A Description Of Enzymatic Catalysis In N-Acetylhexosamine 1-Kinase: Concerted Mechanism Of Two-Magnesium-Ion-Assisted Gicnac Phosphorylation, Flexibility Behavior Of Lid Motif Upon Substrate Recognition, And Water-Assisted Gicnac-1-P Release

The N-acetylhexosamine 1-kinase (NahK) is the typical example of anomeric kinases acting on gluco-type substrate, which catalyzes the phosphorylation of GlcNAc or GalNAc at anomeric Cl position with ATP, playing a crucial role in bifidobacteria metabolic pathway and biosynthesis of sugar 1-phosphates and oligosaccharides. Herein, by using state-of-the art ab initio QM/MM MD and MM MD simulations, one-dimensional and two-dimensional free energy profiles to descript catalytic process have been explored. A concerted mechanism has been recognized for the delivery of phosphate group and proton to product ADP and GlcNAc-1-P with the free energy barrier of similar to 7.0 kcal/mol. The chemical reaction is assisted by two Mg2+ ions with a standing six coordination structure during the enzymatic process. The deficiency of mg(2+) bridging beta-gamma phosphates of ATP may alter the catalytic mechanism and brings higher free energy barriers. The protonation of G1cNAc-1-P is beneficial because its eliminates the Mg2+ ion binding. The water-assisted G1cNAc-1-P cleavage from binding of Mg2+ requires, similar to 9.4 kcal/mol at least, which means that the pi-pi bond breaking in the chemical reaction step is probably not the rate-limiting step in the entire enzymatic process. A strongly exothermic phenomenon and an open-closed structural change of lid motif have been observed upon the GlcNAc binding. The exothermic trend is strongly dependent on the quantity and quality of the hydrogen bond network around the ligand.