Acetate Kinase From Methanosarcina thermophila, a Key Enzyme for Methanogenesis

Materials and procedures are described for the overproduction and purification of acetate kinase from Methanosarcina thermophila. Methods are detailed for large-scale preparation of the unaltered enzyme and for overproduction and one-step purification of the histidine-tagged recombinant acetate kinase. Also included is a method for routine assay of the enzyme, and enzymelinked assay procedures for obtaining kinetic constants in the forward and reverse reaction directions. Finally, a method is included for the overproduction and purification of a variant of the acetate kinase with an altered K_m for acetate. A method is presented utilizing the variant for determination of acetate levels in biological samples.