1. Department of Advanced Fermentation Fusion Science & Technology
발효화학 (Fermentation Chemistry), Bacterial Physiology and Metabolism
Chapter 4. Glycolysis
Yong-Cheol Park (
Department of Advanced Fermentation
Fusion Science & Technology

2. Introduction
A bacterium, Escherichia coli can synthesize all cell constituents using glucose and mineral salts.
Glucose is metabolized to pyruvate through the Embden-Meyerhof-Parnas (EMP) pathway and hexose monophosphate (HMP) pathway.
Pyruvate is decarboxylated oxidatively to acetyl-CoA to be oxidized through the tricarboxylic acid (TCA) cycle.
Twelve intermediates of
these pathways are used
as carbon skeletons for
biosynthesis (Table 4.1).
Content

3. Introduction
Most organisms metabolize glucose though the EMP pathway to generate ATP, pyruvate and NADH, and the HMP pathway is needed to supply the metabolic intermediates not available from the EMP pathway such as pentose-5-phosphate and erythrose-4-phosphate, and NAPDH.
Some prokaryotes metabolize glucose through unique pathways known only in prokaryotes, e.g. the Entner-Doudoroff (ED) pathway and phosphoketolase (PK) pathway.
Some prokaryotes have gens for the ED pathway in addition to the EMP pathway.
Carbohydrates are phosphorylated before they are metabolized in most cases.
It is believed that phosphorylated intermediates are less likely to diffuse away through the cytoplasmic membrane.
Content

4. 4.1 EMP pathway
Many anaerobic and enteric bacteria transport glucose via the PTS in the form of glucose-6-phosphate.
Hexokinase
(1) Phosphorylates glucose which is delivered via active transport.
(2) Able to phosphorylate other hexoses such as mannose.
(3) Requires Mg2+ for activity.
(4) Cannot catalyze the reverse reaction.
Glucose-6-phosphate can also be obtained from glycogen.

5. 4.1 EMP pathway

6. 4.1 EMP pathway
4.1.1 Phosphofructokinase (PFK): key enzyme of the EMP pathway
PFK
(1) Phosphorylates fructose-6-phosphate to fructose-1,6-bisphophate.
(2) Requires Mg2+.
(3) Catalyzes an irreversible reaction, meaning PFK is an key enzyme in the EMP pathway.
(4) Its presence indicates that this organism may catabolize glucose through the EMP pathway.

7. 4.1 EMP pathway 4.1.2 ATP synthesis and production of pyruvate
Triose-phosphate isomerase (TPI)
(1) Equilibrates dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3- phosphate (GAP).
(2) Under standard conditions, the equilibrium shifts to the formation of DHAP (∆Go’=-7.7 kJ/mol GAP).
(3) But the reverse reaction is favored because GAP is continuously consumed in subsequent reactions.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(1) Oxidize GAP to 1,3-diphosphoglycerate (1,3-DPG)
(2) Produces 1 mole NADH and phosphorylates GAP with inorganic phosphate.
(3) Catalyze the endergonic reaction (∆Go’=+6.3 kJ/mol GAP)

8. 4.1 EMP pathway 4.1.2 ATP synthesis and production of pyruvate
3-Phosphoglycerate kinase (PGK)
(1) Catalyzes the exergonic reaction of ATP generation through substrate-level- phosphorylation
(2) Requires Mg2+.
Pyruvate kinase
(1) Catalyze ATP formation via irreversible dephosphorylation of PEP to pyruvate in the presence of Mg2+ and K+.
PTS
(1) PEP supplies energy in the PTS-mediated group translocation
In overall reactions,
(1) Four ATPs are synthesized and two high energy phosphate bonds are consumed in glycolysis, resulting in a net gain of two ATPs per glucose oxidized.
(2) The NADH reduced in the glycolysis is reoxidized in aerobic (Section 5.8) and anaerobic respiration (Chapter 9), and in fermentation (Chapter 8), reducing various electron acceptors depending on the organism and on their availability.

9. 4.1 EMP pathway 4.1.3 Modified EMP pathways
Some prokaryotes, unlike eukaryotes, metabolize glucose through modified EMP pathways depending on the growth conditions.
Methylglyoxal bypass
Under phosphate-limited conditions, Escherichia coli oxidize DHAP to pyruvate though methylglyoxal.
This diversion enables acetyl-CoA synthesis through pyruvate when GAPDH cannot function due to a low concentration of inorganic phosphate (Pi), one of its substrate, with a reduced ATP yield.
Methylglyoxal is very reactive, destroying nucleic acid and proteins, so methylglyoxal synthase activity is regulated tightly by Pi through feed-back inhibition, and is activated by its substrate, DHAP.

10. 4.1 EMP pathway 4.1.3.2 Modified EMP pathways in archaea
Most archaea utilizing carbohydrates employ modified ED pathways (Section ), but a few metabolize sugars in modified EMP pathways.
The halophilic archaeon Haloarcula vallismortis transprots fructose via an active transporter and a ketohexokinase phosphorylates the free sugar to fructose-1- phosphate.

11. 4.1 EMP pathway 4.1.3.2 Modified EMP pathways in archaea (continued)
Hyperthermophilic archaea such as Pyrococcus, Thermococcus, Desulfurococcus employ another modified EMP pathway.
Pyrococcus furiosus does not use glucose, but ferments starch (Figure 4.3).
ADP is more stable than ATP under high temperature conditions.
Ferredoxin as the electron acceptor, has a lower redox potential than NAD+.

12. 4.1 EMP pathway 4.1.4 Regulation of the EMP pathway
The EMP pathway is regulated by the energy status of the cell as well as by the concentration of certain metabolic intermediates.
A parameter, the adenylate energy charge (EC), was devised to describe the energy status in a culture (Section 5.6.2).
Generally,
(1) Low EC values  the glycolytic pathway is activated
(2) High EC values  the glycolytic pathway is repressed
The enzymes that do not catalyze the reverse reaction are regulated.
In the EMP pathway, these enzymes are phosphofructokinase (PFK) and pyruvate kinase. In organisms where glucose is transported by active transport, hexokinase is repressed by its product, glucose-6-phosphate.

13. 4.1 EMP pathway 4.1.4.1 Regulation of phosphofructokinase (PFK)
The EMP pathway is controlled mainly by regulating PFK activity.
In bacteria, PFK is activated by ADP and GDP, and repressed by PEP.
In yeast, PFK is activated by AMP and repressed by ATP and citrate.
In general,
(1) The enzyme is repressed when the EC value and the concentration of intermediates used as precursors for biosynthesis are high.
(2) The enzyme is activated when the organism needs ATP and biosynthetic precursors.
If PFK and fructose-1,6-bisphosphatase (Fructose 1,6-BP F-6-P + Pi in gluconeogenesis) are both activated, ATP is wasted.
This is called a futile cycle and is avoided through an elaborate regulatory mechanism (Section 4.2.4).

14. 4.1 EMP pathway 4.1.4.2 Regulation of pyruvate kinase
Pyruvate kinase is activated when fructose-1,6-bisphosphate is accumulated in the cell. This kind of regulation is termed feedforward activation or precursor activation.
Global regulation
Glycolysis if regulated as a part of global regulation, in addition to regulation of individual enzymes.
In G(+) bacteria such as Bacillus subtilis (Section ), CcpA (catabolite control protein A) activates transcription of the genes fro glycolytic enzymes.
In Escherichia coli, CsrA (carbon storage regulator A) activates the expression of the glycolytic genes, and represses genes fro gluconeogenesis and glycogen synthesis.

15. 4.2 Glucose-6-phosphate synthesis: gluconeogenesis
Hexose-6-phosphates are the precursors of polysaccharide synthesis.
Microbes growing on carbon sources other than sugars (Chapter 7) need to synthesize glucose-6-phosphate from their substrate.
Since PFK and pyruvate kinase catalyze their exergonic reactions and do not catalyze their reverse reaction, hexose-6-phosphate cannot be synthesized by a reversal of the EMP pathway and separate enzymes are used in gluconeogenesis to overcome that the reverse reactions are thermodynamically unfavorable.

16. 4.2 Glucose-6-phosphate synthesis: gluconeogenesis
4.2.1 PEP synthesis
PEP can be produced through pyruvate, oxaloacetate or malate by microbes (Chapter 7) growing on non-carbohydrate compounds.

17. 4.2 Glucose-6-phosphate synthesis: gluconeogenesis
4.2.2 Fructose diphosphatase
PEP can be converted to fructose-1,6-bisphosphate by the EMP pathway enzymes since they catalyze the reverse reactions.
Fructose diphosphatase dephosporylates fructose-1,6-bisphosphate to fructose-6-phoshate and inorganic phosphate (Pi).
Pyrophosphate (Ppi)-dependent phosphofructokinase is found in a hypertheromphilic archaeon Thermoproteus tenax.
This enzyme catalyzes the reversible reaction. Similar reactions are known in plants and bacteria.

18. 4.2 Glucose-6-phosphate synthesis: gluconeogenesis
4.2.4 Regulation of gluconeogenesis
Gluconeogenesis is regulated through the control of fructose diphosphatase activity.
When the energy status is good, this enzyme is activated by ATP to supply carbon skeletons for growth.
AMP represses the enzyme activity when energy status is too low for growth.
A futile cycle is avoided by the opposite controls of glycolysis and gluconeogenesis.

19. 4.3 Hexose monophosphate (HMP) pathway
The HMP pathway is also called the pentose phosphate (PP) pathway.
When Escherichia coli grows on glucose as the sole carbon source, the EMP and HMP pathways consume 72% and 28% glucose, respectively, indicating that the EMP cannot meet all the requirements for biosynthesis.
The HMP pathway provides some precursors (pentose-5-phosphate and erythrose-4-phosphate) and a reducing power (NADPH) to the biosynthetic metabolism.
When glucose is only metabolized through the EMP pathway and TCA cycle, NADPH is produced only by isocitrate dehydrogenase (Section 5.2).
NADH is seldom used in biosynthetic reactions.
Most of the NADPH needed for biosynthesis arises from the HMP pathway.

20. 4.3 Hexose monophosphate (HMP) pathway
4.3.1 HMP pathway in three steps

21. 4.3 Hexose monophosphate (HMP) pathway
4.3.2 Additional functions of the HMP pathway
In addition to supplying precursors and reducing powers, the HMP pathway is the major glycolytic metabolism in microbes that
(1) utilize pentoses,
(2) do not possess other glycolytic activities
The HMP cycle is also employed for the complete oxidation of sugars in bacteria lacking a functional TCA cycle.
4.3.2 Additional functions of the HMP pathway
In addition to supplying precursors and reducing powers, the HMP pathway is the major glycolytic metabolism in microbes that
(1) utilize pentoses,
(2) do not possess other glycolytic activities
The HMP cycle is also employed for the complete oxidation of sugars in bacteria lacking a functional TCA cycle.

22. 4.3 Hexose monophosphate (HMP) pathway
Utilization of pentoses
Pentoses such as xylose and arabinose are phosphorylated and metabolized to fructose-6-phosphate and glyceraldehyde-3-phosphate through steps 2 and 3 of the HMP pathway (Figure 4.4)
Oxidative HMP cycle
Thiobacillus novellus and Brucella abortus oxidize glucose completely although they lack a functional EMP
or ED pathway.
Glucose is oxidized
through the
oxidative
HMP cycle.

23. 4.3 Hexose monophosphate (HMP) pathway
4.3.3 Regulation of the HMP pathway
Regulation of the HMP pathway is exerted through control of glucose-6- phosphate dehydrogenase and 6-phosphogluconate dehydrogenase,
Which are inhibited by the accumulation of NADPH and NADH.
In the HMP and ED pathways, dehydrogenation of glucose-6-phosphate is a common reaction, but is catalyzed by separate enzymes.
The NADP+-dependent HMP pathway enzyme is inhibited by NAD(P)H but not by ATP.
The enzyme in the ED pathway uses NAD+ as the electron acceptor and is inhibited by ATP and PEP.

24. 4.4 Entner-Doudoroff (ED) pathway
4.4.1 Glycolytic pathways in some G(-) bacteria
The ED pathway is an unique glycolytic pathway in prokaryotes.
The ED pathway was first identified in Pseudomonas saccharophila by Entner and Doudoroff.
The ED pathway turned out to be the main glycolytic pathway in prokaryotes that do not possess enzymes of the EMP pathway.
The ED pathway functions as the main glycolytic pathway in other G(-) bacteria such as Zymomonas and Azotobacter species.
Gluconate is metabolized through the ED pathway in some other G(-) bacteria including Escherichia coli and in some coryneform bacteria.

25. 4.4 Entner-Doudoroff (ED) pathway
4.4.1 Glycolytic pathways in some G(-) bacteria (continued)

26. 4.4 Entner-Doudoroff (ED) pathway
4.4.2 Key enzymes of the ED pathway
NAD(P)+-dependent glucose-6-phosphate dehydrogenase converts glucose-6- phosphate to 6-phosphogluconolactone that is converted to 6- phosphogluconate.
6-Phosphogluconate dehydratase removes water molecule from 6- phosphogluconate and produces 2-keto-3-deoxy-phosphogluconate (KDPG).
KDPG aldolases splits KDPG into pyruvate and glyceraldehyde-3-phosphate.
The key enzymes in the ED pathway are 6-phosphogluconate dehydrogenase and KDPG aldolase.

27. 4.4 Entner-Doudoroff (ED) pathway
4.4.2 Key enzymes of the ED pathway (continued)
The ED pathway has a net yield of 1 ATP for every glucose molecules as well as 1 NADH and 1 NADPH.
In comparison, glycolysis has a net yield of 2ATP and 2 NADH for one glucose molecule.
On Wikipedia.com

28. 4.4 Entner-Doudoroff (ED) pathway
4.4.3 Modified ED pathways
Unusually, some prokaryotes oxidize glucose and the intermediates are phosphorylated before being metabolized in a similar manner as in the ED pathway.
Extracellular oxidation of glucose by G(-) bacteria
On Wikipedia.com

29. 4.4 Entner-Doudoroff (ED) pathway
Extracellular oxidation of glucose by G(-) bacteria
Some strains of Pseudomonas oxidize glucose extracellularly when the glucose concentration is high.
These bacteria possess glucose dehydrogenase and gluconate dehydrogenase on the periplasmic face of the cytoplasmic membrane.
When glucose is depleted, gluconate and 2-ketogluconate are transported through specific transporters and phosphorylated, consuming ATP.
2-Keto-6-phosphogluconate is reduced to 6-phosphogluconate by a NADPH- dependent reductase.
Gluconate and 2-ketogluconate are uncommon in nature, and few microbes use these compounds. The ability to oxidize glucose and to use its products might therefore be advantageous for those organisms capable of doing this.

30. 4.4 Entner-Doudoroff (ED) pathway
Modified ED pathway in archaea
Hyperthermophilic archaea (Sulfolobus, Thermoplasma, Thermoproteus) metabolize glucose to pyruvate and glyceraldehyde without phosphorylation in a similar way as the ED pathway.
A halophilic archaeon, Halobacterium saccharovorum oxidize glucose to 2-keto-3-deoxygluconate.
The archaeal glucose dehydrogenase is a NAD(P)+-dependent enzyme.

31. 4.5 Phosphoketolase pathways
Classification of lactic acid bacteria (LAB) according to the final product in glucose metabolism,
(1) Homofermentative LAB : producing lactate only (=homolactic fermentation)
(2) Heterofermentative LAB : producing lactate and ethanol (or acetate) (=heterolactic fermentation)
The homofermentative LAB metabolizes glucose through the EMP pathway.
The phosphoketolase (PK) pathway is employed in the heterofermentative LAB and bifidus bacteria.
For example, a heterofermentative bacterium, Leuconostoc mesenteroides, produces lactate from glucose through the PK pathway, involving one PK active on xylulose-5-phosphate (Figure 4.9).
Lactate and acetate are produced from glucose by Bifidobacterium bifidum with two PKs active on fructose-6-phosphate and xylulose-5-phosphate (Figure 4.10).

32. 4.5 Phosphoketolase pathways
4.5.1 Glucose fermentation by Leuconostoc mesenteroides
Heterofermentative LAB ferment glucose to lactate, ethanol and CO2.
In overall reaction, Glucose  1 lactate +1 ethanol + 1 CO2
Phosphoketolase splits xylulose-5-phosphate to glyceraldehyde-3- phosphate and acetyl- phosphate.

33. 4.5 Phosphoketolase pathways
4.5.1 Glucose fermentation by Leuconostoc mesenteroides (continued)
Since LAB have a restricted electron transport chain, NAD+ from NADH is regenerated by using pyruvate and acetyl-phosphate as electron acceptors in the reactions catalyzed by lactate dehydrogenase, acetaldehyde dehydrogenase and alcohol dehydrogenase.
Thiamine pyrophosphate (TPP) is a prosthetic group in phosphoketolase. TPP binds glycoaldehyde, and the complex is dehydrated before being phosphorylated to acetyl-phosphate.
PK in L. mesenteroides is active on xylulose-5-phosphate, but not fructose-6- phosphate.
Hexose fermentation results in the net gain of 1 ATP.

34. 4.5 Phosphoketolase pathways
4.5.2 Bifidus pathway
Bifidobacterium bifidum has two PK, each active on fructose-6-phosphate and xylulose-5-phosphate, and ferments glucose in the bifidum pathway, which is different from that of L. mesenteroides.
In overall reaction,
2 Glucose  2 lactate acetate + 5 ATP