Steady-state kinetic mechanism of Thermoanaerobium brockii alcohol dehydrogenase: A study of discrimination between alternative kinetic models

Two steady-state kinetic mechanisms have been proposed for the oxidation of 2-propanol catalyzed by Thermoanaerobium brockii alcohol dehydrogenase (TBADH): the sequential ordered BiBi mechanism and the Theorell-Chance BiBi mechanism. Discrimination between these kinetic mechanisms was achieved by analyzing the inhibition produced by acetone, the first product of the reaction sequence in relation to the second substrate of the enzyme (2-propanol). Two types of experimental data were used: a data set combining initial rate experiments in the absence of products with data involving inhibition by acetone in relation to 2-propanol as variable substrate; and, data sets just containing product inhibition experiments by acetone with 2-propanol as variable substrate. The results obtained strongly suggest that the kinetic mechanism of TBADH is described by the Theorell-Chance BiBi kinetic model.