THE HINGED LID OF YEAST TRIOSE-PHOSPHATE ISOMERASE - DETERMINATION OFTHE ENERGY BARRIER BETWEEN THE 2 CONFORMATIONS

Covalent modification of GlU(165) in the catalytic center of triose-ph osphate isomerase with the substrate analogue 3-chloroacetol phosphate traps the complex in two conformations. The two resulting P-31 NMR re sonances at 6.9 and 5.7 ppm appear to reflect conformations in which t he 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 f rom 6.9 to 5.7 ppm also takes place as a function of time and temperat ure. 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 fluorescenc e spectroscopy indicate that the 3-chloroacetol phosphate-modified enz yme does not undergo major structural changes. From the temperature de pendence of this transition, an energy barrier of 144 kJ/mol (34.4 kca l/mol) was calculated for this conversion.