The long-range goal of this research program is to understand the molecular basis of substrate specificity of glutathione transferases. These enzymes are a family of detoxification enzymes which are found in a wide range of species, including plants, insects, and mammals. In humans, glutathione transferases play a role in the resistance towards carcinogens and the development of drug resistance of tumors to chemotherapeutic drugs. An intriguing and functionally important property of these enzymes is their broad substrate specificity towards hydrophobic compounds. A single glutathione transferase is catalytically active on several different substrates and different glutathione transferases display different substrate specificities. The molecular mechanism of substrate specificity will be investigated by testing three, not necessarily exclusive, working hypotheses: 1) broad substrate specificity may result from the existence of several functional hydrophobic binding sites contained within the active site regions, 2) different glutathione transferases may utilize the free energy of substrate binding to alter the free energy of different positions along the reaction coordinate. The storage of free energy in different enzymes will be assessed by measuring the effect of ligand binding on amide exchange kinetics. This information will be correlated with kinetic rate constants to determine the relationship between free-energy storage and catalysis, 3) protein dynamics may play a role in substrate binding and product release by gating access to the active site. Protein dynamics will be investigated by computer modeling, measurement of 15N nuclear relaxation rates and by disulfide crosslinking. Proteins with altered dynamic properties will be generated by genetic and chemical means to confirm the relationship between protein dynamics and catalysis. These experiments will provide a comprehensive molecular description of the relationship between the structure of these enzymes and their ability to function on structurally diverse substrates. This information will be essential in the design of chemotherapeutic drugs that are not inactivated by these enzymes. A key component of the above studies is the use of NMR methods to assess the structure and dynamics of these molecules in solution.
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