Elucidation of glycoside hydrolase mechanisms by measuring kinetic isotope effects using direct NMR method

Enzymes that catalyze the removal of carbohydrate units from biological molecules are called glycoside hydrolases (GH). These enzymes have been categorized into more than 150 different families. This thesis presents an analysis of the mechanistic aspects of three glycoside hydrolases elucidated by measuring kinetic isotope effects (KIEs) by a direct nuclear magnetic resonance (NMR) spectroscopic method. A review of scope and limitations of the NMR method for competitive heavy atom (13C, 18O, 15N) and secondary deuterium KIEs measurement in biological systems is provided. A method for continuous monitoring of isotopically enriched materials is described in detail including the current state of instrumentation and computer programs for data acquisition and analysis. In order to refine the mechanistic understanding of the glycoside hydrolase family 4 (GH4) α-galactosidase from Citrobacter freundii (MelA), leaving group effects were measured with various metal cations and competitive deuterium KIEs were measured with singly and doubly deuterated activated substrates, 2-fluorophenyl and 4-fluorophenyl α-D-galactopyranosides, in the presence of Sr2+, Y3+, and Mn2+. The observations are consistent with hydride transfer at C-3 to the on-board NAD+, deprotonation at C-2, and a non-chemical step contributing to the virtual TS for V/K. The α-D-glucopyranosyl fluoride (α-GlcF) hydrolysis catalyzed by GH15 inverting α-glucoamylases from A. niger and Rhizopus sp. has been studied by use of multiple competitive kinetic isotope effect measurements. The experimental KIEs are consistent with the enzymatic reaction occurring via an SN1-type mechanism, in which the transition state has significant pyranosylium-ion like character and is late with respect to C−F bond cleavage. α-D-Glucopyranosyl fluoride is hydrolyzed by the family 55 inverting exo-1,3-β-glucanase from Trichoderma virens via the Hehre resynthesis–hydrolysis mechanism. The transition state for the Hehre resynthesis-hydrolysis mechanism for the GH55 catalyzed hydrolysis of α-GlcF has been studied by the use of multiple kinetic isotope effect measurements. The transition state for the Hehre resynthesis-hydrolysis reaction is late with respect to both C–F bond cleavage and proton transfer.