Purification and characterization of acetate kinase from Clostridium thermocellum

Acetate kinase (EC 2.7.2.1), an enzyme involved in the wasteful production of acetate during conversion of cellulose to ethanol by Clostridium 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 50 degrees C and between pH 7.2 and 8.0. Acetate kinase is stable to temperatures up to 60 degrees C, but is completely inactivated at 80 degrees C after two h. The enzyme is stable between pH 7.0 and 9.0 when incubated at 50 degrees C for two h. Optimum acetate kinase activity occurs at a MgCl2:ATP ratio of 2.1, which indicates an interaction between Mg2+ and ATP and that between Mg2+ and acetate kinase. Enzyme activity is partially inhibited by KCl, an inorganic salt frequently used in chromatography and fermentation media, losing 60% activity in the presence of 0.2 M KCI. Sigmoidal enzyme kinetics were observed from the velocity plot of acetate kinase when either the acetate (S-0.5 = 285 mM) or ATP (S-0.5 = 11 mM) concentration was varied suggesting cooperative binding of the two substrates. (C) 1998 Elsevier Science Ltd. All rights reserved.