A systematic investigation into the quantitative effect of pH changes on the upper glycolytic enzymes of Escherichia coli and Saccharomyces cerevisiae

Swanepoel, Christiaan Johann (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Kinetic modelling of biological phenomena in an attempt to understand the underlying dynamics and complexity of life is becoming an indispensable tool to systems biology. A new paradigm of mathematical and computational integration of experimental data has shifted the focus in biological sciences from mere characterisation and cataloguing of the components of life, to a more holistic view. The functioning of these components in dynamic interactions in non-linear biochemical networks is now a major field of interest for many biologists. Classically, enzyme kinetic assays are optimised for yielding the maximal activity of the enzyme of interest. This raises the question of how applicable the obtained kinetic parameters are for systems biology, especially when considering how the intracellular reality (in terms of pH and ionic strength and composition) affects the catalytic activity of enzymes in vivo. Another concern is how accurate and predictive the kinetic models, constructed from such obtained data, can be. Much effort has been directed towards the standardisation of enzyme kinetics for systems biology and in vivo-like assay media have been developed for the determination of enzyme kinetic parameters in both Escherichia coli and Saccharomyces cerevisiae. However, the effect of pH changes on kinetic parameters of enzymes, has been somewhat neglected in systems biology studies. With this in mind we investigated the quantitative effects elicited by pH changes on the upper glycolytic enzymes in Escherichia coli and Saccharomyces cerevisiae using NMR spectroscopy. This is especially important as recent studies have shown that intracellular pH, while remaining a tightly homeostatically controlled parameter, is not as constant as once thought and has been shown to vary in response to environmental perturbation. The investigation focused on parameter estimation and the unique identifiability of the estimated parameters. The main aim of this project is the development of a robust, reliable technique for parameter identification from experimental data using mathematical and computational approaches.

AFRIKAANSE OPSOMMING: In ’n poging om die onderliggende dinamika en kompleksiteit van die lewe beter te verstaan, raak kinetiese modellering van biologiese fenomene ’n onontbeerlike instrument vir sisteembiologie. ’n Nuwe paradigma van wiskundige en rekenaarmatige integrasie van eksperimentele data het die fokus in biologiese wetenskappe verskuif van blote karakterisering en katalogisering van die komponente van die lewe, na ’n meer holistiese siening. Die funksionering van hierdie komponente in dinamiese interaksies in nie-lineêre biochemiese netwerke raak ’n belangrike navorsingsveld vir baie bioloë. Histories is ensiem-kinetiese essai’s geoptimeer vir maksimale aktiwiteit van die betrokke ensiem. Dit laat die vraag ontstaan hoe toepasbaar die verkrygde kinetiese parameters is in terme van sisteembiologie, veral as in ag geneem word hoe die intrasellulêre werklikheid (in terme van pH en ioniese sterkte en samestelling) die katalitiese aktiwiteit van ensieme in vivo beïnvloed. ’n Verdere bekommernis is die akkuraatheid en voorspellingsvermoë van kinetiese modelle wat op grond van sulke data saamgestel is. Onlangs is beduidende pogings aangewend om ensiemkinetika vir sisteembiologie te standaardiseer, en essai-media wat in vivo toestande naboots is ontwikkel vir die bepaling van ensiem-kinetiese parameters in beide Escherichia coli en Saccharomyces cerevisiae. Die effek van pH-veranderinge op kinetiese parameters van ensieme is egter ietwat verwaarloos in sisteembiologiese studies. Met hierdie in gedagte het ons die kwantitatiewe effekte van pH veranderinge op die boonste glikolitiese ensieme in Escherichia coli en Saccharomyces cerevisiae met behulp van KMR spektroskopie bepaal. Dit is veral belangrik aangesien onlangse studies getoon het dat intrasellulêre pH, terwyl dit ’n streng homeostatiese beheerde parameter bly, nie so konstant bly as voorheen gedink is nie, en dat dit kan verander in respons op versteurings in die omgewing. Hierdie studie het gefokus op beraming van ensiem-kinetiese parameters asook die bepaling van die unieke identifiseerbaarheid van hierdie parameters. Die hoofdoel van hierdie projek is die ontwikkeling van ’n robuuste, betroubare tegniek vir parameterbepaling vanaf eksperimentele data deur gebruik te maak van wiskundige en rekenaarmatige tegnieke.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/103716
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