Acid-Base Catalysis of the Elimination and Isomerization Reaction of Triose Phosphates

Article abstract of DOI:10.1021/ja00329a050

The nonenzymatic β-elimination, isomerization, and racemization reactions of L-glyceraldehyde 3-phosphate (LGAP) are through a common enediolate intermediate which partitions between leaving group expulsion, C-1 protonation, and C-2 protonation, respectively.The elimination reaction mechanisms of LGAP and dihydroxyacetone phosphate (DHAP) are Elcb because enolate intermediates have been identified in very closely related systems.Strong general base catalysis of elimination demontstrates that the enediolate intermediate is formed essentially irreversibly by rate-determinig substrate deprotonation.The pH rate profile for the elimination of LGAP is first order in hydroxide at pH > 10 due to direct substrate deprotonation by hydroxide, pH independent at pH 6-10 due to intramolecular deprotonation by the C-3 phosphate dianion, and first order in hydroxide at pH < 6 due to a decrease in the reactive phosphate dianion form of the substrate.The Bronsted β values for buffer catalysis of DHAP and LGAP elimination by 3-substituted quinuclinides are 0.48 and 0.45 respectively.The solvent deuterium isotope effect on the second-order rate constant for quinuclidinone catalysis of DHAP elimination is 1.1.At > 0.2 M concentrations of 3-oxo- and 3-hydroxy-substituted quinuclidine buffers there is a curvature in buffer catalysis plots for the elimination reaction of DHAP which is attributed to a change from rate determining substrate deprotonation to partially rate determining leaving group expulsion.The isomerization and racemization reactions of LGAP were followed by coupling the formation of DHAP andDGAP to enzymatic NADH oxidation under conditions where > 90percent of the product is from the elimination reaction.Uncatalyzed racemization is five times slower than uncatalyzed isomerization, and buffer-catalyzed racemization is estimated to be > 20 times slower than buffer-catalyzed isomerization.The observed rate constant for LGAP isomerization is second order in total buffer concentration; the basic form of buffer acts to increase the steady-state concentration of the enediolate, and the acidic form of the buffer increases the fractional partitioning of the enediolate to DHAP.Rate constant ratios k_BH/k_e and k_-o/k_e for partitioning of the enediolate between buffer-catalyzed or uncatalyzed protonation (k_BH or k_-o) and leaving group expulsion (k_e) were obtained from the slopes and the intercepts respectively of linear plots of the isomerization/elimination rate constant ratio against buffer concentration.The Bronsted α value for enediolate protonation at C-1 is 0.47.The (k_BH/k_e)_H2O/(k_BD/k_e)_D2O ratio for quinuclidinonium catalysis in H2O and D2O is 3.2.The pH dependence plot of k_BH/k_e values shows pH-independent regions at pH < 7 and > 10, with a 100-fold greater limiting k_BH/k_e value at pH > 10.The increased k_BH/K_e value at high pH is due to slower leaving group expulsion from the enediolate phosphate dianion compared to that of the enediolate phosphate monoanion.The nonenzymatic reactions of triose ...