SUBSTRATE SPECIFICITY OF GLUTATHIONE TRANSFERASES

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The long range 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 toward 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 toward hydrophobic compounds. A single glutathione transferase is catalytically active on several different substrates and different glutathione transferase display different substrate specificities. The molecular mechanism of substrate specificity will be investigated by testing three, not necessarily exclusive, working hypotheses: Broad substrate specificity may result from the existence of several functional hydrophobic binding sites contained within the active site region. To test this hypothesis residues in contact with different hydrophobic substrates will be identified by magnetization transfer experiments. The potential involvement of certain residues in substrates binding and subsequent catalysis will be tested by site-directed mutagenesis. Different glutathione transferases may utilize the free energy of substrate binding to alter the free energy of different positions along the reaction co-ordinate. 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. 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 N-15 nuclear relaxation rates, and by disulfide cross-linking. Protein 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 inactive by these enzymes.