Glycolytic Enzymes

Paul AM Michels, de Duve Institute, Brussels, Belgium
Christophe LMJ Verlinde, University of Washington, Seattle, Washington, USA
Linda A Fothergill‐Gilmore, University of Edinburgh, Scotland, UK

Published online: March 2010

DOI: 10.1002/9780470015902.a0000621.pub2

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. 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.

Key concepts:

  • Glycolysis is an ancient pathway that is present at least in part in all organisms.
  • Ten enzymes catalyse the reactions of the glycolytic pathway.
  • 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-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase).
  • Phosphofructokinase occurs as two distantly related types that use different phospho donors: ATP or inorganic pyrophosphate.
  • Activities of phosphofructokinase and pyruvate kinase are allosterically regulated.
  • A conserved protein fold of eight parallel -strands and eight parallel 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.
  • Pyruvate kinase activity is sometimes regulated by posttranslational phosphorylation.

Keywords: allosteric regulation; 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; TIM, triosephosphate isomerase. The letter ‘P’ in the chemical structures represents a phospho group.
Figure 2. The reaction catalysed by hexokinase requires major domain movement to open and close the cleft between the domains. (a) Space-filling representations of yeast hexokinase in an open/unligated representation on the left, and in the closed conformation with glucose (yellow) deep within the binding pocket on the right. (b) Schematic view of the phosphorylation of glucose by hexokinase, highlighting the role of ATP, Mg2+, the pentavalent transition state and the inversion of configuration. Ado, adenosine and encircled P represents a phospho group. Figure 2a courtesy of Dr Daniel Rigden, University of Liverpool.
Figure 3. The chimeric ATP-dependent phosphofructokinase from Trypanosoma brucei is an allosterically regulated homo tetrameric 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 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, 1990). Courtesy of Dr Iain McNae, University of Edinburgh.
Figure 4. Glyceraldehyde-3-phosphate dehydrogenase is a homo tetrameric 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. (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 4a courtesy of Dr Daniel Rigden, University of Liverpool.
Figure 5. A cartoon representation of the tetrameric structure of Leishmania mexicana pyruvate kinase shows the positions of bound substrates and effector (in space-filling representation). The foreground is a single subunit consisting of four domains, with the remainder of the tetramer faded behind. The active site is shared between domains A (blue) and B (yellow), and the effector site is contained within domain C (green). The N-terminal domain is coloured red. Domain A is folded into a barrel of eight parallel -strands surrounded by eight helices in the same fundamental topology as triosephosphate isomerase. The allosteric transition of pyruvate kinase involves rotation of the A- and C-domain cores of each of the four subunits. Note that the effector for L. mexicana pyruvate kinase is fructose 2,6-bisphosphate instead of the more usual fructose 1,6-bisphosphate. Courtesy of Dr Hugh Morgan, University of Edinburgh.
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 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.
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    book 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.
    ePath http://www.wwpdb.org/ An Information Portal to Biological Macromolecular Structures (including all glycolytic enzymes).
    book Price NC and Nairn J (2009) Exploring Proteins. Oxford: Oxford University Press.
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    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.

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Enzymes: Structure and Action Mechanism | Bioenergetics
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Article Title: Glycolytic Enzymes
 
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Michels, Paul AM, Verlinde, Christophe LMJ, and Fothergill‐Gilmore, Linda A(Mar 2010) Glycolytic Enzymes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000621.pub2]