The long range goal of this research program is to determine the molecular basis of the enzymatic mechanism of a class of enzymes called glutathione transferases. These proteins form a family of detoxification enzymes that function by conjugating glutathione to a wide variety of potentially harmful hydrophobic compounds. They have been shown to play an important role in the initiation of tumor growth, as well as in the development of resistance to chemotherapeutic drugs. Collectively, these enzymes show a remarkably broad range of substrate specificities. The goal of this proposal is to determine the relationship between the molecular dynamics and enzyme function for these enzymes. The x-ray derived structures of six classes are currently known. Although the overall fold of these enzymes are similar, each class possesses a distinct molecular architecture which affects both the substrate specificity as well as the enzyme mechanism. For three of these classes (alpha, mu, and pi), a large number of x-ray derived structures of these proteins have been determined. In some cases, these structures show considerable change in the conformation of the enzyme due to ligand binding. In other cases, substrate binding causes little change in structure. Since all of these studies have been performed in the crystalline lattice, the extent and importance of ligand induced changes on the structure and dynamics of these enzymes in solution is unknown. A more comprehensive understanding of these enzymes will be useful in the development of more useful chemotherapeutics. The specific aims of this proposal are to investigate substrate induced changes in the dynamics of human class mu, pi, alpha, and theta enzymes by NMR spectroscopy. The first hypothesis to be tested is that molecular dynamics of the backbone plays an important role in the enzymatic mechanism of these enzymes by gating substrate accessibility and product release. The dynamic properties of the backbone atoms in these enzymes in the presence and absence of various substrates and products will be investigated with measurements of amide exchange kinetics, residual dipolar coupling, chemical exchange, and 15N nuclear relaxation. The second hypothesis to be tested is that the dynamic properties of side-chain residues play an important role in the recognition of different substrates by the same enzyme. Side chain dynamics of wild-type and mutant proteins will be characterized by '3C, 2H, and 19F nuclear spin relaxation.