Structural and regulatory properties of pyruvate kinase from the Cyanobacterium synechococcus PCC 6301.
Pyruvate kinase (PK) from the cyanobacterium Synechococcus PCC 6301 was purified 1,300-fold to electrophoretic homogeneity and a final specific activity of 222 micromol of pyruvate produced/min/mg of protein. The enzyme was shown to have a pI of 5.7 and to exist as a 280-kDa homotetramer composed of 66-kDa subunits. This PK appears to be immunologically related to Bacillus PK and a green algal chloroplast PK, but not to rabbit muscle PK, or vascular plant cytosolic and plastidic PKs. The N-terminal amino acid sequence of the Synechococcus PK exhibited maximal (67%) identity with the corresponding region of a putative PK-A sequence deduced from the genome of the cyanobacterium, Synechocystis PCC 6803. Synechococcus PK was relatively heat-labile and displayed a broad pH optimum around pH 7.0. Its activity was not influenced by K(+), but required high concentrations of Mg(2+), and was relatively nonspecific with respect to the nucleoside diphosphate substrate. Potent allosteric regulation by various effectors was observed (activators: hexose monophosphates, ribose 5-phosphate, glycerol 3-phosphate, and AMP; inhibitors: fructose 1,6-bisphosphate, inorganic phosphate, ATP, and several Krebs' cycle intermediates). The enzyme exhibited marked positive cooperativity for phosphoenolpyruvate, which was eliminated or reduced by the presence of the allosteric activators. The results are discussed in terms of the phylogeny and probable central role of PK in the control of cyanobacterial glycolysis.
Knowles
V L
VL
Departments of Biology and Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Smith
C S
CS
Smith
C R
CR
Plaxton
W C
WC
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D000458:Cyanobacteria / Q000201:enzymology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D046911:Macromolecular Substances; D021122:Protein Subunits; D011770:Pyruvate Kinase; D017868:Cyclic AMP-Dependent Protein Kinases