title: Slow proton transfer from the hydrogen-labelled carboxylic acid side chain (Glu-165) of triosephosphate isomerase to imidazole buffer in D2O.
abstract: The catalytic base at the active site of triosephosphate isomerase (TIM) was labelled with -H by abstraction of a proton from substrate d-glyceraldehyde 3-phosphate to form an enzyme-bound enediol(ate) in D2O solvent. The partitioning of this labelled enzyme between intramolecular transfer of -H to form dihydroxyacetone phosphate (DHAP), and irreversible exchange with -D from solvent was examined by determining the yields of H- and D-labelled products by 1H NMR spectroscopy. The yield of hydrogen-labelled product DHAP remains constant as the concentration of the basic form of imidazole buffer is increased from 0.014 to 0.56 M. This shows that the active site of free TIM, which has an open conformation needed to allow substrate binding, adopts a closed conformation at the enediolate-complex intermediate where the catalytic side chain is sequestered from interaction with imidazole dissolved in D2O.
authors.affiliation: Department of Chemistry, University Science Laboratories, South Road, Durham, United Kingdom DH1 3LE. annmarie.odonoghue@durham.ac.uk
authors.forename: AnnMarie C
authors.initials: AC
authors.lastname: O'Donoghue
authors.forename: Tina L
authors.initials: TL
authors.lastname: Amyes
authors.forename: John P
authors.initials: JP
authors.lastname: Richard
mesh_terms: D001665:Binding Sites; D002021:Buffers; D002264:Carboxylic Acids / Q000737:chemistry*; D002384:Catalysis; D017666:Deuterium Oxide / Q000737:chemistry*; D005986:Glyceraldehyde 3-Phosphate / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D015394:Molecular Structure; D011522:Protons; D014305:Triose-Phosphate Isomerase / Q000737:chemistry*
chemical_list: D002021:Buffers; D002264:Carboxylic Acids; D007093:Imidazoles; D011522:Protons; D005986:Glyceraldehyde 3-Phosphate; C029899:imidazole; D006859:Hydrogen; D014305:Triose-Phosphate Isomerase; D017666:Deuterium Oxide