Date of Award
Doctor of Philosophy (PhD)
The human detoxification enzyme glutathione s-transferase alpha 1 (GSTA1-1) is known for its ability to conjugate a variety of different hydrophobic xenobiotics to the tripeptide glutathione. The conjugation can be catalyzed through either a substitution or an addition reaction. Which reaction mechanism is being used affects the cooperativity of the enzyme. GST is also capable of catalyzing isomerization reactions of particular substrates without conjugation to GSH which further extends its repertoire. The C-terminal of the enzyme transitions from a random loop to an α-helix that localizes over the active site as a ligand binds. The α-helix is also involved in product release. We hypothesize that the broad substrate specificity and the catalytic flexibility of GST is a result of a heterogeneous protein population where multiple conformers with different properties coexist. To identify different conformers in a population a single molecule approach is needed. Fluorescently tagged substrates and products of GSTA1-1 have been imaged as they bind immobilized GSTA1-1 using TIRF microscopy. Single molecule binding events can be analyzed to characterize different binding states of the enzyme. The observation of at least two average occupancy times suggests that there are multiple binding states and conformations of the enzyme. One of these binding states is dominating the population and a large number of binding events has to be sampled to pick up the more rare states. To further characterize the behavior of GSTA1-1 FRET and DEER have been used to study the active site dynamics during binding of a ligand. Unlike X-ray crystallography, DEER and FRET provide a distribution in a distance between groups, and not just an average. Our data suggest that binding of S-hexylglutathione does not localize the C-terminal helix which is contrary to what can be observed in the X-ray crystal structure. Glutathione alone is sufficient to localize the helix. This highlights the importance of using proteins in solution to study their conformation.
Pettersson, John, "Single Molecule Studies of hGSTA1-1: Binding Kinetics and Active Site Dynamics" (2015). Dissertations. 620.