title: Interactions between the catalytic centers and subunit interface of triosephosphate isomerase probed by refolding, active site modification, and subunit exchange.
abstract: The effects of unfolding, refolding, and hybridization of triosephosphate isomerase (TPI) subunits from different species and subunits which have been specifically modified at the active site have been examined. These effects have been evaluated in terms of changes in catalytic parameters, CD spectra, and susceptibility to denaturation. Dissociation followed by reassociation yields an active dimer but with increased Km, reduced kcat, and increased susceptibility to inactivation and unfolding in denaturants. These data suggest that while the general structure of the refolded dimer is similar to the native enzyme, its complete original structure is not restored. Covalent reaction of the active site Glu165 with the substrate analogue 3-chloroacetol phosphate (CAP) results in dimers with increased susceptibility to unfolding and inactivation by denaturants (i.e. the rates of inactivation and unfolding are (TPICAP)2 greater than (TPI-TPICAP) greater than (TPI)2). These data point to the interactions between the catalytic center and the subunit interface. Subunits of TPI from different species, in spite of structural differences at the subunit interface, hybridized to active heterodimers. Subunit hybridization was random among monomers from different mammals, preferential between yeast and mammalian or avian monomers. Hybridization did not occur between avian and mammalian monomers under these conditions. These data provide information on the elements in the interface of the dimer and the relationship of the catalytic center with the subunit interface.
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: R W
authors.initials: RW
authors.lastname: Gracy
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002645:Chickens; D002942:Circular Dichroism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D009943:Organophosphorus Compounds / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013045:Species Specificity; D013552:Swine; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D009943:Organophosphorus Compounds; C100168:chloroacetol phosphate; D014305:Triose-Phosphate Isomerase