Dynamic, Structural, and Mechanistic Study of Glutathione Transferases

Abstract

This project involved the investigation of both the dynamic features of the dimer interface of the Mu class GSH transferase rGSTM1-1, as well as, the structural and mechanistic characteristics of HCCA Isomerase, an enzyme related to the mitochondrial (Kappa) GSH transferase family. The dynamics along the dimer interface of rGSTM1-1 were probed by using site-directed mutagenesis and hydrogen-deuterium exchange mass spectrometry. This work led to hypotheses about the roles of both hydrophobic and electrostatic motifs along the interface. It also resulted in more clear understanding of the regions within each monomer that are important for the stability of individual subunits. Crystallography of HCCA Isomerase in conjunction with activity assays on the native substrates allowed us to conclude that this enzyme is definitely a Kappa GSH transferase. Transient state kinetic measurements with native substrates and analogs as well as crystallography with one of the analogs allowed us to propose a global pathway for HCCA Isomerase’s catalytic mechanism.