Mechanisms of protein disulphide isomerase catalyzed disulphide bond formation
1University of Oulu, Faculty of Science, Department of Biochemistry
2University of Oulu, Biocenter Oulu
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|Persistent link:|| http://urn.fi/urn:isbn:9789514262753
Oulu : University of Oulu,
|Publish Date:|| 2010-09-14
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic dissertation to be presented with the assent of the Faculty of Science of the University of Oulu for public defence in OP-sali (Auditorium L10), Linnanmaa, on 24 September 2010, at 12 noon
Professor Lloyd Ruddock
Professor Lars Ellgaard
Doctor David Ferrari
Professor Robert Freedman
Protein folding of outer membrane and secreted proteins, including receptors, cytokines and antibodies is often linked to disulphide bond formation. Native disulphide bond formation is complex and is usually the rate limiting step in the folding of such proteins. The enzymes which catalyse the slow steps in disulphide bond formation belong to the protein disulphide isomerase (PDI) family. PDI catalyses formation, reduction and isomerization of newly synthesized disulphide bonds. The mechanisms of action of the PDIs are currently poorly understood and this not only inhibits our understanding of the biogenesis of a range of medically important proteins, and hence associated disease states, but also prevents the effective manipulation of the cellular environment by the biotechnology industry for the production of high value therapeutic proteins. Hence, understanding the mechanism of action of these enzymes is vital for a wide range of medically important processes and therapies.
In this study the role of a conserved arginine residue in the catalytic activity of PDI was shown. The movement of this residue into and out of the active site locale of PDI was shown to modulate the pKa of the C-terminal active site cysteine of PDI and by that way to allow the enzyme to act efficiently as catalyst both of oxidation and isomerization reactions.
The possible role of hydrogen peroxide produced by sulphydryl oxidases during disulphide bond formation was studied in an oxidative protein refolding assay. Analysis showed that hydrogen peroxide can be used productively to make native disulphide bonds in folding proteins with minimal side reactions.
In addition, the kinetics of oxidation and reduction of the a domains of PDI and Pdi1p by glutathione was studied in this thesis. The kinetics obtained with stopped-flow and quenched-flow experiments showed the reactions to be more rapid and complex than previously thought. Significant differences exist between the kinetics of PDI and Pdi1p. This implies that the use of yeast systems to predict physiological roles for mammalian PDI family members should be treated cautiously.
Acta Universitatis Ouluensis. A, Scientiae rerum naturalium
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