Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties.
Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.
D002262:Carboxy-Lyases / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D008293:Malates / Q000494:pharmacology; D008970:Molecular Weight; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D013696:Temperature
D046911:Macromolecular Substances; D008293:Malates; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase