Redesign of GSTA1-1 for mechanistic studies: dynamics of the C-terminus

GSTA1-1 contains within the C-terminal 13 residues a helix that undergoes a ligand-dependent disorder-to-order transition. In the apo enzyme, the C-te rminus is mobile or statically heterogeneous. In the presence of GSH conjug ates, the C-terminus forms a helix that restricts access to the active site . In order to explore the function of this helix and the point of the react ion cycle at which it becomes ordered, X-ray crystallography, stopped-flow kinetics, and titration calorimetry were performed with mutant proteins. Th e results indicate that aromatic-aromatic interactions between Phe-10, Tyr- 9, and Phe-220 are critical for the C-terminal transition. The catalytic Ty r-9 also plays a previously unappreciated role in orchestrating the dynamic s of the C-terminus, which becomes ordered at a point downstream of GSH bin ding. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.