The hinged lid of yeast triose-phosphate isomerase. Determination of the energy barrier between the two conformations.

Covalent modification of Glu165 in the catalytic center of triose-phosphate isomerase with the substrate analogue 3-chloroacetol phosphate traps the complex in two conformations. The two resulting 31P NMR resonances at 6.9 and 5.7 ppm appear to reflect conformations in which the hinged lid (residues 167-176) is in the open and closed positions. The conformation represented by the 5.7-ppm resonance is more stable, and unfolding and refolding in guanidine converts all of the molecules to the 5.7-ppm conformation. The complete conformational transition from 6.9 to 5.7 ppm also takes place as a function of time and temperature. Under these conditions the native enzyme retains more than 80% of the catalytic activity, indicating that this conversion is not due to thermal denaturation of the enzyme. Circular dichroic and fluorescence spectroscopy indicate that the 3-chloroacetol phosphate-modified enzyme does not undergo major structural changes. From the temperature dependence of this transition, an energy barrier of 144 kJ/mol (34.4 kcal/mol) was calculated for this conversion.