Purification and properties of pyruvate kinase from Mycobacterium smegmatis

Pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S 0.5 v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest K m value. The enzyme showed an absolute requirement for divalent cations (either Mg 2+ or Mn 2+), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg 2+-activated enzyme to varying degrees (Ni 2+ > Zn 2+ > Cu 2+ > Ca 2+ > Ba 2+). The differences in the kinetic responses of the enzyme to Mg 2+ and Mn 2+ are discussed.