Comparison of progress curve analysis and initial rate kinetics for the estimation of identifiable enzyme kinetic parameters : a case study using two glycolytic enzymes from Sulfolobus solfataricus

Louw, Carla (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Obtaining accurate parameter value estimations is imperative to studying enzyme kinetics. It is also important that these parameter values be identifiable. The nonidentifiability of parameter estimations is usually indicative of parameter correlation, but may also be caused by other factors. These factors include the quality and quantity of the data used for parameter characterisation, the method of data analysis used as well as the model or rate equation to which the data is fitted. The non-identifiability of parameter estimations can either be structural or practical non-identifiability. Structural non-identifiability is caused by the model structure and is an indication of parameter correlations while practical non-identifiability is an indication of qualitatively or quantitatively insufficient data. The choice in kinetic assay and the type of data that will be collected is often dictated by the ligands, the enzyme itself or the availability of equipment. However, in the absence of these limitations, either progress curve or initial rate analysis may be selected. There are advantages and limitations to using either of these methods. The aim of this study is to try and determine if either of these two methods returns a greater number of identifiable parameter estimations for specific enzyme kinetic attributes. Experimental progress curve and initial rate data are collected for two different enzymes, GAPN and PGI, of Sulfolobus solfataricus. The GAPN and PGI enzymes represent different enzyme kinetic characteristics. Parameter value estimations are then obtained from the data fitting and an identifiability analysis for all possible combinations of the parameters are completed. When considering all the combinations of parameter fits it is possible to see trends in the combinations that return identifiable or non-identifiable parameters. With the identifiability analysis approach used in this study it is possible to determine if the non-identifiability of a parameter is structural or practical. Thereafter it is possible to speculate if the non-identifiability of the parameter estimations are due to parameter correlation or other factors and which method is superior for the analysis of certain enzyme kinetic attributes.

AFRIKAANS OPSOMMING: Die verkryging van akkurate parameterwaarde skattings is noodsaaklik vir die studie van ensiem kinetika. Dit is ook belangrik dat hierdie parameterwaardes identifiseerbaar is. Die nie-identifiseerbaarheid van parameterwaardes is gewoonlik ’n aanduiding van parameter korrelasie, maar kan ook veroorsaak word deur ander faktore. Hierdie faktore sluit in die kwaliteit en kwantiteit van die data wat gebruik is vir parameter karakterisering, die data analise metode wat gebruik is asook die model of vergelyking waarop die datapassing gedoen is. Die nie-identifiseerbaarheid van parameterwaardes kan òf strukturele of praktiese nie-identifiseerbaarheid wees. Strukturele nie-identifiseerbaarheid word veroorsaak deur die modelstruktuur en is ’n aanduiding van parameter korrelasie terwyl praktiese nie-identifiseerbaarheid ’n aanduiding is van kwalitatiewe of kwantitatiewe onvoldoende data. Die keuse in kinetiese toets en die tipe data wat ingesamel sal word, word dikwels bepaal deur die ligande, die ensiem self of die beskikbaarheid van toerusting. Egter, in die afwesigheid van hierdie beperkinge, is dit moontlik om van òf vorderingskurwe òf inisiële snelheid analise gebruik te maak. Daar is voordele en beperkings met die gebruik van enige een van hierdie twee metodes. Die doel van hierdie studie is om te probeer bepaal of enige van hierdie twee metodes meer identifiseerbare parameterwaardes lewer vir spesifieke ensiem kinetika eienskappe. Eksperimentele vorderingskurwe en inisiële snelheid data vir twee verskillende ensieme, GAPN en PGI, van Sulfolobus solfataricus is verkry. Die GAPN en PGI ensieme verteenwoordig verskillende ensiem kinetika eienskappe. Parameterwaardes is verkry deur middel van die datapassing en ’n identifiseerbaarheids-analise vir alle moontlike kombinasies van die parameters is daarna voltooi. Met die inagneming van alle kombinasies van parameter passings is dit moontlik om tendense te sien in die kombinasies wat identifiseerbare en nie-identifiseerbare parameters lewer. Die identifiseerbaarheids-analise benadering wat in hierdie studie gebruik is maak dit moontlik om te bepaal of die nie-identifiseerbaarheid van ’n parameter struktureel of prakties is. Dit is dan moontlik om te spekuleer of die nie-identifiseerbaarheid van die parameterwaardes is as gevolg van parameter korrelasie of ander faktore en watter metode beter is vir die analise van spesifieke ensiem kinetika eienskappe.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100311
This item appears in the following collections: