title: Effects of active site modification and reversible dissociation on the secondary structure of triosephosphate isomerase.
abstract: Binding of ligands to the catalytic center of mammalian triosephosphate isomerase (TPI) induces a conformational change(s) that enhances the specific deamidation of Asn71 at the subunit interface. Deamidation initiates dissociation and degradation of the enzyme in vivo and in vitro. We have utilized circular dichroism spectroscopy to examine the conformational changes in the enzyme upon ligand binding and subunit dissociation/reassociation. Native TPI from rabbit, chicken, and yeast exhibit similar spectra at pH 7.5, but are substantially different at pH 9.5. Covalent reaction of the active site Glu 165 with the substrate analogue 3-chloroacetol phosphate results in a conformational change (decrease in beta-sheet) which is similar in TPI from all three species. Reversible dissociation of the dimeric enzyme in guanidine followed by dialysis, although permitting full recovery of catalytic activity, results in refolded dimers with decreased alpha-helix. These conformational changes induced by ligand binding, pH, or reversible dissociation explain, in part, the differences in the chemical and physical properties of the enzyme from the three species at alkaline pH, the increased lability of the dissociated/reassociated enzyme, and corroborate 31P NMR data on substrate-induced conformational changes. These studies also support the concept of molecular wear and tear whereby ligand binding at the catalytic center induces conformational changes that increase the probability of covalent modification and ultimate degradation of the protein.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of North Texas/Texas College of Osteopathic Medicine, Fort Worth 76107.
authors.forename: A Q
authors.initials: AQ
authors.lastname: Sun
authors.forename: K U
authors.initials: KU
authors.lastname: Yüksel
authors.forename: G S
authors.initials: GS
authors.lastname: Rao
authors.forename: R W
authors.initials: RW
authors.lastname: Gracy
mesh_terms: D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D014305:Triose-Phosphate Isomerase / Q000737:chemistry*
chemical_list: D014305:Triose-Phosphate Isomerase