Glycolytic Enzymes


All cells must burn fuels to drive the myriad of cellular processes necessary for life. The most important organic fuel is glucose, a stable and soluble sugar that is particularly well suited for its role in biology. Cellular combustion of glucose occurs in 10 well‐controlled steps in which six‐carbon glucose molecules are broken apart (literally ‘glycolysis’) into three‐carbon compounds. In the same process, chemical energy is captured through the production of ATP (adenosine triphosphate), the hydrolysis of which powers many cellular processes. The 10 enzymes which catalyse the steps of glycolysis are exceptionally well characterised with regard to structure, catalytic mechanism and activity regulation. They provide a fascinating array of enzymes that have been perfected over long evolution to carry out their tasks swiftly, efficiently and with finely tuned control. Glycolytic enzymes are recognised as promising targets in health and disease.

Key Concepts

  • Glycolysis is an ancient pathway comprising 10 enzymes that are present at least in part in all organisms.
  • Many enzymes occur as isoenzymes in different tissues or in response to different metabolic conditions.
  • Hexokinase activity is regulated by product inhibition.
  • A hinge‐bending motion of the two‐lobed structures of hexokinase and phosphoglycerate kinase is induced by substrate binding and is required to position the substrates correctly for catalysis.
  • Several reactions of the glycolytic pathway can be catalysed by pairs of nonhomologous enzymes (i.e. glucose‐6‐phosphate isomerase, aldolase, glyceraldehyde‐3‐phosphate dehydrogenase and phosphoglycerate mutase).
  • Phosphofructokinase occurs as two distantly related types that use different phosphodonors: ATP or inorganic pyrophosphate.
  • Activities of phosphofructokinase and pyruvate kinase are allosterically regulated.
  • A conserved protein fold of eight parallel β‐strands and eight parallel α helices, first identified in triosephosphate isomerase and therefore known as the ‘TIM barrel’, is found in several glycolytic enzymes and occurs in approximately 10% of all enzymes.
  • The Rossmann fold that occurs in many nucleotide‐binding proteins was first identified in glyceraldehyde‐3‐phosphate dehydrogenase.
  • Activities of glycolytic enzymes are sometimes regulated by posttranslational modification.

Keywords: allosteric regulation; drug discovery; enzyme evolution; hinge‐bending motion; isoenzymes; Rossmann fold; substrate‐level phosphorylation; TIM barrel

Figure 1. (a) Glycolysis and (b) the glycerol phosphate shuttle. The abbreviations are as follows: ALD, aldolase; DHAP, dihydroxyacetone phosphate; ENO, enolase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GPDH, glycerol‐3‐phosphate dehydrogenase; Gly 3‐P, glycerol 3‐phosphate; HK, hexokinase; LDH, lactate dehydrogenase; PFK, phosphofructokinase; PGAM, phosphoglycerate mutase; PGI, glucose‐6‐phosphate isomerase: PGK, phosphoglycerate kinase; PYK, pyruvate kinase and TIM, triosephosphate isomerase. The letter ‘P’ in the chemical structures represents a phospho group.
Figure 2. The chimeric ATP (adenosine triphosphate)‐dependent phosphofructokinase from Trypanosoma brucei is an allosterically regulated homotetrameric enzyme. A cartoon representation shows a single subunit consisting of three domains in the foreground, with the remainder of the tetramer faded behind. ATP molecules at the active sites are shown in stick representation. Domains B and C (except the inserted loop) are characteristic of ATP‐dependent phosphofructokinases, whereas Domain A and a large, mostly α helical insertion (not shown) in the position of the inserted loop pertain to PPi‐dependent phosphofructokinases. The enzymes from protists such as T. brucei possess a unique inserted loop that forms an important part of the active site and an embracing arm as well as a C‐terminal helix that help to stabilise the quaternary structure. Comparison of the ligated structure with that of the unligated apoenzyme shows that the allosteric transition is characterised by an opening of the active sites to accommodate the substrates (primarily by the lifting of the inserted loops), together with a dramatic ordering of the C‐terminal helices which stabilise the tetramer in an active conformation. This mechanism is fundamentally different from that of bacterial phosphofructokinases. Those enzymes are arranged as dimers of dimers that rotate approximately 7° with respect to each other during the allosteric transition and thereby increase the affinity for ATP and fructose 6‐phosphate by replacing an unfavourable glutamic acid side chain at the active site by a favourable arginine side chain (Schirmer and Evans, ). Courtesy of Dr Iain McNae, The University of Edinburgh.
Figure 3. Glyceraldehyde‐3‐phosphate dehydrogenase is a homotetrameric enzyme that requires the participation of the cofactor NAD+. (a) Cartoon representation of one subunit of human glyceraldehyde‐3‐phosphate dehydrogenase with bound NAD+ in stick representation. The two Rossmann folds in the cofactor‐binding domain are shown in two shades of purple, with the remainder of the domain in green. The catalytic domain is shown in blue. Courtesy of Dr Daniel Rigden, University of Liverpool. (b) In the first step of the reaction pathway, a hemithioacetal is formed between the substrate glyceraldehyde 3‐phosphate and the sulfhydryl group of the cysteine residue at the active site. This leads to the conversion of the carbonyl group into an alcohol, which is oxidised by NAD+ (2). The energy‐rich thioester thus formed reacts with orthophosphate to produce 1,3‐bisphosphoglycerate (4). The acyl transfer in step 4 is very slow unless NAD+ is bound to the enzyme. The replacement of NADH by NAD+ (3) is therefore an essential step in the reaction sequence. R1 and R2 symbolise the rest of the substrate and NAD+ molecules, respectively.
Figure 4. Cofactor‐independent phosphoglycerate mutase undergoes large domain movements during catalysis. (a) The phosphatase domains (green) of the apoenzyme from Leishmania mexicana and the substrate‐bound enzyme from T. brucei were superposed and shown to have nearly identical conformations. However, a comparison of the transferase domains (with coloured helices) shows that the open conformation of the T. brucei enzyme (increased transparency) is very different from the closed conformation of the L. mexicana enzyme. This can be most easily seen by the faded red helix of the open conformation top right. The dashed red arrow indicates the movement of this helix to its position in the closed conformation. Rotation of the enzymes through 90° is shown in the right of panel (a). It can be seen that closure of the domains involves a rotation of 67° and translation of the centre of gyration (from grey to black dots). The substrate and metal ion (represented as spheres) are shown bound to the active site, which forms when both the transferase and phosphatase domains meet. (b) The schematic representation of these movements shows that the active site is exposed in the open form, allowing for substrate binding and product release. A rigid body rotation of the transferase domain closes the active site, allowing for the interconversion of 3‐ and 2‐phosphoglycerates. Reproduced from Blackburn et al. 2014 © Elsevier.
Figure 5. Human pyruvate kinase. (a) A cartoon representation of the homotetrameric structure of human pyruvate kinase shows the positions of bound substrates and the effectors fructose 1,6‐bisphosphate (FBP) and serine (in space‐filling representation). Domains of one subunit are coloured [N‐terminal domain (red, residues 14–43); A‐domain (blue, residues 25–116 and 220–402); B‐domain (yellow, residues 117–219) and C‐domain (green, residues 403–531)]. The other three subunits are shown in grey. The cartoon is mainly based on structures of human pyruvate kinase M2 (PDB code: 4B2D and 4FXF) (Chaneton et al., and Morgan et al., ). (b) The regulation of human pyruvate kinase by metabolites. The blue solid arrow shows the transformation of pyruvate kinase between active tetramer (green) and inactive tetramer (orange). Green dotted arrows show that FBP and serine stabilise pyruvate kinase in the active form and thereby activate its enzymic activity. In contrast, phenylalanine, tryptophan and alanine inhibit its enzymic activity by stabilising it in an inactive form (shown with orange dotted arrows). Autophagic alanine secretion from neighbouring cells (Sousa et al., ) is shown in purple. Courtesy of Dr Meng Yuan, Scripps Institute, USA.


Albery WJ and Knowles JR (1976) Evolution of enzyme function and the development of catalytic efficiency. Biochemistry 15: 5631–5640.

Anastasiou D, Yu Y, Israelsen WJ, et al. (2012) Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nature Chemical Biology 10: 839–847.

Bakker BM, Overkamp KM, van Maris AJ, et al. (2001) Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiology Reviews 25: 15–37.

Banner DW, Bloomer AC, Petsko GA, et al. (1975) Structure of chicken muscle triose phosphate isomerase determined crystallographically at 2.5 angstrom resolution using amino acid sequence data. Nature 255: 609–614.

Bernstein BE, Michels PAM and Hol WGJ (1997) Synergistic effects of substrate induced conformational changes in phosphoglycerate kinase activation. Nature 385: 275–278.

Berrisford JM, Hounslow AM, Akerboom J, et al. (2006) Evidence supporting a cis‐enediol‐based mechanism for Pyrococcus furiosus phosphoglucose isomerase. Journal of Molecular Biology 358: 1353–1366.

Blackburn EA, Fuad FAA, Morgan HP, et al. (2014) Trypanosomatid phosphoglycerate mutases have multiple conformational and oligomeric states. Biochemical and Biophysical Research Communications 450: 936–941.

Brimacombe KR, Walsh MJ, Liu L, et al. (2013) Identification of ML251, a potent inhibitor of Trypanosoma brucei and T. cruzi phosphofructokinase. ACS Medicinal Chemistry Letters 5: 12–17.

Cardenas ML, Cornish‐Bowden A and Ureta T (1998) Evolution and regulatory role of the hexokinases. Biochimica et Biophysica Acta 1401: 242–264.

Chaneton B, Hillmann P, Zheng L, et al. (2012) Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature 491: 458–462.

Choi KH, Shi J, Hopkins CE, et al. (2001) Snapshots of catalysis: the structure of fructose‐1,6‐(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate. Biochemistry 40: 13868–13875.

Conejo MS, Thompson S and Miller BG (2010) Evolutionary bases of carbohydrate recognition and substrate discrimination in the ROK protein family. Journal of Molecular Evolution 70: 545–556.

Cristescu ME and Egbosimba EE (2009) Evolutionary history of d‐lactate dehydrogenases: a phylogenomic perspective on functional diversity in the FAD binding oxidoreductase/transferase type 4 family. Journal of Molecular Evolution 69: 276–287.

Fothergill‐Gilmore LA and Michels PAM (1993) Evolution of glycolysis. Progress in Biophysics and Molecular Biology 59: 1055–1235.

Gómez‐Arreaza A, Acosta H, Quiñones W, et al. (2014) Extracellular functions of glycolytic enzymes of parasites: unpredicted use of ancient proteins. Molecular and Biochemical Parasitology 193: 75–81.

Hall ER and Cottam GL (1978) Isozymes of pyruvate kinase in vertebrates: their physical, chemical, kinetic and immunological properties. International Journal of Biochemistry 9: 785–793.

Hol WGJ, Halie LM and Sander C (1981) Dipoles of the alpha‐helix and beta‐sheet: their role in protein folding. Nature 294: 532–536.

João HC and Williams RJP (1993) The anatomy of a kinase and the control of phospho transfer. European Journal of Biochemistry 216: 1–18.

Johnson LN and Barford D (1990) Glycogen phosphorylase. Journal of Biological Chemistry 265: 2409–2412.

Kawai S, Mukai T, Mori S, et al. (2005) Hypothesis: structures, evolution, and ancestor of glucose kinases in the hexokinase family. Journal of Bioscience and Bioengineering 99: 320–330.

Kim JW and Dang CV (2005) Multifaceted roles of glycolytic enzymes. Trends in Biochemical Sciences 30: 142–150.

Liu Y, Ray WJ and Baranidharan S (1997) Structure of rabbit muscle phosphoglucomutase refined at 2.4 Å resolution. Acta Crystallographica Section D 53: 392–405.

Madern D (2002) Molecular evolution within the l‐malate and l‐lactate dehydrogenase super‐family. Journal of Molecular Evolution 54: 825–840.

Marsh JJ and Lebherz HG (1992) Fructose‐bisphosphate aldolases: an evolutionary history. Trends in Biochemical Sciences 17: 110–113.

McNae IW, Martinez‐Oyanedel J, Keillor JW, et al. (2009) The crystal structure of ATP‐bound phosphofructokinase from Trypanosoma brucei reveals conformational transitions different from those of other phosphofructokinases. Journal of Molecular Biology 385: 1519–1533.

Monod J, Wyman J and Changeux JP (1965) On the nature of allosteric transitions: a plausible model. Journal of Molecular Biology 12: 88–118.

Moras D, Olsen KW, Sabesan MN, et al. (1975) Studies of asymmetry in the three‐dimensional structure of lobster d‐glyceraldehyde‐3‐phosphate dehydrogenase. Journal of Biological Chemistry 250: 9137–9162.

Moreno F and Herrero P (2002) The hexokinase 2‐dependent glucose signal transduction pathway of Saccharomyces cerevisiae. FEMS Microbiology Reviews 26: 83–90.

Morgan HP, McNae IW, Nowicki MW, et al. (2010) Allosteric mechanism of pyruvate kinase from Leishmania mexicana uses a rock and lock model. Journal of Biological Chemistry 285: 12892–12898.

Morgan HP, O'Reilly FJ, Wear MA, et al. (2013) M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation. Proceedings of the National Academy of Sciences of the United States of America 110: 5881–5886.

Morgan HP, Zhong W, McNae IW, et al. (2014) Structures of pyruvate kinases display evolutionarily divergent allosteric strategies. Royal Society Open Science 1: 140120.

Muirhead H and Watson H (1992) Glycolytic enzymes: from hexose to pyruvate. Current Opinion in Structural Biology 2: 870–876.

Nakamura T, Kashima Y, Mine S, et al. (2012) Characterization and crystal structure of the thermophilic ROK hexokinase from Thermus thermophilus. Journal of Bioscience and Bioengineering 114: 150–154.

de A S Navarro MV, Gomes Dias SM, Mello LV, et al. (2007) Structural flexibility in Trypanosoma brucei enolase revealed by X‐ray crystallography and molecular dynamics. FEBS Journal 274: 5077–5089.

Nowicki M, Kuaprasert B, McNae IW, et al. (2009) Crystal structures of Leishmania mexicana phosphoglycerate mutase suggest a one‐metal mechanism and a new enzyme subclass. Journal of Molecular Biology 394: 535–543.

Nukui M, Mello LV, Littlejohn JE, et al. (2007) Structure and molecular mechanism of Bacillus anthracis cofactor‐independent phosphoglycerate mutase: a crucial enzyme for spores and growing cells of Bacillus species. Biophysical Journal 92: 977–988.

Read JA, Winter VJ, Eszes CM, et al. (2001) Structural basis for altered activity of M‐ and H‐isoenzyme forms of human lactate dehydrogenase. Proteins 43: 175–185.

Rigden DJ (2008) The histidine phosphatase superfamily: structure and function. Biochemical Journal 409: 333–348.

Schirmer T and Evans PR (1990) Structural basis of the allosteric behaviour of phosphofructokinase. Nature 343: 140–145.

Schöneberg T, Kloos M, Brüser A, et al. (2013) Structure and allosteric regulation of eukaryotic 6‐phosphofructokinases. Biological Chemistry 394: 977–993.

Skarzynski T, Moody PCE and Wonacott AJ (1987) Structure of holo‐glyceraldehyde‐3‐phosphate dehydrogenase from Bacillus stearothermophilus at 1.8 Å resolution. Journal of Molecular Biology 193: 171–187.

Solomons JTG, Zimmerly EM, Burns S, et al. (2004) The crystal structure of mouse phosphoglucose isomerase at 1.6 Å resolution and its complex with glucose 6‐phosphate reveals the catalytic mechanism of sugar ring opening. Journal of Molecular Biology 342: 847–860.

Sousa CM, Biancur DE, Wang X, et al. (2016) Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature 536: 479–483.

Wigley DB, Gamblin SJ, Turkenburg JP, et al. (1992) Structure of a ternary complex of an allosteric lactate dehydrogenase from Bacillus stearothermophilus at 2.5 Å resolution. Journal of Molecular Biology 223: 317–335.

Winter VJ, Cameron A, Tranter R, et al. (2003) Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus. Molecular & Biochemical Parasitology 131: 1–10.

Yuan M (2016) Allosteric regulation of human pyruvate kinase M2. FEBS Journal 282 (Suppl. 1, abstract P35‐010): 326.

Zhu L, Xu X, Wang L, et al. (2015a) The D‐lactate dehydrogenase from Sporolactobacillus inulinus also possessing reversible deamination activity. PLoS One 10 (9): e0139066.

Zhu L, Xu X, Wang L, et al. (2015b) NADP+‐preferring D‐lactate dehydrogenase from Sporolactobacillus inulinus. Applied and Environmental Microbiology 81: 6294–6301.

Further Reading

Bakker BM, Westerhoff HV, Opperdoes FR and Michels PAM (2000) Metabolic control analysis of glycolysis in trypanosomes as an approach to improve selectivity and effectiveness of drugs. Molecular and Biochemical Parasitology 106: 1–10.

Branden C and Tooze J (1999) Introduction to Protein Structure, 2nd edn. New York: Garland Publishing.

Cornish‐Bowden A (1995) Fundamentals of Enzyme Kinetics. London: Portland Press Ltd.

Creighton TE (1993) Proteins, Structures and Molecular Properties, 2nd edn. New York: WH Freeman and Company.

Erlandsen H, Abola EE and Stevens RC (2000) Combining structural genomics and enzymology: completing the picture in metabolic pathways and enzyme active sites. Current Opinion in Structural Biology 10: 719–730.

Fell D (1997) Understanding the Control of Metabolism, Frontiers in Metabolism 2. London: Portland Press Ltd.

Gatenby RA and Gillies RJ (2007) Glycolysis in cancer: a potential target for therapy. International Journal of Biochemistry and Cell Biology 39: 1358–1366.

Price NC and Nairn J (2009) Exploring Proteins. Oxford, UK: Oxford University Press.

Scopes RK (1977) Multiple enzyme purifications from muscle extracts by using affinity‐elution‐chromatographic procedures. Biochemical Journal 161: 265–277.

Verhees CH, Kengen SW, Tuininga JE, et al. (2003) The unique features of glycolytic pathways in Archaea. Biochemical Journal 375: 231–246.

Verlinde CLMJ, Hannaert V, Blonski C, et al. (2001) Glycolysis as a target for the design of new anti‐trypanosome drugs. Drug Resistance Updates 4: 50–65.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Michels, Paul AM, and Fothergill‐Gilmore, Linda A(Jul 2017) Glycolytic Enzymes. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0000621.pub3]