Presentation on theme: "The control of glycolysis: inside or outside of the pathway ? Demand vs supply and the unending quest for the rate limiting step."— Presentation transcript:
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):
... 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: Koebmann BJ, Westerhoff HV, Snoep JL, Nilsson D, Jensen PR. 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. 29:41-5Koebmann BJ, Westerhoff HV, Snoep JL, Solem C, Pedersen MB, Nilsson D, Michelsen O, Jensen PR. 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 F 1 -ATPase code for the cytoplasmic F1 part of the (F 1 F 0 ) 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
(F 1 F 0 ) 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 dellATP
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) Range 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
Un controllo effettivo da parte del demand richiede che ε demand sia circa 20 volte più piccola di ε supply Siccome difficilmente ε supply può essere >4, allora ε demand dovrà essere <0.2
Se è quindi il demand ad avere il completo controllo sul flusso, lentità della variazione di P (omeostasi di P) dipenderà solo da ε sup …the functions of flux and concentration control are mutually exclusive. …the higher ε supply, 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 v demand e misurare ε e C J Flusso in funzione di ATP/ADP Log(J) in funzione di Log(ATP/ADP)
The full line represents C e2 J1 based on the fitted polynomium for the relative growth rates whereas the dotted line represents C e2 J1 based on a linear fit for the relative growth rates ΔG p : cellular energy state Elasticità di supply e demand (growth) in funzione di ATP/ADP C J 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 lidrolisi 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. Brevetti Patent number: Filing date: May 22, 1997 Issue 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, ReviewHofmeyr, J.S. Cornish-Bowden, A. 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.Anaerobic Energy Metabolism in Yeast as a Supply-Demand System,New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge Kroukamp O, Rohwer JM, Hofmeyr JH, Snoep JL. (2002) Experimental supply-demand analysis of anaerobic yeast energy metabolism. Mol Biol Rep. 29:203-9.Kroukamp O, Rohwer JM, Hofmeyr JH, Snoep JL. Hofmeyr JH, Kacser H, van der Merwe KJ.(1986) Metabolic control analysis of moiety-conserved cycles. Eur J Biochem. 155:631-41Hofmeyr JH, Kacser H, van der Merwe KJ. 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: