Structural and kinetic properties of high and low molecular mass phosphoenolpyruvate carboxylase isoforms from the endosperm of developing castor oilseeds.
Phosphoenolpyruvate carboxylase (PEPC) is believed to play an important role in producing malate as a substrate for fatty acid synthesis by leucoplasts of the developing castor oilseed (COS) endosperm. Two kinetically distinct isoforms of COS PEPC were resolved by gel filtration chromatography and purified. PEPC1 is a typical 410-kDa homotetramer composed of 107-kDa subunits (p107). In contrast, PEPC2 exists as an unusual 681-kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated 64-kDa polypeptide (p64) that is structurally and immunologically unrelated to p107. Relative to PEPC1, PEPC2 demonstrated significantly enhanced thermal stability and a much lower sensitivity to allosteric activators (Glc-6-P, Glc-1-P, Fru-6-P, glycerol-3-P) and inhibitors (Asp, Glu, malate) and pH changes within the physiological range. Nondenaturing PAGE of clarified extracts followed by in-gel PEPC activity staining indicated that the ratio of PEPC1:PEPC2 increases during COS development such that only PEPC1 is detected in mature COS. Dissimilar developmental profiles and kinetic properties support the hypotheses that (i) PEPC1 functions to replenish dicarboxylic acids consumed through transamination reactions required for storage protein synthesis, whereas (ii) PEPC2 facilitates PEP flux to malate in support of fatty acid synthesis. Interestingly, the respective physical and kinetic properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low M(r) Class 1 and heteromeric high M(r) Class 2 PEPC isoforms of unicellular green algae.
Blonde
James D
JD
Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Plaxton
William C
WC
D000595:Amino Acid Sequence; D000460:Chlorophyta / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012278:Ricinus / Q000201:enzymology* / Q000254:growth & development; D012639:Seeds / Q000201:enzymology; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D010730:Phosphoenolpyruvate Carboxylase