title: Functional significance of protein conformational isomerisation in the glyceraldehyde-3-phosphate dehydrogenase-catalysed reaction.
abstract: The tetrameric mung bean glyceraldehyde-3-phosphate dehydrogenase is found to bind approximately four moles substrate, glyceraldehyde-3-phosphate, per mole enzyme with Kdiss equal to or less than 9.6 microM at pH 7.3, showing a slight positive cooperativity. Addition of excess substrate to a solution of the enzyme and excess NAD+ leads to a "burst" of NADH formation followed by a slow linear increase (monitored spectrophotometrically). Amount of NADH formed in the burst phase is pH-dependent and is equal to 3.6 moles per mole enzyme at pH 8.6 and above. Presuming four equivalent and independent sites per enzyme molecule (i.e. D2-symmetry), consistent values were obtained for the equilibrium constant of the oxidation-reduction step at different pH and most substrate concentrations. At lower pH (7.3) and high [NAD+]/[substrate] ratios, favouring the C2- symmetry conformation of the enzyme, the magnitude of the burst phase was negligibly small; practically no oxidation reduction reaction took place. Combining these with earlier results on the group transfer step, it is suggested that the oxidation-reduction and group transfer steps of the reaction catalysed by this enzyme require the D2 and C2 symmetry conformations of the enzyme, respectively.
authors.affiliation: Department of Chemistry, Banaras Hindu University, Varanasi, India.
authors.forename: O P
authors.initials: OP
authors.lastname: Malhotra
authors.lastname: Srinivasan
authors.forename: K
authors.initials: K
authors.lastname: Tikoo
authors.forename: A M
authors.initials: AM
authors.lastname: Kayastha
mesh_terms: D002384:Catalysis; D007887:Fabaceae / Q000201:enzymology; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000378:metabolism*; D010946:Plants, Medicinal; D011487:Protein Conformation
chemical_list: D010940:Plant Proteins; D009243:NAD; D005986:Glyceraldehyde 3-Phosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases