The induced-fit model of substrate specificity adequately explains many enzyme-substrate interactions but fails to explain the diversity of substrates of many drug-metabolizing enzymes. Glutathione S-transferase A1-1, an example of a drug-metabolizing enzyme with broad substrate specificity, has a highly dynamic C-terminal helix that may help it accommodate a variety of substrates. To better understand the mechanism of GST A1-1 catalysis - and, in doing so, attempt to answer broader enzymological questions about how dynamic regions of proteins catalyze reactions - we are studying how GST A1-1 conjugates glutathione to the substrate monobromobimane, a reaction that demonstrates burst kinetics.
Viscosity titrations and other experiments show that the burst is due to a slow conformational change localized to the C-terminus. We are using stopped-flow fluorescence to determine the rates of individual steps of the reaction with monobromobimane, and we plan to determine the reaction rates with a series of C-terminal mutants as well. We then plan to measure the order parameter of the wildtype protein and the mutants using time-resolved fluorescence. Linear free-energy relationships will be used to probe the relative order vs. disorder of the C-terminus during the transition state. We currently are in the process of making the C terminal mutants and plan to begin making anisotropy measurements soon.