Feedforward activation in metabolic systems

Coetzee, Marnette (2016-02-22)

Thesis (MSc)--Stellenbosch University, 2016

rs201605

Thesis

ENGLISH ABSTRACT : This thesis describes an analytical and quantitative analysis of the regulatory phenomenon of feedforward activation in metabolic pathways. The necessary background in kinetic modelling of metabolic pathways, enzyme kinetics of allosteric enzymes, metabolic control analysis and supply-demand analysis are provided. A few selected examples of feedforward activated enzymes are discussed, focussing on their classification into the two major mechanistic classes, namely K-enzymes, for which the allosteric activator acts by increasing the affinity for the enzyme substrate (specific activation), and V-enzymes, for which the allosteric activator acts by increasing the limiting rate (Vf) of the enzyme (catalytic activation). Feedforward activation is then studied by means of metabolic control analysis and supply-demand analysis of a minimal system subject to feedforward activation. An initial control analysis of the full system suggests that saturation of the allosteric enzyme with its substrate would allow it to control the flux through the demand pathway for the allosteric activator. The enzyme kinetics of K-enzymes however show that under these conditions the allosteric effect is abolished, and other conditions should be sought under which the allosteric enzyme controls its demand flux. This was done using supply-demand analysis, which showed that the allosteric enzyme would have the necessary control of the activator demand flux if the nested supply flux for its substrate was near equilibrium. V-enzymes do not exhibit this problem, and the catalytic allosteric effect operates under conditions of substrate saturation of the allosteric enzyme. A kinetic model of feedforwardregulated system was constructed and used to provide data for a graphical analysis of the theoretical results. The last part of the study is concerned with a particular allosteric enzyme, lactate dehydrogenase (LDH) in glucose fermentation metabolism in Lactococcus lactis, which is activated through feedforward action by fructose- 1,6-bisphosphate (FBP), with the interesting twist that it also has an absolute requirement for FBP. An existing kinetic model of this metabolic pathway contained a rate equation for LDH that only incorporated a non-cooperative V-effect of FBP, but omitted other potentially important effects that have been described in the literature, such as the competitive inhibition of FBP binding by inorganic phosphate (Pi), cooperative binding of both FBP and Pi, and the alteration of the KM-values of both the substrates pyruvate and NADH (Keffects). A new rate equation for LDH that incorporated these effects was developed and parameterised with data from the literature. The kinetic model with the original and one with the new rate equation were compared in terms of their steady-state behaviour as the external glucose concentration was increased from 0 to 2mM. The only observable differences occurred at glucose concentrations below 50mM and are probably of physiological significance only in the very last stage of glucose depletion. With our new LDH rate equation there was a decrease in the mixed acid fermentation fluxes as compared to the original model. We were able to relate the observed differences to the different types of allosteric effects through a series of ‘what-if’ experiments in which we compared the effects of four forms of our rate equation: the full equation, one which was completely desensitised to FBP, one with V-effects only and one with K-effects only. We also studied the effects of binding cooperativity of FBP and Pi-binding, and of Pi-inhibition of FBP-binding. We found that the activating V-effect of FBP on LDH operated mostly at very low glucose concentrations, while the K-effect of FBP on LDH operated only at higher glucose concentrations. The K-effect still dominated in the region between exclusively V-effect and exclusively K-effect, and it is only in this region that the cooperative binding of FBP and Pi and the Pi-inhibition of FBP-binding had any visible effect.

AFRIKAANSE OPSOMMING : Hierdie tesis beskryf ’n analitiese and kwantitatiewe analise van die regulatoriese verskynsel van allosteriese vooruitvoeraktivering in metaboliese paaie. Die nodige agtergrond in kinetiese modellering van metaboliese paaie, ensiemkinetica van allosteriese ensieme, metaboliese kontrole analise en aanbod- aanvraag analise word verskaf. ’n Paar uitgesoekte voorbeelde van allosteriese ensieme word bespreek, met spesifieke fokus op hul klassifikasie in twee hoof meganistiese klasse, naamlik K-ensieme, waar die allosteriese aktiveerder die affiniteit van die ensiemsubstraat verhoog (spesifieke aktivering), en V-ensieme, waar die allosteriese aktiveerder die limiterende snelheid (Vf) van die ensiem verhoog (katalitiese aktivering). Vooruitvoeraktivering word dan bestudeer met behulp van metaboliese kontrole analise and aanbod-aanvraag analise van ’n kernmodel wat aan vooruitvoeraktivering onderwerp is. ’n Aanvanklike kontrole analise van die model suggereer dat versadiging van die allosteriese ensiem met sy substraat die ensiem in staat stel om die fluksie deur die aanvraagpad vir die allosteriese aktiveerder te beheer. Die ensiemkinetika van K-ensieme toon egter dat die allosteriese effek onder hierdie kondisies opgehef word, sodat ander kondisies gevind moet word wat ook die allosteriese ensiem in staat stel om sy aanvraag fluksie te beheer. Dit is gedoen met aanbod-aanvraag analise, wat getoon het dat die allosteriese ensiem die nodige beheer oor sy aanvraagfluksie sou hˆe wanneer die aanbodfluksie vir sy subtraat naby ewewig is. V-ensieme het nie hierdie probleem nie, en die katalitiese allosteriese effek funksioneer wanneer die allosteriese ensiem met substraat versadig is. ’n Kinetiese model van die vooruitvoergereguleerde sisteem is gebou en gebruik om data te verskaf vir ’n grafiese analise van die teoretiese resultate. Die laaste gedeelte van die studie het gekonsentreer op ’n bepaalde allosteriese ensiem, naamlik laktaatdehidrogenase (LDH) in glukose metabolisme van Lactococcus lactis, wat deur vooruitvoer deur glukose-1,6-bisfosfaat (FBP) geaktiveer word, en interessant genoeg ’n absolute vereiste vir FBP het. ’n Bestaande kinetiese model van hierdie metaboliese pad het ’n snelheidsvergelyking vir hierdie ensiem bevat wat slegs ’n nie-ko¨operatiewe V-effek van FBP ge¨ınkorporeer het, en ander potensieel belangrike effekte wat in die literatuur beskryf word uitgelaat het, soos kompeterende inhibisie van FBPbinding deur anorganiese fosfaat (Pi), ko¨operatiewe binding van beide FBP en Pi, en die wysiging van die KM-waardes van beide die substrate pirovaat en NADH (K-effekte). ’n Nuwe snelheidsvergelyking vir LDH wat hier die effekte inkorporeer is ontwikkel en geparameteriseer met data uit die literatuur. Die kinetiese model met die oorspronklike en ’n model met die nuwe snelheidsvergelyking is in terme van hulle bestendige toestandsgedrag vergelyk soos wat die eksterne glukose konsentrasie toeneem van nul tot 2mM. Die enigste waarneembare verskille het by glukose konsentrasies laer as 50mM voorgekom en is waarskynlik net fisiologies belangrik in die heel laaste stadium van glukose uitputting. Met ons nuwe LDH snelheidsvergelyking was daar in vergelyking met die oorspronklike model ’n afname in die gemengde suurfermentasie fluksies. Ons kon hierdie waargenome verskille toewys aan die verskillende tipes allosteriese effekte deur ’n reeks van ‘wat-as’ eksperimente waarin ons die effekte van vier vorms van ons snelheidsvergelyking vergelyk het: die volle vergelyking, een wat volledig vir FBP gedesensiteer is, een met alleenlik V-effekte, en een met alleenlik K-effekte. Ons het ook die effekte van ko¨operatiewe binding van FBP en Pi, en van kompeterende inhibisie van FBP-binding deur Pi ondersoek. Ons het gevind dat die aktiverende V-effek van FBP op LDH slegs by baie lae glukose konsentrasies gefunksioneer het, terwyl die K-effek slegs by ho¨er glukose konsentrasies gefunksioneer het. In die gebied tussenin het die K-effek steeds gedomineer, en dit was slegs in hierdie gebied wat die ko¨operatiewe binding van FBP en Pi, en die kompeterende inhibisie van FBP-binding deur Pi waarneembare effekte gehad het.

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