Studies on enzymatic and non-enzymatic proton transfer in aqueous solutions

Abstract

The following proton transfer systems in aqueous solutions were studied. The non-enzymatic reaction of 5'-deoxypyridoxal with L-alanine was investigated. DPL reacts with alanine to form the following products: a Claisen addition product of DPL and 5'-deoxypyridoxamine (DPM), and pyruvate. The interactions of TIM and the phosphate arm of the substrate that are important for catalysis but are not directly involved in the chemistry of TIM-catalyzed reactions were studied. The effect of the phosphite dianion on the product partitioning of the enediolate intermediate in the TIM-catalyzed reactions of the two-part substrate, glycoaldehyde and phosphite, using [1- 13 C]-glycoaldehyde ([1- 13 C]-GA) was determined. The contribution of the interaction between Lysine-12 and the substrate on the transition-state stabilization energy on TIM-catalyzed reactions by preparing a K12G mutant TIM and compared its kinetic parameters with wild-type TIM was investigated. The activation of K12G mutant TIM using small alkylammonium cations was also investigated. Finally, the effect of removing the interaction of TIM and the phosphate arm of the substrate on the mutation on TIM by comparing the product distribution of the mutant TIM-catalyzed reactions of GAP and [1- 13 C]-GA with the product distribution of wild-type TIM-catalyzed reactions of GAP and [1- 13 C]-GA, respectively was studied. The mutant TIM enzymes used were the loop-deletion mutant and the K12G mutant.