Browsing by Author "Van Schalkwyk, Clara Elizabeth"

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    Understanding yeast glycolysis dynamics using HPLC analysis and mathematical modelling.
    (Stellenbosch : Stellenbosch University, 2023-03) Van Schalkwyk, Clara Elizabeth; Van Niekerk, David Douglas; Snoep, Jacob Leendert ; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Glycolysis is a universally conserved metabolic pathway that forms the backbone of carbon metabolism and energy generation. A comprehensive understanding of this pathway may lead to a more complete understanding of energy metabolism. A novel HPLC-based method was recently developed for the detection of metabolite, cofactor and coenzyme concentrations during metabolic studies. The purpose of the current study is to implement this novel method to generate data which can be used to eluci- date dynamics of glycolytic intermediates and cofactors in conjunction with a existing enzyme kinetic model of yeast glycolysis in Saccharomyces cerevisiae, a model organism for eukariotic biology. The HPLC method was used to study the dynamic behaviour of three key glycolytic intermediates in response to a glucose pulse in cell free extracts of S. cerevisiae. It was found that in response to a glucose pulse, glucose is consumed, fructose-1,6- bisphosphate accumulates prior to glucose depletion and ethanol accumulates. Sub- sequently, the dynamic behaviour of the adenine nucleotides in response to a glucose pulse was investigated. It was found that while glycolysis is active, AMP accumulates and ATP depletes while ADP remains at a constant low concentration. Once glucose is depleted ATP increases and AMP decreases. This is the result of the combined action of adenylate kinase, glycolytic enzymes and ATPases. Finally, the effect of phosphate on glycolytic intermediate dynamics and specific enzyme activity was investigated. It was found that phosphate influences the rate of glucose consumption and degree of F16BP accumulation. The inhibitory effect of phosphate on pyruvate kinase activity was char- acterised and incorporated into the model, however, the adapted model could not yet describe the observed phenomena and further investigation is required.