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.
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Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth 76107.
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D005971:Glutamates; D018698:Glutamic Acid; D006860:Hydrogen Bonding; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009943:Organophosphorus Compounds; D010758:Phosphorus; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000737:chemistry*
D005971:Glutamates; D009943:Organophosphorus Compounds; C100168:chloroacetol phosphate; D010758:Phosphorus; D018698:Glutamic Acid; D014305:Triose-Phosphate Isomerase