Feedforward activation in metabolic systems

[摘要] ENGLISH ABSTRACT : This thesis describes an analytical and quantitative analysis of the regulatoryphenomenon of feedforward activation in metabolic pathways. The necessarybackground in kinetic modelling of metabolic pathways, enzyme kineticsof allosteric enzymes, metabolic control analysis and supply-demandanalysis are provided. A few selected examples of feedforward activatedenzymes are discussed, focussing on their classification into the two majormechanistic classes, namely K-enzymes, for which the allosteric activator actsby increasing the affinity for the enzyme substrate (specific activation), andV-enzymes, for which the allosteric activator acts by increasing the limitingrate (Vf) of the enzyme (catalytic activation).Feedforward activation is then studied by means of metabolic controlanalysis and supply-demand analysis of a minimal system subject to feedforwardactivation. An initial control analysis of the full system suggeststhat saturation of the allosteric enzyme with its substrate would allow it tocontrol the flux through the demand pathway for the allosteric activator. Theenzyme kinetics of K-enzymes however show that under these conditions theallosteric effect is abolished, and other conditions should be sought underwhich the allosteric enzyme controls its demand flux. This was done usingsupply-demand analysis, which showed that the allosteric enzyme wouldhave the necessary control of the activator demand flux if the nested supplyflux for its substrate was near equilibrium. V-enzymes do not exhibit thisproblem, and the catalytic allosteric effect operates under conditions of substratesaturation of the allosteric enzyme. A kinetic model of feedforwardregulatedsystem 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 inLactococcus lactis, which is activated through feedforward action by fructose-1,6-bisphosphate (FBP), with the interesting twist that it also has an absoluterequirement for FBP. An existing kinetic model of this metabolic pathwaycontained a rate equation for LDH that only incorporated a non-cooperativeV-effect of FBP, but omitted other potentially important effects that have beendescribed in the literature, such as the competitive inhibition of FBP bindingby inorganic phosphate (Pi), cooperative binding of both FBP and Pi, and thealteration of the KM-values of both the substrates pyruvate and NADH (Keffects).A new rate equation for LDH that incorporated these effects was developedand parameterised with data from the literature. The kinetic modelwith the original and one with the new rate equation were compared in termsof their steady-state behaviour as the external glucose concentration was increasedfrom 0 to 2mM. The only observable differences occurred at glucoseconcentrations below 50mM and are probably of physiological significanceonly in the very last stage of glucose depletion. With our new LDH rate equationthere was a decrease in the mixed acid fermentation fluxes as comparedto the original model. We were able to relate the observed differences to thedifferent types of allosteric effects through a series of 'what-if' experimentsin which we compared the effects of four forms of our rate equation: the fullequation, one which was completely desensitised to FBP, one with V-effectsonly and one with K-effects only. We also studied the effects of binding cooperativityof FBP and Pi-binding, and of Pi-inhibition of FBP-binding. Wefound that the activating V-effect of FBP on LDH operated mostly at verylow glucose concentrations, while the K-effect of FBP on LDH operated onlyat higher glucose concentrations. The K-effect still dominated in the regionbetween exclusively V-effect and exclusively K-effect, and it is only in thisregion that the cooperative binding of FBP and Pi and the Pi-inhibition ofFBP-binding had any visible effect.

[发布机构] Stellenbosch University