Acetate kinase (EC 2.7.2.1), an enzyme involved in the wasteful production of acetate during conversion of cellulose to ethanol by Clostridlum thermocellum, was purified 144-fold. The enzyme has an Mr of 84 kD by non-denaturing gradient gel electrophoresis, and an Mr of 46 kD when estimated with a denaturing gel; thus it appears to be a homodimer. Optimum enzyme activity occurs at 50Β°C and between pH 7.2 and 8.0. Acetate kirm~c,e is stable to temperatures up to 60Β°C, but is completely inactivated at 80Β°C after two h. The enzyme is stable between pI-I 7.0 and 9.0 when incubated at 50Β°C for two h. Optimum acetate kinase activity occurs at a MgCI2:ATP ratio of 2:1, which indicates an interaction between Mg 2+ and ATP and that between Mg 2+ and acetate klna~. Enzyme activity is partially inhibited by KCI, an inorganic salt frequently used in chromatography and fermentation media, losing 60% activity in the presence of 0.2 M KCI. Signmidal enzyme kinetics were observed from the velocity plot of acetate trin~Lqe when either the acetate (So5 = 285 raM) or ATP (So.5 --11 raM) concentration was varied, suggesting cooperative binding of the two substrates.