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Oulun yliopiston väitöskirjat

MECHANISMS OF PROTEIN DISULPHIDE ISOMERASE CATALYZED DISULPHIDEBOND FORMATION, ACTA UNIVERSITATIS OULUENSIS A Scientiae Rerum Naturalium 561

ISBN-13:	978-951-42-6274-6
Kieli:	englanti
Kustantaja:	Oulun yliopisto
Oppiaine:	Luonnontieteet
Painosvuosi:	2010
Sidosasu:	pehmeäkantinen
Sijainti:	Print Tietotalo
Sivumäärä:	138
Tekijät:	LAPPI ANNA-KAISA

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Protein folding of outer membrane and secreted proteins, including receptors, cytokines andantibodies is often linked to disulphide bond formation. Native disulphide bond formation iscomplex and is usually the rate limiting step in the folding of such proteins. The enzymes whichcatalyse the slow steps in disulphide bond formation belong to the protein disulphide isomerase(PDI) family. PDI catalyses formation, reduction and isomerization of newly synthesizeddisulphide bonds. The mechanisms of action of the PDIs are currently poorly understood and thisnot 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 cellularenvironment 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 ofmedically 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 modulatethe pKa of the C-terminal active site cysteine of PDI and by that way to allow the enzyme to actefficiently as catalyst both of oxidation and isomerization reactions. The possible role of hydrogen peroxide produced by sulphydryl oxidases during disulphidebond formation was studied in an oxidative protein refolding assay. Analysis showed thathydrogen peroxide can be used productively to make native disulphide bonds in folding proteinswith minimal side reactions. In addition, the kinetics of oxidation and reduction of the a domains of PDI and Pdi1p byglutathione was studied in this thesis. The kinetics obtained with stopped-flow and quenched-flowexperiments 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 ofyeast systems to predict physiological roles for mammalian PDI family members should be treatedcautiously.

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