Date of Award

3-10-2019

Publication Type

Master Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

Supervisor

Bulent Mutus

Rights

info:eu-repo/semantics/openAccess

Abstract

Hydrogen sulfide was known as a toxic, flammable gas, until 1996 when it was shown that H¬2S plays an active role within the body. Cystathionine γ-lyase (CSE) is one of the enzymes responsible for the production of H2S within the body. Little to nothing is understood about the regulation of CSE, specifically two conserved CxxC residues found in each monomer of the enzyme. The work aimed to identify the possible regulatory or catalytic role of each CxxC motif within CSE. Site-directed mutagenesis revealed the effect of each cysteine residue on the catabolism of cystathionine, as well as its effect on varying pH. The Cys252 residue was shown to be of genetic importance; point mutation of this residue rendered the enzyme inactive. Cys255, Cys307, and Cys310 showed little to no impact on the function of the enzyme. Cys307-X-X-Cys310 is located on the periphery of the enzyme, while Cys252-X-X-Cys255 is located in the interior, at the dimer-dimer interface. This suggests a structural role for the 200 CxxC and a more catalytic role for the 300 motif. The catalytic potential of the 300 motif was interrogated with synthesized free thiol fluorescent probes, which mimic an already established one in the Mutus lab. The Cys307-X-X-Cys310 motif exhibited a specificity toward the FITC2-homocysitne probe, due to the nature of the dihedral disulfide bond angle. This was further shown through fluorescent kinetics, as well as a fluorescent labeling and imaging system based around SDS-PAGE. Lastly, mass spectrometry was employed to detect the modification site, but no positive result has been observed thus far. However, holoenzyme mass spectrometry has shown an addition of homocystine to the Cys307 and Cys310, as well as the WT enzyme. Together, this provides a strong indication of a secondary active site responsible for the reduction of homocystine to feed downstream pathways which CSE is implicated in.

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