Rethinking glycolysis: on the biochemical logic of metabolic pathways

Article metrics

Abstract

Metabolic pathways may seem arbitrary and unnecessarily complex. In many cases, a chemist might devise a simpler route for the biochemical transformation, so why has nature chosen such complex solutions? In this review, we distill lessons from a century of metabolic research and introduce new observations suggesting that the intricate structure of metabolic pathways can be explained by a small set of biochemical principles. Using glycolysis as an example, we demonstrate how three key biochemical constraints—thermodynamic favorability, availability of enzymatic mechanisms and the physicochemical properties of pathway intermediates—eliminate otherwise plausible metabolic strategies. Considering these constraints, glycolysis contains no unnecessary steps and represents one of the very few pathway structures that meet cellular demands. The analysis presented here can be applied to metabolic engineering efforts for the rational design of pathways that produce a desired product while satisfying biochemical constraints.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The Embden-Meyerhof-Parnas glycolytic pathway.
Figure 2: The reduction potentials, E′, of half-reactions between functional groups composed of only carbon, oxygen and hydrogen.
Figure 3: Sequential assembly of the lower glycolytic reaction sequence according to biochemical constraints: the basic energetic and mechanistic constraints.
Figure 4: Sequential assembly of the glycolytic reaction sequence according to biochemical constraints: redox carriers, feasible mechanisms and energy conservation.
Figure 5: Proposed pathways for the production of 3-hydroxypropionate from pyruvate.

References

  1. 1

    Barnett, J.A. A history of research on yeasts 5: the fermentation pathway. Yeast 20, 509–543 (2003).

  2. 2

    Ray, L.B. Metabolism is not boring. Science 330, 1337 (2010).

  3. 3

    Reaves, M.L. & Rabinowitz, J.D. Metabolomics in systems microbiology. Curr. Opin. Biotechnol. 22, 17–25 (2011).

  4. 4

    Vander Heiden, M.G. Targeting cancer metabolism: a therapeutic window opens. Nat. Rev. Drug Discov. 10, 671–684 (2011).

  5. 5

    Klitgord, N. & Segre, D. Ecosystems biology of microbial metabolism. Curr. Opin. Biotechnol. 22, 541–546 (2011).

  6. 6

    Heinemann, M. & Sauer, U. Systems biology of microbial metabolism. Curr. Opin. Microbiol. 13, 337–343 (2010).

  7. 7

    Molenaar, D., van Berlo, R., de Ridder, D. & Teusink, B. Shifts in growth strategies reflect tradeoffs in cellular economics. Mol. Syst. Biol. 5, 323 (2009).

  8. 8

    Jones, R.G. & Thompson, C.B. Tumor suppressors and cell metabolism: a recipe for cancer growth. Genes Dev. 23, 537–548 (2009).

  9. 9

    Hsu, P.P. & Sabatini, D.M. Cancer cell metabolism: Warburg and beyond. Cell 134, 703–707 (2008).

  10. 10

    Kroemer, G. & Pouyssegur, J. Tumor cell metabolism: cancer's Achilles' heel. Cancer Cell 13, 472–482 (2008).

  11. 11

    Folger, O. et al. Predicting selective drug targets in cancer through metabolic networks. Mol. Syst. Biol. 7, 501 (2011).

  12. 12

    McInerney, M.J., Sieber, J.R. & Gunsalus, R.P. Syntrophy in anaerobic global carbon cycles. Curr. Opin. Biotechnol. 20, 623–632 (2009).

  13. 13

    Dolfing, J., Jiang, B., Henstra, A.M., Stams, A.J. & Plugge, C.M. Syntrophic growth on formate: a new microbial niche in anoxic environments. Appl. Environ. Microbiol. 74, 6126–6131 (2008).

  14. 14

    Lin, L.H. et al. Long-term sustainability of a high-energy, low-diversity crustal biome. Science 314, 479–482 (2006).

  15. 15

    Yim, H. et al. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat. Chem. Biol. 7, 445–452 (2011).

  16. 16

    Dellomonaco, C., Clomburg, J.M., Miller, E.N. & Gonzalez, R. Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355–359 (2011).

  17. 17

    Bond-Watts, B.B., Bellerose, R.J. & Chang, M.C. Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat. Chem. Biol. 7, 222–227 (2011).

  18. 18

    Shen, C.R. et al. Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl. Environ. Microbiol. 77, 2905–2915 (2011).

  19. 19

    Romano, A.H. & Conway, T. Evolution of carbohydrate metabolic pathways. Res. Microbiol. 147, 448–455 (1996).

  20. 20

    Fothergill-Gilmore, L.A. The evolution of the glycolytic pathway. Trends Biochem. Sci. 11, 47–51 (1986).

  21. 21

    Fothergill-Gilmore, L.A. & Michels, P.A. Evolution of glycolysis. Prog. Biophys. Mol. Biol. 59, 105–235 (1993).

  22. 22

    Heinrich, R., Montero, F., Klipp, E., Waddell, T.G. & Melendez-Hevia, E. Theoretical approaches to the evolutionary optimization of glycolysis: thermodynamic and kinetic constraints. Eur. J. Biochem. 243, 191–201 (1997).

  23. 23

    Sel'kov, E.E. Self-oscillations in glycolysis. 1. A simple kinetic model. Eur. J. Biochem. 4, 79–86 (1968).

  24. 24

    Hynne, F., Dano, S. & Sorensen, P.G. Full-scale model of glycolysis in Saccharomyces cerevisiae. Biophys. Chem. 94, 121–163 (2001).

  25. 25

    Stephani, A., Nuno, J.C. & Heinrich, R. Optimal stoichiometric designs of ATP-producing systems as determined by an evolutionary algorithm. J. Theor. Biol. 199, 45–61 (1999).

  26. 26

    Teusink, B. et al. Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry. Eur. J. Biochem. 267, 5313–5329 (2000).

  27. 27

    Meléndez-Hevia, E., Waddell, T.G., Heinrich, R. & Montero, F. Theoretical approaches to the evolutionary optimization of glycolysis—chemical analysis. Eur. J. Biochem. 244, 527–543 (1997).

  28. 28

    Noor, E., Eden, E., Milo, R. & Alon, U. Central carbon metabolism as a minimal biochemical walk between precursors for biomass and energy. Mol. Cell 39, 809–820 (2010).

  29. 29

    Weber, A.L. Energy from redox disproportionation of sugar carbon drives biotic and abiotic synthesis. J. Mol. Evol. 44, 354–360 (1997).

  30. 30

    Weber, A.L. Chemical constraints governing the origin of metabolism: the thermodynamic landscape of carbon group transformations under mild aqueous conditions. Orig. Life Evol. Biosph. 32, 333–357 (2002).

  31. 31

    Guihard, G., Benedetti, H., Besnard, M. & Letellier, L. Phosphate efflux through the channels formed by colicins and phage T5 in Escherichia coli cells is responsible for the fall in cytoplasmic ATP. J. Biol. Chem. 268, 17775–17780 (1993).

  32. 32

    Peralta-Yahya, P.P. & Keasling, J.D. Advanced biofuel production in microbes. Biotechnol. J. 5, 147–162 (2010).

  33. 33

    Frey, P.A. & Hegeman, A.D. Enzymatic Reaction Mechanisms (Oxford University Press, 2007).

  34. 34

    Fersht, A. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. (W. H. Freeman, New York, 1998).

  35. 35

    Frey, P.A. Radical mechanisms of enzymatic catalysis. Annu. Rev. Biochem. 70, 121–148 (2001).

  36. 36

    Buckel, W. & Golding, B.T. Radical enzymes in anaerobes. Annu. Rev. Microbiol. 60, 27–49 (2006).

  37. 37

    Feierberg, I. & Aqvist, J. Computational modeling of enzymatic keto-enol isomerization reactions. Theor. Chem. Acc. 108, 71–84 (2002).

  38. 38

    Mills, S.G. & Beak, P. Solvent effects on keto-enol equilibria: tests of quantitative models. J. Org. Chem. 50, 1216–1224 (1985).

  39. 39

    D'Ari, R. & Casadesus, J. Underground metabolism. Bioessays 20, 181–186 (1998).

  40. 40

    Aoshima, M. & Igarashi, Y. A novel oxalosuccinate-forming enzyme involved in the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6. Mol. Microbiol. 62, 748–759 (2006).

  41. 41

    Bar-Even, A., Noor, E., Flamholz, A., Buescher, J.M. & Milo, R. Hydrophobicity and charge shape cellular metabolite concentrations. PLoS Comput. Biol. 7, e1002166 (2011).

  42. 42

    Bar-Even, A. et al. The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters. Biochemistry 50, 4402–4410 (2011).

  43. 43

    Kerfeld, C.A., Heinhorst, S. & Cannon, G.C. Bacterial microcompartments. Annu. Rev. Microbiol. 64, 391–408 (2010).

  44. 44

    Ovádi, J. & Srere, P.A. Macromolecular compartmentation and channeling. Int. Rev. Cytol. 192, 255–280 (2000).

  45. 45

    Huang, X., Holden, H.M. & Raushel, F.M. Channeling of substrates and intermediates in enzyme-catalyzed reactions. Annu. Rev. Biochem. 70, 149–180 (2001).

  46. 46

    Marinoni, I. et al. Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis. FEBS J. 275, 5090–5107 (2008).

  47. 47

    O'Brien, P.J., Siraki, A.G. & Shangari, N. Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health. Crit. Rev. Toxicol. 35, 609–662 (2005).

  48. 48

    Ukeda, H., Hasegawa, Y., Ishi, T. & Sawamura, M. Inactivation of Cu,Zn-superoxide dismutase by intermediates of Maillard reaction and glycolytic pathway and some sugars. Biosci. Biotechnol. Biochem. 61, 2039–2042 (1997).

  49. 49

    Brownlee, M., Vlassara, H. & Cerami, A. Nonenzymatic glycosylation and the pathogenesis of diabetic complications. Ann. Intern. Med. 101, 527–537 (1984).

  50. 50

    Thornalley, P.J., Langborg, A. & Minhas, H.S. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose. Biochem. J. 344, 109–116 (1999).

  51. 51

    Kalapos, M.P. Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications. Toxicol. Lett. 110, 145–175 (1999).

  52. 52

    Marnett, L.J. Lipid peroxidation-DNA damage by malondialdehyde. Mutat. Res. 424, 83–95 (1999).

  53. 53

    Marnett, L.J. Oxy radicals, lipid peroxidation and DNA damage. Toxicology 181182, 219–222 (2002).

  54. 54

    Marnett, L.J. et al. Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104. Mutat. Res. 148, 25–34 (1985).

  55. 55

    Kalapos, M.P. The tandem of free radicals and methylglyoxal. Chem. Biol. Interact. 171, 251–271 (2008).

  56. 56

    Weber, A.L. Kinetics of organic transformations under mild aqueous conditions: implications for the origin of life and its metabolism. Orig. Life Evol. Biosph. 34, 473–495 (2004).

  57. 57

    Banzon, J.A. et al. Mechanism-based inactivation of phosphotriesterase by reaction of a critical histidine with a ketene intermediate. Biochemistry 34, 743–749 (1995).

  58. 58

    Foroozesh, M. et al. Aryl acetylenes as mechanism-based inhibitors of cytochrome P450–dependent monooxygenase enzymes. Chem. Res. Toxicol. 10, 91–102 (1997).

  59. 59

    Schräder, T. & Andreesen, J.R. Purification and characterization of protein PC, a component of glycine reductase from Eubacterium acidaminophilum. Eur. J. Biochem. 206, 79–85 (1992).

  60. 60

    Andreesen, J.R. Glycine reductase mechanism. Curr. Opin. Chem. Biol. 8, 454–461 (2004).

  61. 61

    Buckel, W. Unusual dehydrations in anaerobic bacteria: considering ketyls (radical anions) as reactive intermediates in enzymatic reactions. FEBS Lett. 389, 20–24 (1996).

  62. 62

    Kim, J., Hetzel, M., Boiangiu, C.D. & Buckel, W. Dehydration of (R)-2-hydroxyacyl-CoA to enoyl-CoA in the fermentation of alpha-amino acids by anaerobic bacteria. FEMS Microbiol. Rev. 28, 455–468 (2004).

  63. 63

    Bennett, B.D. et al. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 5, 593–599 (2009).

  64. 64

    Qian, H., Beard, D.A. & Liang, S.D. Stoichiometric network theory for nonequilibrium biochemical systems. Eur. J. Biochem. 270, 415–421 (2003).

  65. 65

    Beard, D.A. & Qian, H. Relationship between thermodynamic driving force and one-way fluxes in reversible processes. PLoS ONE 2, e144 (2007).

  66. 66

    Flamholz, A., Noor, E., Bar-Even, A. & Milo, R. eQuilibrator—the biochemical thermodynamics calculator. Nucleic Acids Res. 40, D770–D775 (2012).

  67. 67

    Meléndez-Hevia, E. & Isidoro, A. The game of the pentose phosphate cycle. J. Theor. Biol. 117, 251–263 (1985).

  68. 68

    Mittenthal, J.E., Clarke, B., Waddell, T.G. & Fawcett, G. A new method for assembling metabolic networks, with application to the Krebs citric acid cycle. J. Theor. Biol. 208, 361–382 (2001).

  69. 69

    Machajewski, T.D. & Wong, C.H. The catalytic asymmetric aldol reaction. Angew. Chem. Int. Edn Engl. 39, 1352–1375 (2000).

  70. 70

    Fersht, A. Electrophilic catalysis by Schiff base formation in Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding 77–79 (W. H. Freeman, 1999).

  71. 71

    Palm, K., Luthman, K., Ros, J., Grasjo, J. & Artursson, P. Effect of molecular charge on intestinal epithelial drug transport: pH-dependent transport of cationic drugs. J. Pharmacol. Exp. Ther. 291, 435–443 (1999).

  72. 72

    Chakrabarti, A.C. & Deamer, D.W. Permeability of lipid bilayers to amino acids and phosphate. Biochim. Biophys. Acta 1111, 171–177 (1992).

  73. 73

    Chakrabarti, A.C. & Deamer, D.W. Permeation of membranes by the neutral form of amino acids and peptides: relevance to the origin of peptide translocation. J. Mol. Evol. 39, 1–5 (1994).

  74. 74

    Finkelstein, A. Water and nonelectrolyte permeability of lipid bilayer membranes. J. Gen. Physiol. 68, 127–135 (1976).

  75. 75

    Winiwarter, S. et al. Correlation of human jejunal permeability (in vivo) of drugs with experimentally and theoretically derived parameters. A multivariate data analysis approach. J. Med. Chem. 41, 4939–4949 (1998).

  76. 76

    Davis, B.D. On the importance of being ionized. Arch. Biochem. Biophys. 78, 497–509 (1958).

  77. 77

    Westheimer, F.H. Why nature chose phosphates. Science 235, 1173–1178 (1987).

  78. 78

    Walter, A. & Gutknecht, J. Permeability of small nonelectrolytes through lipid bilayer membranes. J. Membr. Biol. 90, 207–217 (1986).

  79. 79

    Paula, S., Volkov, A.G., Van Hoek, A.N., Haines, T.H. & Deamer, D.W. Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness. Biophys. J. 70, 339–348 (1996).

  80. 80

    Hassett, A., Blattler, W. & Knowles, J.R. Pyruvate kinase: is the mechanism of phospho transfer associative or dissociative? Biochemistry 21, 6335–6340 (1982).

  81. 81

    Pharkya, P., Nikolaev, E.V. & Maranas, C.D. Review of the BRENDA Database. Metab. Eng. 5, 71–73 (2003).

  82. 82

    Chang, A., Scheer, M., Grote, A., Schomburg, I. & Schomburg, D. BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009. Nucleic Acids Res. 37, D588–D592 (2009).

  83. 83

    Holliday, G.L. et al. MACiE: exploring the diversity of biochemical reactions. Nucleic Acids Res. 40, D783–D789 (2012).

  84. 84

    Schink, B. Energetics of syntrophic cooperation in methanogenic degradation. Microbiol. Mol. Biol. Rev. 61, 262–280 (1997).

  85. 85

    McInerney, M.J. et al. Physiology, ecology, phylogeny, and genomics of microorganisms capable of syntrophic metabolism. Ann. NY Acad. Sci. 1125, 58–72 (2008).

  86. 86

    Bagramyan, K. & Trchounian, A. Structural and functional features of formate hydrogen lyase, an enzyme of mixed-acid fermentation from Escherichia coli. Biochemistry (Mosc.) 68, 1159–1170 (2003).

  87. 87

    Garrigues, C., Loubiere, P., Lindley, N.D. & Cocaign-Bousquet, M. Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio. J. Bacteriol. 179, 5282–5287 (1997).

  88. 88

    Fuhrer, T., Fischer, E. & Sauer, U. Experimental identification and quantification of glucose metabolism in seven bacterial species. J. Bacteriol. 187, 1581–1590 (2005).

  89. 89

    Conway, T. The Entner-Doudoroff pathway: history, physiology and molecular biology. FEMS Microbiol. Rev. 9, 1–27 (1992).

  90. 90

    Noguchi, K. & Yoshida, K. Interaction between photosynthesis and respiration in illuminated leaves. Mitochondrion 8, 87–99 (2008).

  91. 91

    Bustos, D.M., Bustamante, C.A. & Iglesias, A.A. Involvement of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in response to oxidative stress. J. Plant Physiol. 165, 456–461 (2008).

  92. 92

    Zhang, X., Harrison, D.H. & Cui, Q. Functional specificities of methylglyoxal synthase and triosephosphate isomerase: a combined QM/MM analysis. J. Am. Chem. Soc. 124, 14871–14878 (2002).

  93. 93

    Hopper, D.J. & Cooper, R.A. The regulation of Escherichia coli methylglyoxal synthase; a new control site in glycolysis? FEBS Lett. 13, 213–216 (1971).

  94. 94

    Ahmed, H. et al. The semi-phosphorylative Entner-Doudoroff pathway in hyperthermophilic archaea: a re-evaluation. Biochem. J. 390, 529–540 (2005).

  95. 95

    Berg, I.A. et al. Autotrophic carbon fixation in archaea. Nat. Rev. Microbiol. 8, 447–460 (2010).

  96. 96

    Jiang, X., Meng, X. & Xian, M. Biosynthetic pathways for 3-hydroxypropionic acid production. Appl. Microbiol. Biotechnol. 82, 995–1003 (2009).

  97. 97

    Sato, K., Nishina, Y., Setoyama, C., Miura, R. & Shiga, K. Unusually high standard redox potential of acrylyl-CoA/propionyl-CoA couple among enoyl-CoA/acyl-CoA couples: a reason for the distinct metabolic pathway of propionyl-CoA from longer acyl-CoAs. J. Biochem. 126, 668–675 (1999).

  98. 98

    Frey, P.A. The role of radicals in enzymatic processes. Chem. Rec. 1, 277–289 (2001).

Download references

Acknowledgements

We thank D. Tawfik for helpful discussions, scientific support and critique regarding the manuscript. We also would like to thank D. Arlow, R. Burton, D. Fraenkel, R. Last, W. Liebermeister, A. Weber and members of the Milo laboratory for helpful comments. A.B.-E. is supported by the Adams Fellowship Program of the Israel Academy of Sciences and Humanities. E.N. is grateful to the Azrieli Foundation for the award of an Azrieli Fellowship. This study was supported by the European Research Council (grant 260392–SYMPAC) and by the Israel Science Foundation (Grant 750/09).

Author information

Correspondence to Ron Milo.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bar-Even, A., Flamholz, A., Noor, E. et al. Rethinking glycolysis: on the biochemical logic of metabolic pathways. Nat Chem Biol 8, 509–517 (2012) doi:10.1038/nchembio.971

Download citation

Further reading