Molecular forms and kinetic properties of phosphoenolpyruvate carboxylase from barnyard grass (Echinochloa crus-galli(L.) Beauv.: Poaceae)

摘要

The thermal, kinetic, and electrophoretic properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were analyzed in plants from two ecotypes of barnyard grass (Echinochloa crus-galli(L.) Beauv.: Poaceae) originated from sites of contrasting climates in Quebec (QUE) and Mississippi (MISS). The thermostability, cold inactivation, the apparent energy of activation (Ea), the Michaelis-Menten constant (Km), andVmax/Kmratios for phosphoenolpyruvate (PEP) and Mg2+were analyzed with desalted Sephadex G-25 crude PEPC extracts, with partially purified PEPC from the polyethylene glycol (PEG) 13 fraction and with purified PEPC obtained after elution from DEAE-Sepharose affinity chromatography. PEPC from illuminated leaves from both ecotypes consisted of one isomorph with the same electrophoretic mobility in polyacrylamide gels, similar molecular masses for the native enzyme (400 kDa) and for each subunit of the tetramer (100 kDa), and a same isoelectric point (pI) of 4.95. The only kinetic property for PEPC for which differences of physiological importance among ecotypes were observed at the three levels of purification wasKmfor PEP for which values for QUE plants were significantly lower at low assay temperatures. Differences among ecotypes for thermostability were only observed in assays with crude and partially purified PEPC extracts, while no differences were found for cold inactivation rates,Km(Mg2+) estimates at any level of purification or forVmax/Kmratios (PEP or Mg2+) from purified PEPC. Significant differences among the two ecotypes were found for catalytic constant (Kcat) estimates obtained with purified PEPC. However, results show higher catalytic efficiency for PEPC from MISS plants at high assay temperatures but no indication of an improved catalytic efficiency for PEPC from QUE plants at low assay temperatures. The lack of ecotypic differences for most thermal and kinetic properties observed with purified PEPC casts doubts about the evolutionary interpretations of results obtained in previous kinetic comparative analyses, which were based on crude or partially purified enzymatic preparations of PEPC extracted fromE. crus-galliplants.Key words: phosphoenolpyruvate carboxylase, enzyme kinetics, thermal adaptation, barnyard grass, electrophoresis,Echinochloa crus-galli.