The control of glycolysis:inside or outside of the pathway ?

Demand vs supply and the unending quest for the ‘rate limiting step’

What is the ‘rate limiting step’ of glycolysis?

Gli enzimi glicolitici NON sembrano essere limitanti in lievito...

J Biomed Biotechnol. (2008): 597913.

... e neanche negli altri organismi

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.

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. 184:3909-16

• 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

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

(F1F0) H-ATP synthase

ATP synthesis and proton translocation can be uncoupled

Pictures from Gruissem B&MBoP

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.

The transformants have more ATPase activity when assayed in vitro

pCP44 (wt)

Increasing ATPase

Correlation between specific ATPase activity and specific -galactosidase activity.

Galactosidase activity is a good indicator for engineered ATPase activity

Correlation between specific ATPase activity with ATP, ADP, ATPADP pools and [ATP]/[ADP] ratios.

Introducing an ATPase activity has a measurable effect on ATP and ADP concentration

ATP

ADP

ATP/ADP

ATP+ADP

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.

pCP44 (wt)Increasing ATPase

- Slower growth

- Lower final cell density

Glucose consumptions in E. coli BOE270 derivatives with F1-ATPase activities.

pCP44 (wt)

Increasing ATPase

- Faster glucose consumtption despite a reduced growth rate

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

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.

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.

Metabolismo dell’ATP

Come possiamo indicare lo stato di carica del sistema ATP?

Tra gli indicatori possibili ricordiamo:

1) Energy Charge (ec)

2) Adenilati carichi/Adenilati scarichi (c/u)

[AMP] [ADP] [ATP]

0.5[ADP] [ATP]

ec

0.5[ADP] [AMP]

0.5[ADP] [ATP]

1/

ec

ecuc

Range 0 - 1

Range 0 - +

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.

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

supply

demand

1supply

supply

/

/

v

uc

v

uc

supplydemand

demand

//

/Supply v

uc

v

uc

v

ucJC

supplydemand

supply

//

/Demand v

uc

v

uc

v

ucJC

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

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.”

supplydemand

//

/

Demand

/

Supply

1v

uc

v

uc

ucuc CC

…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)

Se introduciamo un leak (es. una ATPasi gratuita)

The relative glycolytic fluxes and growth rates

Dependence of glycolytic flux and growth rate on the [ATP]/[ADP] ratio, and calculation of elasticity and flux control coefficients.

Logarithmic (scaled) relative fluxes

Possiamo modulare vdemand e misurare ε e CJ

Flusso in funzione di ATP/ADP Log(J) in funzione di Log(ATP/ADP)

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

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)

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)

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.

Altri esempi di ciclo futile:

Due casi di enzimi glicolitici e gluconeogenetici che funzionano contemporaneamente

PFK e PBPase; PyrK e PEPCK

Se i due enzimi sono attivi contemporaneamente, il risultato netto è l’idrolisi di ATP

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

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.

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.

BrevettiPatent number: 5968790Filing date: May 22, 1997Issue date: Oct 19, 1999

Same approach, different organism

Lactobacillus lactis epresssing F1 ATPase activity

Correlation between specific -galactosidase activities and biomass yield for the F1-ATPase library.

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.

Effect of uncoupled F1-ATPase on the intracellular energy level

ATP

ADP

ATP/ADP

ATP+ADP

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

Increasing ATPase

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

Increasing ATPase

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

Intracellular [ATP]/[ADP] ratios in resuspended cells

Effect of uncoupled F1-ATPase in nongrowing cells

glycolytic flux increases up to its limit

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

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

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…

Referenze addizionali• Hofmeyr, J.S. Cornish-Bowden, A. (2000) Regulating the cellular economy of supply and demand. FEBS Lett., 476, 47-51 Review

• Hofmeyr (1997) "Anaerobic Energy Metabolism in Yeast as a Supply-Demand System, pp. 225-242 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: 597913.