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Many studies have been done on the glyoxalase system due to its potential implications for the design of new anti-cancer and anti-protozoal drugs. The glyoxalase system plays a significant role in cellular chemical detoxification. Tumor cells, as well as cells affected by the malaria parasite and certain complications of diabetes have increased levels of glyoxalase activity; therefore, inhibition of these enzymes could potentially serve as an effective treatment for these diseases. Despite sharing conserved amino acids known to bind zinc, different forms of glyoxalase II have been shown to exhibit differing metal binding preferences. A study published in 2000 found that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center essential for activity and suggested an overall reaction mechanism (Zang, et al., 2001). The next step in the effort to provide information about glyoxalase II that can be used for the rational design of inhibitors for human treatment is to study the human form of glyoxalase II to find out if it possesses these same characteristics. In 1999, Cameron, et al. did a study on the crystal structure of human glyoxalase 2-2 and claimed it has a dizinc nuclear binding site; however, they never accounted for the 0.7 equivalents of iron they observed in the sample. Thus, there were questions concerning the metal binding properties of human glyoxalase 2-2. The research project discussed here involved the over-expression and purification of the human GLX 2-2 enzyme in order to characterize its kinetic properties, substrate specificity, and metal binding properties. Human glyoxalase 2-2 was found to have a dinuclear zinc binding site as was proposed by Cameron et al. Human glyoxalase 2-2 grown in the presence of manganese also yielded high activity enzyme. Growing the enzyme with only iron added to minimal media resulted in inactive enzyme. |
