Enzyme kinetics studies have shown that Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH) and other drosophilid alcohol dehydrogenases function by a compulsory-ordered mechanism where the coenzyme binds to the free enzyme, and that a proton is released upon formation of the binary enzyme-NAD(+) complex. A proton relay mechanism for the proton abstraction has been suggested that includes an eight-membered chain of water molecules connecting the active site with the bulk solvent. Thr114 bridges between two water molecules in the water chain. In a previous structural and enzyme kinetic study of a Thr114 Val mutant of SlADH, we showed that an intact water chain is essential for full enzyme activity. In the present study, comparative molecular dynamic (MD) simulations of the wild type and the SlADH(T114V) were performed. The simulations showed differences in hydrogen bonding properties and dynamics between the wild type and the SlADH(T114V). Differences in molecular dynamical behaviour were seen in the loop of importance for binding the nicotinamide part of NAD(+), in the region important for binding the adenine part of NAD(+), and in the region of the amino acid at position 114. The substrates also had more freedom for conformational changes in active site of the wild type SlADH than of the SlADH(T114V). The differences in hydrogen bonding properties and MDs between the wild type and mutant could not have been observed from the X-ray crystal structures only.