Browsing by Author "Coetzee, Marnette"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Feedforward activation in metabolic systems
    (Stellenbosch : Stellenbosch University, 2016-02-22) Coetzee, Marnette; Hofmeyr, Jan-Hendrik Servaas; Rohwer, Johann Martin; Stellenbosch University. Faculty of Science. Department of Biochemistry
    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.