Browsing by Author "Park, Sophia"
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Item Identifying key residues in the active site of 2SL (2-succino lyase)(2021) Park, SophiaSuccination is a chemical reaction that occurs spontaneously at a rate directly related to fumarate, an intermediate in the citric acid cycle. It has been observed that high fumarate levels, and consequently increased succination, in humans contributes directly to the pathogenesis of diabetes, obesity, and certain cancers through deleterious effects on proteins. The stable adduct S-(2-succino)cysteine (2SC) is commonly formed by succination of fumarate with cysteinyl residues of proteins; this 2SC adduct is an oncometabolite—a product of metabolism that accumulates in cancers—as well as a biomarker for other human diseases. The first breakdown pathway for 2SC was discovered in Bacillus subtilis. This pathway contains an enzyme that exhibits 2-succino lyase ability (2SL) in Enterococcus italicus and Dickeya dadantii. These enzymes are all members of the large ‘lyase I-like’ superfamily of lyase enzymes, many of which cannot be functionally differentiated based on sequence alone. My project aimed to determine whether certain residues in the 2SL active site are important for 2SC lyase activity. We modeled the 2SL enzyme using https://swissmodel.expasy.org/ and PyMOL. By comparing 2SL to related enzymes, we made predictions about which residues are functional significant to 2SL (including residue N13). Using primers with a mismatch at position 13, we introduced a mutation converting polar asparagine to non-polar isoleucine (N13I). We were able to express and purify both mutant and WT 2SL proteins to near homogeneity. The HPLC assay of the reverse reaction seems to suggest that the mutant N13I enzyme is more efficient (product peak area = 9251.5) than the WT (product peak area = 6998.4). The disparity between the product peaks in the forward reaction was smaller. Upon a second HPLC assay, the results of the forward reaction were not replicated. Only one run of the HPLC assay is shown, more assays will need to be done in the forward direction to determine whether the results are conclusive. The understanding of 2SL active sites will contribute insight to the ongoing efforts to identify more 2SC catabolic pathways and, more broadly, understand and treat diseases such as diabetes and cancer.Item The importance of the N13 residue to the activity of the metabolite repair enzyme 2-succino lyase (2SL)(2022) Park, Sophia;A 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.