1. The control of glycolysis: inside or outside of the pathway ?
Demand vs supply and the unending quest for the ‘rate limiting step’

3. What is the ‘rate limiting step’ of glycolysis?
Gli enzimi glicolitici NON sembrano essere limitanti in lievito...
J Biomed Biotechnol. (2008):

5. ... e neanche negli altri organismi

6. A long standing question…
Is the control due to a single glycolytic enzyme?
Is the control shared by many gl. enzymes?
Does the control lie outside of glycolysis?
Experimental evidence in several cases…
If the control lies outside, the most sensible candidate is ATP consumption. The authors therefore sought ways to increase ATP consumption.

7. Relevant papers:
Koebmann BJ, Westerhoff HV, Snoep JL, Nilsson D, Jensen PR. (2002) The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J. Bacteriol. 184:
Koebmann BJ, Westerhoff HV, Snoep JL, Solem C, Pedersen MB, Nilsson D, Michelsen O, Jensen PR. (2002) The extent to which ATP demand controls the glycolytic flux depends strongly on the organism and conditions for growth. Mol Biol Rep. 29:41-5
Koebmann BJ, Solem C, Pedersen MB, Nilsson D, Jensen PR (2002) Expression of Genes Encoding F1-ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis. Appl. Envir. Microb.68:4274–4282

8. Plasmid for expression of F1-ATPase in E. coli
Plasmid for expression of F1-ATPase in E. coli. The boxes indicate the specific genes or origin of replication. “CPX” indicates synthetic promoters.
The A,G and D subunits of F1-ATPase code for the cytoplasmic F1 part of the (F1F0) H+-ATP synthase which possesses the catalytic site for ATP synthesis and/or hydrolysis. The combination of the α, β and γ subunits exerted the strongest ATPase activity

9. ATP synthesis and proton translocation can be uncoupled
(F1F0) H-ATP synthase
Pictures from Gruissem B&MBoP
ATP synthesis and proton translocation can be uncoupled

10. Introduction of ATPase activity by overexpression of F1 genes
Measuring in vitro ATPase activities:
change in ATP concentration related to the
total protein level is shown as a function of
time after the addition of cellular extracts.
pCP44 (wt)
Increasing ATPase
The transformants have more ATPase activity when assayed in vitro

11. Galactosidase activity is a good indicator for engineered ATPase activity
Correlation between specific ATPase activity and specific -galactosidase activity.

12. ATP
ADP
ATP/ADP
ATP+ADP
Introducing an ATPase activity has a measurable effect on ATP and ADP concentration
Correlation between specific ATPase activity with ATP, ADP, ATPADP pools and [ATP]/[ADP] ratios.

13. - Lower final cell density
Increasing ATPase
pCP44 (wt)
- Slower growth
- Lower final cell density
Growth curves of E. coli BOE270 derivatives with F1-ATPase activities. Cell density (i.e., the OD450 value) is shown as a function of time of the cultures.

14. - Faster glucose consumtption despite a reduced growth rate
pCP44 (wt)
Increasing ATPase
- Faster glucose consumtption despite a reduced growth rate
Glucose consumptions in E. coli BOE270 derivatives with F1-ATPase activities.

15. Summarizing:
The effect on ATP concentration (or growth rate) is not as big as the effect on Glucose consumption (glycolysis)
or as the effect on biomass yield

16. (ed. A. Cornish-Bowden), Universitat de València, Valencia, Spain.
Anaerobic energy metabolism in yeast as a supply-demand system Jan-Hendrik S. Hofmeyr (1997)
Capitolo del libro:
New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge
(ed. A. Cornish-Bowden), Universitat de València, Valencia, Spain.

17. Metabolismo dell’ATP
Fig. 1. The main reactions involved in ATP production and consumption in a fermenting yeast cell. Abbreviations: HK: hexokinase; PFK: phosphofructokinase; PGK: phosphoglycerate kinase; PK; pyruvate kinase; AK: adenylate kinase. The reaction catalysed by adenylate kinase is depicted with a dotted line to indicate that it is considered to be in equilibrium, therefore carrying no net flux. The number associated with the adenylate kinase reaction indicates reaction stoichiometry. The block designated "Demand" symbolizes the set of non-glycolytic ATP-consuming reactions.

18. Come possiamo indicare lo stato di carica del sistema ATP?
Tra gli indicatori possibili ricordiamo:
Energy Charge (ec)
Adenilati carichi/Adenilati scarichi (c/u)
Range 0 - 1
Range 0 - +

19. Usando come variabile la Energy charge (ec) invece che la concentrazione di ATP, il sistema si semplifica e può essere descritto come sistema di supply-demand di tipo ciclico (Fig. A)
Usando come variabile c/u (o il rapporto molare ATP/ADP), il sistema si semplifica ulteriormente (B) e diventa un sistema di supply demand lineare.

20. Ricadiamo quindi in un classico sistema Supply-demand trattato in precedenza

21. demand
supply

22. Un controllo effettivo da parte del demand richiede che εdemand sia circa 20 volte più piccola di εsupply
Siccome difficilmente εsupply può essere >4, allora εdemand dovrà essere <0.2

23. Buona omeostasi di P, cioè piccole variazioni di (c/u)
Se è quindi il demand ad avere il completo controllo sul flusso, l’entità della variazione di P (omeostasi di P) dipenderà solo da εsup
“…the functions of flux and concentration control are mutually exclusive.”
…the higher εsupply , the more effective the buffering of the c/u ratio.
Cambiamenti nel demand cambiano il flusso, cambiamenti nel supply non cambiano il flusso…
Buona omeostasi di P, cioè piccole variazioni di (c/u)

24. Se introduciamo un leak (es. una ATPasi gratuita)

25. Possiamo modulare vdemand e misurare ε e CJ
Flusso in funzione di ATP/ADP
Log(J) in funzione di Log(ATP/ADP)
The relative glycolytic fluxes and growth rates
Logarithmic (scaled) relative fluxes
Dependence of glycolytic flux and growth rate on the [ATP]/[ADP] ratio, and calculation of elasticity and flux control coefficients.

26. CJ del demand (calcolato in base a due diversi fitting della curva)
ΔGp : cellular energy state
Elasticità di supply e demand (growth) in funzione di ATP/ADP
CJ del demand (calcolato in base a due diversi fitting della curva)
The full line represents Ce2J1 based on the fitted polynomium for the relative growth rates whereas the dotted line represents Ce2J1 based on a linear fit for the relative growth rates

27. Main conclusions (I)
In wild type cells, catabolic reactions (glycolysis) have little flux control
In other words, the glycolytic flux is controlled by the ATP demand (this ensures metabolite homeostasis)
In cells with a high ATPase level, the control is more in the catabolic reactions
This would account for the evidence (yeast, coli…) that glycolityc reactions have no flux control and explains the difference between the effect on growth rate and yield (ATP/ADP vs ATP hydrolysis)

28. Altri esempi di ciclo futile:
Due casi di enzimi glicolitici e gluconeogenetici che funzionano contemporaneamente
PFK e PBPase; PyrK e PEPCK
Glycolysis & Gluconeogenesis pathways are both spontaneous. If both pathways were simultaneously active within a cell it would constitute a "futile cycle" that would waste energy.
Se i due enzimi sono attivi contemporaneamente, il risultato netto è l’idrolisi di ATP

29. La velocità di crescita rallenta e la resa di cellule (g di cellule prodotte per g di glucosio) diminuisce.

30. La velocità di produzione di EtOH aumenta del 22%
[per il calcolo: 100 x (50.5 – 41.3) / 41.3 ]
Purtroppo la maggiore resa non compensa il rallentamento della crescita.

31. Brevetti
Patent number: Filing date: May 22, 1997 Issue date: Oct 19, 1999
1) A method to increase the production of carbon dioxide by Saccharomyces cerevisiae … with a strain of Saccharomyces cerevisiae genetically modified so as to conduct at least two futile cycles in the anaerobic glycolytic pathway which results in increased …
2) The method of claim 1 wherein said two futile cycles are effected by modifying said Saccharomyces cerevisiae to constitutively express the gene for fructose-1,6-biphosphatase and the gene for phosphoenolpyruvate carboxykinase.

32. Same approach, different organism
Lactobacillus lactis epresssing F1 ATPase activity
Correlation between specific -galactosidase activities and biomass yield for the F1-ATPase library.

33. Correlation between specific -galactosidase activities and biomass yield for the F1-ATPase library. The specific –galactosidase activities and biomass yields were measured for overnight cultures of L. lactis strains grown in SA medium supplemented with 1.5 g of glucose per liter and 5 g of erythromycin per ml.

34. Effect of uncoupled F1-ATPase on the intracellular energy level
ATP+ADP
ATP
ATP/ADP
ADP

35. Increasing ATPase
Cultures were grown in batches without aeration at 30°C in SA medium supplemented with 1 g of glucose per liter

36. Increasing ATPase
Steady-state consumption of glucose in L. lactis strains with uncoupled F1-ATPase during batch fermentation.

37. The effect on growth rate (or ATP concentration) is as big as the effect on biomass yield
No effect on glycolytic flux
Le misure fin qui descritte sono state fatte su cellule in crescita

38. glycolytic flux increases up to its limit
Effect of uncoupled F1-ATPase in nongrowing cells
glycolytic flux increases up to its limit
Intracellular [ATP]/[ADP] ratios in resuspended cells

39. Misure dalle cellule in crescita
Dependence of glycolytic flux and growth rate on the [ATP]/[ADP] ratio and calculation of elasticity and flux control coefficients.
The relative glycolytic fluxes and growth rates
Logarithmic (scaled) relative fluxes
Misure dalle cellule in crescita

40. Elasticities of glycolytic flux and growth rate
Flux control by the demand for
ATP on the glycolytic flux
ATP consumption has a very low control coefficient (<0.1) in growing Lactobacillus cells

42. Conclusions (II)
Glycolysis is close to its maximum capacity in growing Lactobacillus cells
ATP demand is not limiting glycolytic flux
Large difference between E. coli and Lactobacillus lactis
This can be interpreted in terms of the physiology (ATP yield: 2 vs 10, flux: 24 vs 7)
In non growing Lactococcus cells, glycolysis is limited by ATP demand untill…

43. Referenze addizionali
Hofmeyr, J.S. Cornish-Bowden, A. (2000) Regulating the cellular economy of supply and demand. FEBS Lett., 476, Review
Hofmeyr (1997) "Anaerobic Energy Metabolism in Yeast as a Supply-Demand System, pp in New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge (ed. A. Cornish-Bowden), Universitat de València, Valencia, Spain.
Kroukamp O, Rohwer JM, Hofmeyr JH, Snoep JL. (2002) Experimental supply-demand analysis of anaerobic yeast energy metabolism. Mol Biol Rep. 29:203-9.
Hofmeyr JH, Kacser H, van der Merwe KJ.(1986) Metabolic control analysis of moiety-conserved cycles. Eur J Biochem. 155:631-41
Oliver S. (2002) Demand managment in cells Nature 418:33-34 (Commentary)
Moreno-Sánchez R, Saavedra E, Rodríguez-Enríquez S, Olín-Sandoval V. (2008) Metabolic Control Analysis: A Tool for Designing Strategies to Manipulate Metabolic Pathways J Biomed Biotechnol. 2008: