Date of Award

10-5-2017

Publication Type

Master Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

Keywords

Enzymology, Kinetics, Redox Biology, Regulation

Supervisor

Mutus, Bulent

Rights

info:eu-repo/semantics/openAccess

Abstract

Despite its study since the 1960's, very little is known regarding the regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI). A variety of conserved residues have been implicated as either important or vital for activity. This work ventures to identify a functional role for the highly conserved CGHC-flanking residues Lys57 and Lys401 of human PDI in vitro. Site-directed mutagenesis studies have revealed a role for the active site lysine residues in modulating the oxidoreductase activity of PDI in a pH-dependent manner. The effects of mutagenesis indicated that, along with the oxidoreductase activity, the kinetics of thiol-reductase and thiol-oxidase catalysis were also attenuated. Substitution of the aforementioned residues to glutamine, alanine or glutamic acid resulted in an enzyme variant 20 to 35% as efficient as wild type. This was found to translate to the decreased rate of electron shuttling between PDI and ERO1α to as little as 54% that of wild type. Consistent with this, molecular dynamic simulations suggest a role for Lys57 and Lys401 in differentially mediating the accessibility of the nucleophilic cysteine of each active site. The possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using Aspirin, coupled with mass spectrometry. A total of 18 acetyllysine (acK) residues were identified reproducibly, including acK57 and acK401. Altered enzyme kinetics as a result of acetylation by Aspirin corroborate with site-directed mutagenesis data. This provides a strong indication that acetylation of residues Lys57 and Lys401 is a potential modulator of the catalytic activities of PDI.

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