Browsing by Author "Kotze, Sone"
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- ItemThe role of a positive feedforward loop in regulating yeast glycolytic intermediate dynamics(Stellenbosch : Stellenbosch University, 2021-12) Kotze, Sone; Snoep, Jacob Leendert; Van Niekerk, David; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Yeast glycolysis is a well-studied model for metabolic pathways and fundamental aspects of regulation. Whereas negative feedback is common in metabolic networks; positive feedforward regulation is much less often observed. The aim of this project was to elucidate the function of feedforward loops (FFLs); investigating why they occur and to discern their potential role in disease states. A well-known FFL in glycolysis was investigated, namely that of pyruvate kinase (PK) which is known to be strongly regulated. One of its most important activators is fructose 1,6-bisphosphate (FBP), a metabolite found upstream in the pathway. Certain cancer cell lines have been shown to induce a particular isoform of PK, PKM2, which is not activated by FBP, hinting at an advantage that the absence of this FFL may confer to cancerous cells. By defining the fundamental function of FFLs, greater insights into metabolism can be obtained by investigating the effects of its absence on glycolytic flux. To probe this question, we used a systems biology approach of experimentation in conjunction with mathematical modelling. Yeast PK was kinetically characterised using cell-free extracts and was found to be activated by FBP via an FFL and inhibited by inorganic phosphate (Pi). This kinetic data was used to analyse several PK rate equations that incorporate allosteric models for its regulation by FBP and Pi. Based on these results a Hill-type equation was selected to extend an existing detailed kinetic model for yeast glycolysis. In addition, core model simulations were performed on systems with increasing complexity to formulate the core model hypothesis that the function of FFLs in metabolic networks is to buffer the intermediate metabolites between the regulating metabolite and regulated reaction when flux through the pathway changes, thereby preventing metabolic imbalance. We tested this hypothesis via experimental analysis of yeast glycolytic intermediates and cofactors over time after a glucose pulse to the pathway. The results showed interesting cofactor dynamics; with ATP being fully converted to AMP during glucose exhaustion and also indicated that Pi could not function as a switch-off mechanism for the FFL. These glycolytic intermediate dynamics also served as an independent experimental data-set that successfully validated the full glycolytic model that was adapted with the Hill equation to better describe PK activity and flux through the pathway as a whole.