Park, Sophia2022-06-032022-06-032022https://hdl.handle.net/11299/227805A wide variety of metabolite damage reactions exist alongside long-established biochemical pathways. Succination is a spontaneous damage reaction that can occur between fumarate, an intermediate in the citric acid cycle, and cysteinyl residues of proteins. Succination of cysteinyl residues produces the toxic protein modification S-(2-succino) cysteine (2SC). High cellular levels of 2SC are found in association with mitochondrial stress, diabetes, and cancer, directly contributing to the pathogenesis of human diseases through their various deleterious effects on important metabolic proteins. Recently, a catabolic pathway to repair 2SC damage has been identified in the bacterial species Enterococcus italicus and Dickeya dadantii. The breakdown step in this pathway is performed by 2-succino lyase (2SL), a member of the lyase I-like superfamily of enzymes. The metabolite repair function of 2SL is clear. However, the key active site residues that enable 2SL activity, as opposed to the many other diverse functions of lyase Ilike superfamily enzymes, are unknown. Here we explore the significance of one candidate residue, N13, to 2SL activity by introducing a point mutation (N13I) and comparing the mutant 2SL activity to the wildtype 2SL (WT). A spectrophotometric coupled enzyme assay was performed to determine the kinetic parameters of both the N13I and WT. N13I had a significantly lower Vmax and a significantly higher Km. However, this extreme difference in parameters contradicts the preliminary HPLC data we collected which indicated a much smaller activity difference between the WT and N13I enzymes. We anticipate that the continuation of this research will contribute to the efforts to functionally characterize other lyase I-like superfamily members as 2SC lyases. More broadly, we hope that the characterization of 2SC lyases will uncover their associated—and potentially clinically significant—metabolite repair pathways.enCollege of Biological SciencesBiologySumma Cum LaudeThesis or Dissertation