Molecular and regulatory properties of leucoplast pyruvate kinase from Brassica napus (rapeseed) suspension cells.
Plastidic pyruvate kinase (PK(p)) from Brassica napus suspension cells was purified 431-fold to a final specific activity of 28 micromol phosphoenolpyruvate (PEP) utilized/min/mg protein. SDS-PAGE, immunoblot and gel filtration analyses indicated that this PK(p) exists as a 380-kDa heterohexamer composed of equal proportions of 64- (alpha-subunit) and 58-kDa (beta-subunit) polypeptides. The N-terminal sequence of the PK(p) alpha- and beta-subunits exhibited maximal identity with the corresponding regions deduced from putative PK genes of Arabidopsis thaliana and Methylobacterium extorquens, respectively. B. napus PK(p) displayed a sharp pH optimum of pH 8.0, and hyperbolic saturation kinetics with PEP and ADP (K(m) = 0.052 and 0.14 mM, respectively). 6-Phosphogluconate functioned as an activator (K(a) = 0.12 mM) by increasing V(max) by approximately 35% while decreasing the K(m)(PEP) and K(m)(ADP) values by 40 and 50%, respectively. 2-Oxoglutarate and oxalate were the most effective inhibitors (I(50) = 8.3 and 0.23 mM, respectively). A model is presented which highlights the role of 6-phosphogluconate in coordinating stromal NADPH and ATP production for anabolic processes of B. napus leucoplasts.
Plaxton
William C
WC
Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6. plaxton@biology.queensu.ca
Smith
Christopher R
CR
Knowles
Vicki L
VL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D029688:Brassica napus / Q000201:enzymology*; D002412:Cations; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010070:Oxalates / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010728:Phosphoenolpyruvate / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D002412:Cations; D007656:Ketoglutaric Acids; D008670:Metals; D010070:Oxalates; D010455:Peptides; D001224:Aspartic Acid; D018698:Glutamic Acid; D009249:NADP; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase