Structural and regulatory properties of pyruvate kinase from the cyanobacterium Synechococcus PCC 6301

Pyruvate kinase (PK) from the cyanobacterium Synechococcus PCC 6301 was pur ified 1,300-fold to electrophoretic homogeneity and a final specific activi ty of 222 mu mol of pyruvate produced/min/mg of protein. The enzyme was sho wn to have a pi of 5.7 and to exist as a 280-kDa homotetramer composed of 6 6-kDa subunits, This PK appears to be immunologically related to Bacillus P K and a green algal chloroplast PK, but not to rabbit muscle PK, or vascula r plant cytosolic and plastidic PKs, The N-terminal amino acid sequence of the Synechococcus PK exhibited maximal (67%) identity with the correspondin g region of a putative Ph-A sequence deduced from the genome of the cyanoba cterium, Synechocystis PCC 6803, Synechococcus PK was relatively heat-labil e and displayed a broad pH optimum around pH 7.0. Its activity was not infl uenced by K+, but required high concentrations of Mg2+ and was relatively n onspecific with respect to the nucleoside diphosphate substrate. Potent all osteric regulation by various effecters was observed (activators: hexose mo nophosphates, ribose 5-phosphate, glycerol 3-phosphate, and AMP; inhibitors : fructose 1,6-bisphosphate, inorganic phosphate, ATP, and several Krebs' c ycle intermediates). The enzyme exhibited marked positive cooperativity for phosphoenolpyruvate, which was eliminated or reduced by the presence of th e allosteric activators. The results are discussed in terms of the phylogen y and probable central role of PK in the control of cyanobacterial glycolys is.