboxylation within the cycle (catalysed by isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex). Therefore the acetyl unit is oxidized in the cycle and cannot form oxaloacetate de novo. The fact that the stoichiometry of the citric acid cycle commits the acetyl unit of acetyl-CoA to oxidation (to two molecules CO2) is frequently dealt with in some depth in undergraduate textbooks. An equally important facet of the stoichiometry of the citric acid cycle appears, however, to be frequently ovedooked. This is that the acetyl unit of acetyl-CoA is the only substrate that the cycle can oxidize completely. Goldstein and Newsholme x and Vinay et al. 2 have made this point previously but it warrants reiteration given its fundamental importance to intermediary metabolism. T. Norman Palmer and Mary C. Sugden are at the Department of Biochemistry, Chafing Cross Hospital Medical School, London, W6 8RF, U.K.
Textbooks, in consequence, are in error when they imply that conversion of the carbon skeletons of certain amino acids to citric acid cycle intermediates results in complete oxidation per se. These amino acids are glutamate, glutamine, histidine, proline and arginine (cycle entry at 2-oxoglutarate), isoleucine, methionine, threonine and valine (cycle entry at succinyl-CoA), phenylalanine and tyrosine (cycle entry at fumarate), and aspartate and asparagine (cycle entry at oxaloacetate) (Fig. 1). For example, entry of 1 mol of glutamate into the cycle as 2-oxoglutarate
and its metabolism through one revolution of the cycle results in: oxoglutarate + 3NAD + + FAD + GDP + Pi + 2I-hO + acetyl-CoA ---, oxoglutarate + 2CO~ + 3NADH + FADI-h + 2H ÷ + GTP + CoA. It is clear that, provided no intermediates leave the cycle, oxoglutarate is regenerated whilst 1 mol acetate (as acetyl-CoA) is oxidized. Needless to say, the entry of 2-oxoglutarate into the cycle, whilst resulting in no net oxidationperse, would lead to a continuous increase in the concentration of cycle intermediates. The amino acids specified above are obviously capable of being oxidized in vivo. The question is how this is achieved. Given that the acetyl unit of acetyl-CoA is the only substrate oxidized quantitatively by the citric acid cycle, logic dictates that
Acetyl CoA Asparagine ~ Aspartate ] • /Oxaloacetate Malate
Citrate
[rosine 1 l
1
Phenylalanine I ~-Fumarate
Isocitrate
Succinate
C02 [
Isoleucine Methionine Threonine Valine
~ Succinyl CoA 2-Oxoglutarate ,,F-~~ - - - ~ " CO2
~
Glutamate Glutamine Histidine Proline Arginine
Fig. 1. Fates of the carbon skeletons of relevant amino acids. ,~: 1983, Elsevier Science Publishers B.V.. Amsterdam
0376 - 5067/83/$01.00
162
TIBS - May 1983
the amino acids in question must be converted to acetyl-CoA as a preliminary to their oxidation in the cycle. In skeletal muscle there is evidence that phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK) and the pyruvate dehydrogenase complex (PDH) together catalyse net conversion of oxaloacetate to acetyl-CoA L3.4. This allows cycle intermediates (including the products of amino acid metabolism) to be completely oxidized (Fig. 2). For example, 2-oxoglutarate (derived from glutamate, ghitamine, histidine, proline or arginine metabolism) is metabolized via the pathway: 2.-oxoglutarate
(.~) succinyl CoA .__~ (2)
COs oxaloacetate ~ ) phosphoenolpyruate C02 (4_,)pyruvate ~ acetyl-CoA .(6) 2CO~
I~/ * Pyruvate (5)'~",-.~CO2 Phosphoenolpyruvate '~ AcetylCoA
C0207 °acetate
Citrate
Malate Fumarate
Is0citrate
SuccinyleoA I1) 2-Oxoqlutarate CO2 Fig. 2. Pathway o f oxidation o f glutamate (and related amino acids) via phosphoenolpyruvate (see text for key to enzymes).
rate ~ 5CO2. The pathway involves five decarboxylation reactions, three within the citric acid cycle [ 2 0 G H D (twice) and CO2 ICDH] and two outside (PEPCK and [key to steps: (1) 2-oxoglutarate dehy- PDH). Oxaloacetate metabolism via PEP drogenase complex (OGDH); (2) succinyl' does not imply a commitment to oxidation: CoA synthase, succinate dehydrogenase, pyruvate may be converted to alanine fumarase and malate dehydrogenase; (3) (reviewed by SnelP) or, at least in theory, PEPCK; (4) PK; (5) PDH; and (6) citrate phosphoenolpyruvate may act as a synthase, aconitase and isocitmte dehydro- glyconeogenic precursor6. It might be presumed that the pathway(s) genase (ICDH)]. The overall stoichiometry is: oxogluta- of oxidation of citric acid cycleintermed-
iates (including those produced by amino acid metabolism) would be well established in liver and kidney. Surprisingly, this is far from the case. These tissues contain PEPCK and it might appear reasonable to assume that the pathway detailed in Fig. 2 applies. This is an assumption and alternative pathways may existT-L It is not established whether liver and kidney have the ability to completely oxidize citric acid cycle intermediates: amino acid metabolism may be coupled to gluconeogenesis 1°or lipogenesis. References 1 Goldstein, L. and Newsholme, E. A. (1976) Biochem. J. 154,555--558 2 Vinay,P., Mapes, J. P. and Krebs, H. A. (1978) Am. J. Physiol. 235, FI23--FI29 3 Snell, K. and Duff, D. A. (1977) Biochem. J. 162,399--403 4 Duff, D. A. and Snell, K. (1982) Biochem. J. 206, 147-152 5 Snell, K. (1980)Biochem. Soc. Trans. 8, 203--213 6 Odedra, B. R. and Palmer, T. N. (1981) Biosci. Rep. 1,157-165 7 Rognstad, R. (1979) Biochim. Biophys. Acta 586, 242-249 8 Janssens, P., Hems, R. and Ross, B. (1980) Biochem. J. 190, 27-37 9 McLean, P., Brown, J. and Greenbaum, A. L. (1968) in Carbohydrate Metabolism and its Disorders (Dickens, F., Randle, P. J. and Whelan, W. J., eds), Vol. 1, pp. 497-525 AcademicPress, London 10 H~iussinger,D., Gerok, W. and Sies, H. (1982) Eur. J. Biochem. 126,69--76
-
161
May 1983
and Dawson, R. M. C., eds), Ch. 16, Elsevier/North-Holland Biomedical Press, Amsterdam 4 0 p den Kamp, J. A. F. (1979) Annu. Rev. Biochem. 48, 47-71 5 Bloj, B. and Zilversmit, D. B. (1981)Mol. Cell. Biochem. 40, 163-192 6 Wirtz, K. W. A. (1982)inLipid-Protein Interact/ons (Jost, P. C. and Griffith, O. Hayes, eds), Vol. 1, Ch. 6, John Wiley and Sons, New York 7 Stein, O. and Stein, Y. (1971)Adv. LipidRes. 9, 1-72 8 Gould, R. M. and Dawson, R. M. C. (1976) J. Cell Biol. 68,480--496 9 Pagano, R. E., Longmuir, K. J., Martin, O. C. and Struck, D. K. (1981)J. CellBiol. 91,872-877 10 Struck, D. K. and Pagano, R. E. (1980)J. Biol. Chem. 255, 5405-5410
11 Pagano, R. E. and Weinstein, J. N. (1978)Annu. Rev. Biophys. Bioeng. 7,435---468 12 Pagano, R. E., Schroit, A. J. and Struck, D. K. in Liposomes: From Physical Structure to Therapeutic Applications (Knight, C. G., ed.), Ch. 11, Elsevier/Noah-Holland Biomedical Press, Amsterdam 13 Huang, L. in The Liposomes (Ostro, M. J., ed.), Ch. 3, Marcel Dekker, New York (in press) 14 Szoka, F., Jacobson, K., Derzko, Z. and Papahadjopoulos, D. (1980) Biochim. Biophys. Acta 600, 1-18 15 Virtanen, I., Ekblom, P. and Laurila, P. (1980) J. Cell Biol. 85,429-434 16 Johnson, L. V., Walsh, M. L., Bockus, B. J. and Chen, L. B. (1981)J. Cell Biol. 88, 526-535 17 Pagano, R. E., Longmuir, K. J. and Martin, O. C. (1983)J. Biol. Chem. 258, 2034-2040
18 Spector, A. A., Mathur, S. N., Kaduce, T. L. and Hyman B. T. (1981) Prog. Lipid Res. 19, 155-186 19 Edidin, M. (1981) in New Comprehensive Biochemistry (Finean, J. B. and Michell, R. H., eds), Vol. 1, Elsevier/North-Holland Biomedical Press, Amsterdam 20 Small, D. M. (1981) in Proceedings of the International Conference on Biological Membranes (Bloch, K., Bolis, L. and Tosteson, D. C., eds), Ch. 2, pp. 11-34, PSG Publishing Company, Boston 21 Van Golde, L. M. G. and Van den Bergh, S. G. (1977) in Lipid Metabolism in Mammals (Snyder, F., ed. ), Vol. 1, pp. 1-33, Plenum Press, New York 22 Bell, R. M. and Coleman, R. A. (1980) Annu. Rev. Biochem. 49, 459-487
Textbook Errors The stoichiometry of the citric acid cycle T. NormanPalmerandMaryC. Sugden Students are often asked to describe the pathway whereby palmitate (or related long-chain fatty acids containing an even number of carbon atoms) is converted to glucose in mammalian liver. Needless to say, this is a 'trick question'. Mammalian liver and kidney are unable to convert palmitate into glucose. This fact is related to the stoichiometry of the citric acid cycle. The entry of the two-carbon acetyl unit of acetyl CoA (the product of/3-oxidation of palmitate) into the cycle is balanced by the loss of two carbon atoms by decarboxylation within the cycle (catalysed by isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex). Therefore the acetyl unit is oxidized in the cycle and cannot form oxaloacetate de novo. The fact that the stoichiometry of the citric acid cycle commits the acetyl unit of acetyl-CoA to oxidation (to two molecules CO2) is frequently dealt with in some depth in undergraduate textbooks. An equally important facet of the stoichiometry of the citric acid cycle appears, however, to be frequently ovedooked. This is that the acetyl unit of acetyl-CoA is the only substrate that the cycle can oxidize completely. Goldstein and Newsholme x and Vinay et al. 2 have made this point previously but it warrants reiteration given its fundamental importance to intermediary metabolism. T. Norman Palmer and Mary C. Sugden are at the Department of Biochemistry, Chafing Cross Hospital Medical School, London, W6 8RF, U.K.
Textbooks, in consequence, are in error when they imply that conversion of the carbon skeletons of certain amino acids to citric acid cycle intermediates results in complete oxidation per se. These amino acids are glutamate, glutamine, histidine, proline and arginine (cycle entry at 2-oxoglutarate), isoleucine, methionine, threonine and valine (cycle entry at succinyl-CoA), phenylalanine and tyrosine (cycle entry at fumarate), and aspartate and asparagine (cycle entry at oxaloacetate) (Fig. 1). For example, entry of 1 mol of glutamate into the cycle as 2-oxoglutarate
and its metabolism through one revolution of the cycle results in: oxoglutarate + 3NAD + + FAD + GDP + Pi + 2I-hO + acetyl-CoA ---, oxoglutarate + 2CO~ + 3NADH + FADI-h + 2H ÷ + GTP + CoA. It is clear that, provided no intermediates leave the cycle, oxoglutarate is regenerated whilst 1 mol acetate (as acetyl-CoA) is oxidized. Needless to say, the entry of 2-oxoglutarate into the cycle, whilst resulting in no net oxidationperse, would lead to a continuous increase in the concentration of cycle intermediates. The amino acids specified above are obviously capable of being oxidized in vivo. The question is how this is achieved. Given that the acetyl unit of acetyl-CoA is the only substrate oxidized quantitatively by the citric acid cycle, logic dictates that
Acetyl CoA Asparagine ~ Aspartate ] • /Oxaloacetate Malate
Citrate
[rosine 1 l
1
Phenylalanine I ~-Fumarate
Isocitrate
Succinate
C02 [
Isoleucine Methionine Threonine Valine
~ Succinyl CoA 2-Oxoglutarate ,,F-~~ - - - ~ " CO2
~
Glutamate Glutamine Histidine Proline Arginine
Fig. 1. Fates of the carbon skeletons of relevant amino acids. ,~: 1983, Elsevier Science Publishers B.V.. Amsterdam
0376 - 5067/83/$01.00
162
TIBS - May 1983
the amino acids in question must be converted to acetyl-CoA as a preliminary to their oxidation in the cycle. In skeletal muscle there is evidence that phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK) and the pyruvate dehydrogenase complex (PDH) together catalyse net conversion of oxaloacetate to acetyl-CoA L3.4. This allows cycle intermediates (including the products of amino acid metabolism) to be completely oxidized (Fig. 2). For example, 2-oxoglutarate (derived from glutamate, ghitamine, histidine, proline or arginine metabolism) is metabolized via the pathway: 2.-oxoglutarate
(.~) succinyl CoA .__~ (2)
COs oxaloacetate ~ ) phosphoenolpyruate C02 (4_,)pyruvate ~ acetyl-CoA .(6) 2CO~
I~/ * Pyruvate (5)'~",-.~CO2 Phosphoenolpyruvate '~ AcetylCoA
C0207 °acetate
Citrate
Malate Fumarate
Is0citrate
SuccinyleoA I1) 2-Oxoqlutarate CO2 Fig. 2. Pathway o f oxidation o f glutamate (and related amino acids) via phosphoenolpyruvate (see text for key to enzymes).
rate ~ 5CO2. The pathway involves five decarboxylation reactions, three within the citric acid cycle [ 2 0 G H D (twice) and CO2 ICDH] and two outside (PEPCK and [key to steps: (1) 2-oxoglutarate dehy- PDH). Oxaloacetate metabolism via PEP drogenase complex (OGDH); (2) succinyl' does not imply a commitment to oxidation: CoA synthase, succinate dehydrogenase, pyruvate may be converted to alanine fumarase and malate dehydrogenase; (3) (reviewed by SnelP) or, at least in theory, PEPCK; (4) PK; (5) PDH; and (6) citrate phosphoenolpyruvate may act as a synthase, aconitase and isocitmte dehydro- glyconeogenic precursor6. It might be presumed that the pathway(s) genase (ICDH)]. The overall stoichiometry is: oxogluta- of oxidation of citric acid cycleintermed-
iates (including those produced by amino acid metabolism) would be well established in liver and kidney. Surprisingly, this is far from the case. These tissues contain PEPCK and it might appear reasonable to assume that the pathway detailed in Fig. 2 applies. This is an assumption and alternative pathways may existT-L It is not established whether liver and kidney have the ability to completely oxidize citric acid cycle intermediates: amino acid metabolism may be coupled to gluconeogenesis 1°or lipogenesis. References 1 Goldstein, L. and Newsholme, E. A. (1976) Biochem. J. 154,555--558 2 Vinay,P., Mapes, J. P. and Krebs, H. A. (1978) Am. J. Physiol. 235, FI23--FI29 3 Snell, K. and Duff, D. A. (1977) Biochem. J. 162,399--403 4 Duff, D. A. and Snell, K. (1982) Biochem. J. 206, 147-152 5 Snell, K. (1980)Biochem. Soc. Trans. 8, 203--213 6 Odedra, B. R. and Palmer, T. N. (1981) Biosci. Rep. 1,157-165 7 Rognstad, R. (1979) Biochim. Biophys. Acta 586, 242-249 8 Janssens, P., Hems, R. and Ross, B. (1980) Biochem. J. 190, 27-37 9 McLean, P., Brown, J. and Greenbaum, A. L. (1968) in Carbohydrate Metabolism and its Disorders (Dickens, F., Randle, P. J. and Whelan, W. J., eds), Vol. 1, pp. 497-525 AcademicPress, London 10 H~iussinger,D., Gerok, W. and Sies, H. (1982) Eur. J. Biochem. 126,69--76