An investigation into the effects of macromolecular crowding on the kinetics of upper glycolytic enzymes in Saccharomyces cerevisiae

Thesis (MSc)--Stellenbosch University, 2020.ENGLISH ABSTRACT: In order for mathematical models of metabolism to accurately emulate experimental data the conditions in which parameter values are obtained must be close to the actual in vivo environment. However, this is traditionally not the case, with enzyme kinetic studies usually taking place in conditions which are ideal for the enzyme being studied and can be far removed from the actual native conditions the enzyme would be found in. An aspect of the intracellular environment which has not been extensiely covered is the large quantity of different macromolecules which occupy it, known as macromolecular crowding. The space occupied by these macromolecules has thermodynamic and kinetic consequences which are not taken into consideration. In this study we mimicked a crowded environment by using the inert polymers PEG 8000 and Ficoll 70 and studied how they affected enzyme kinetic parameter estimates at different concentrations. NMR spectroscopy was used to obtain timecourse data for the upper glycolytic enzymes, phosphoglucose isomerase (PGI) and phosphofructokinase (PFK), in cell lysate. Parameter estimates were obtained by fitting NMR time-course data to a kinetic model based on rate equations for the two enzymes. The identifiability of each parameter was also determined and could be used to analyse the accuracy of parameter estimation. The aim of this study was to determine the effects of macromolecular crowding on enzyme kinetics and to explore if these effects should be considered when trying to simulate in vivo-like conditions when studying enzyme kinetics. In our results macromolecular crowding was shown to affect the parameter estimates for both enzymes, in particular decreasing their maximal activity, increasing the binding affinity of PFK for fructose-6-phosphate (F6P), and decreasing its affinity for adenosine tri-phosphate (ATP).AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.Master