The Effects of Straight Chain Amino Acid Derivatives of the Kinetics of γ-Glutamylcysteineligase
Chemistry, Dr. Kelly
About My Research:
Enzymes are important proteins within the body that allow chemical reactions to proceed quickly and effectively. Enzymes can stick two molecules together, pull them apart, and so much more. γ-glutamylcysteineligase (γ-GCL) is an enzyme that, with the help of another enzyme, puts together three amino acids to make a molecule called Glutathione. Glutathione acts like an antioxidant in the body because it neutralizes some potentially harmful biological byproducts (like free radicals). Unfortunately, glutathione is often present in cancer cells, so when a doctor gives a patient chemotherapy treatment, glutathione will actually attack the chemotherapeutic drugs so that they will no longer be effective. My research project deals with slowing down γ-GCL so that it makes less glutathione; with less glutathione, chemotherapy may be more effective when used as a cancer treatment. We have identified a series of molecules, called non-substrate analogs (NSAs) to slow down γ-GCL . We measure the effectiveness of these NSAs by monitoring the rate at which our enzyme makes its product in the presence of NSAs and how tightly the γ-GCL binds the various NSAs. So far, our data suggests that straight-chain, sulfur containing NSAs are the most effective at slowing γ-GCL activity. By varying NSA structure in small ways, we can determine the ideal structure for a γ-GCL inhibitor, as well as gaining a better understanding the inner workings of the enzyme.
In the student's own words :
"The connections between class and research are so numerous that it would take many pages of type to explain them all. One simple example is all of the basic mathematics from general chemistry (i.e. stoichiometry, acid/base, molarity, etc.) all come back and are really important in making detailed measurements of compounds and solutions. I would also say that many topics from organic chemistry helped me understand various mechanisms with the enzyme that I was studying."
In all eukaryotic biological systems, the enzyme γ-Glutamylcysteineligase (γ-GCL) catalyzes the first, rate limiting step of glutathione biosynthesis. Glutathione, found in cancer cells at high concentrations, has antioxidant properties and has been implicated in chemotherapeutic resistance. Inhibition of γ-GCL would theoretically reduce concentrations of glutathione within cells, and theoretically, chemotherapeutic resistance should be greatly reduced. In our research, γ-GCL activity was monitored via an indirect Lactate dehydrogenase/Pyruvate kinase assay in which oxidation of NADH was monitored via UV absorbance at 340nm. The activity of γ-GCL was also measured in the presence of non-substrate analogs (NSAs) such as D,L-Buthionine, D- Homocysteine, or D-Norleucine. For further analysis, fluorescence spectroscopy of γ-GCL allowed us to determine the binding affinity of the various NSAs to γ-GCL. Preliminary data showed that straight-chain, sulfur containing NSAs were the most effective at γ-GCL inhibition; D,L-Buthionine in particular reduced enzyme activity by half at low concentrations and also had a significantly lower kD than the other NSAs. At this point it is unclear why these derivatives inhibit the enzyme, but future studies of γ-GCL, specifically binding affinity studies and stoiceometric analyses’, will yield significant insight into this fascinating enzyme.
More student thoughts:
"The number one advantage of doing research at Gustavus is that you can start early. I am a rising sophomore and I am starting research already. At a larger institution, many students would not even hope to start a research project until their junior or even senior year. I hope to take advantage of the opportunity that was given to me and build upon my research over my next three years at Gustavus."
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