Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: It is widely accepted that insufficient insulin-stimulated activation of muscle glycogen
synthesis is one of the major components of non-insulin-dependent (type 2)
diabetes mellitus. Glycogen synthase, a key enzyme in glycogen synthesis, is extensively
regulated, both allosterically (by glucose-6-phosphate, ATP, and other ligands)
and covalently (by phosphorylation). Although glycogen synthase has been
a topic of intense study for more than 50 years, its kinetic characterization has been
confounded by its large number of phosphorylation states. Questions remain regarding
the function of glycogen synthase regulation and the relative importance
of allosteric and covalent modification in fulfilling this function. The regulation
of glycogen synthase and glycogen phosphorylase, the enzyme that catalyses the
degradation of glycogen chains, are reciprocal in many respects.
In the present research, using mathematical modelling, we aim to establish the
function of the allosteric and covalent regulation of glycogen synthase and glycogen
phosphorylase in muscle and, in the case of glycogen synthase, the relative importance
of these two mechanisms in performing this function. In order to realize
these aims it is essential that a detailed kinetic model of glycogen metabolism is
constructed.
We begin with a thorough review of the kinetics and regulation of glycogen synthase
inwhich we propose that both allosteric and covalent modification of glycogen
synthase can be described by a Monod-Wyman-Changeux model in terms of apparent
changes to L0, the equilibrium constant between the T and R conformers. We
then proceed to develop a rate equation according to the proposed Monod-Wyman-Changeux model and determine values for its kinetic parameters from published experimental data using non-linear least-squares regression. We show that the application
of the Monod-Wyman-Changeux model to glycogen synthase kinetics also
has important implications for the rate equations of enzymes that catalyse the phosphorylation
and dephosphorylation of glycogen synthase. We formalize these implications for a generic protein that follows Monod-Wyman-Changeux-type conformational
change and then also show how the findings apply to glycogen synthase. Taking
into account the kinetic model of glycogen synthase and how it also influences
the covalent regulation of the enzyme, we proceed to construct a detailed mathematical
model of glycogen synthesis that includes the glycogen synthase phosphorylation
cascade. A variation of this model in which glycogen synthase phosphorylation
is described with a single parameter is also provided. We reuse an existing model of
muscle glycogenolysis and also combine these models in an overall model of glycogen
metabolism. Finally, we employ the theoretical frameworks of metabolic control
analysis, supply-demand analysis, and co-response analysis to investigate the function
of glycogen synthase and glycogen phosphorylase regulation. We show that
the function of glycogen synthase regulation is not flux control, as assumed in the
textbook view, but rather the maintenance of glucose-6-phosphate within a narrow
range far from equilibrium. Similarly, we show that regulation of glycogen phosphorylase
functions to minimize variation in cellular energy charge in the face of
highly variable energy demand. We conclude with an appeal for a renewed interest
in the enzyme kinetics of muscle glycogen metabolism. / AFRIKAANSE OPSOMMING: Daar word wyd aanvaar dat onvoldoende insulien-gestimuleerde aktivering van
spierglikogeensintese een van die hoofkomponente van insulien-onafhanklike (tipe
2) diabetes mellitus is. Glikogeensintase, ’n sleutelensiem in glikogeensintese is
onderworpe aan breedvoerige regulering, beide allosteries (deur glukose-6-fosfaat,
ATP, en ander ligande) en kovalent (deur fosforilering). Alhoewel glikogeensintase
reeds vir meer as 50 jaar deeglik bestudeer word, word die kinetiese karakterisering
daarvan bemoeilik deur die groot aantal fosforilasiestate waarin die ensiem
voorkom. Daar is steeds vrae betreffende die funksie van die regulering van glikogeensintase
en die relatiewe bydrae van allosteriese en kovalente regulering in die
vervulling van hierdie funksie. Die regulering van glikogeensintase en glikogeenfosforilase,
die ensiem wat die afbraak van glikogeenkettings kataliseer, is in baie
opsigte resiprook.
In hierdie studie beoog ons om met die hulp van wiskundige modellering vas
te stel watter funksie die regulering van glikogeensintase en glikogeenfosforilase
vervul en, in die geval van glikogeensintase, wat die relatiewe belang is van allosteriese
en kovalente regulering in die vervulling van hierdie funksie. Om hierdie oogmerke
te verwesentlik is dit nodig dat ’n kinetiese model van glikogeenmetabolisme
ontwikkel word.
Ons begin met ’n omvattende oorsig van die kinetika en regulering van glikogeensintase
waarin ons voorstel dat beide die allosteriese en kovalente regulering
van glikogeensintase beskryf kan word met die Monod-Wyman-Changeux model
in terme van oënskynlike veranderings aan L0, die ekwilibriumkonstante tussen
die T en R konformasies. Ons gaan dan voort om ’n snelheidsvergelyking te ontwikkel
volgens die voorgestelde Monod-Wyman-Changuex-model en bepaal ook
die waardes van hierdie vergelyking se parameters vanaf gepubliseerde eksperimentele
data deur middel van nie-lineêre kleinste-vierkantsregressie. Ons wys dat
die toepassing van die Monod-Wyman-Changuex-model op glikogeensintase-kinetika belangrike gevolge het vir die snelheidsvergelykings van die ensieme wat die fosforilering
en defosforilering van glikogeensintase kataliseer. Ons formaliseer hierdie
gevolge vir ’n generiese Monod-Wyman-Changeux-tipe proteïen en wys dan ook
hoe die bevindings op glikogeensintase van toepassing is. Met inagneming van die
kinetiese model vir glikogeensintase en hoe dit die kovalente regulering van die
ensiem be¨ınvloed, gaan ons voort om ’n gedetaileerde wiskundige model van glikogeensintese,
wat ook die glikogeensintase-fosforileringskaskade insluit, te ontwikkel.
’n Variasie op hierdie model waarin die fosforilering van glikogeensintase deur
’n enkele parameter beskryf word, word ook voorsien. Ons herbruik ’n bestaande
model van spierglikogenolise en kombineer ook hierdie modelle in ’n oorkoepelende
model van glikogeenmetabolisme. Uiteindelik span ons die teoretiese raamwerke
van metaboliese kontrole-analise, vraag-aanbod-analise, en ko-responsanalise in om
die funksie van die regulering van glikogeensintase en glikogeenfosforilase te ondersoek.
Ons wys dat die funksie van die regulering van glikogeensintase nie fluksiekontrole,
soos algemeen in handboeke aangeneem word, is nie, maar liewer dat
dit glukose-6-fosfaat handhaaf binne ’n noue band ver vanaf ekwilibrium. Insgelyks
wys ons dat die regulering van glikogeenfosforilase funksioneer om variasie
in sellulˆere energielading te beperk ten spyte van hoogs wisselende vlakke van
energie-aanvraag. Ons sluit af met ’n pleidooi vir hernieude belangstelling in die
ensiemkinetika van glikogeenmetabolisme in die spier. / National Research Foundation
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/80183 |
| Date | 03 1900 |
| Creators | Palm, Daniel Christiaan |
| Contributors | Hofmeyr, J.-H. S., Rohwer, J. M., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xvi, 268 p. : col. ill. |
| Rights | Stellenbosch University |
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