Purification and some properties of phosphoenolpyruvate carboxylase from Brevibacterium flavum and its aspartate-overproducing mutant.
Phosphoenolpyruvate (PEP) carboxylases (PC) were purified from a wild strain and an aspartate-producing mutant of Brevibacterium flavum to electrophoretic homogeneity. The molecular weights of the enzymes were determined to be 4.1 X 10(5) by the gel-filtration technique. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme gave only one protein band with a molecular weight of 1.07 X 10(5). The enzyme was labile and stabilized by substrate PEP, activators, metallic cofactors, an allosteric inhibitor and ammonium sulfate. The mechanism for the PC reaction was rapid equilibrium random Bi Bi with a dead end complex, enzyme-bicarbonate-Pi. The KmS for PEP and bicarbonate were 2.5 and 0.63 mM, respectively, and the apparent KmS were not affected by the secondary substrate concentrations. Dissociation constants for Pi of enzyme-Pi and the dead end complex were 5.0 and 16 mM, respectively. Aspartate inhibition was completely competitive with both the substrates, PEP and bicarbonate, with an inhibitor constant of 0.044 mM. An activator, acetyl-CoA, did not alter the apparent Km for bicarbonate but decreased that for PEP. The activator constants for the enzyme-PEP complex and free enzyme were 6.3 and 40 microM, respectively. Double reciprocal plots of reaction rate against PEP concentration were not linear at lower PEP concentrations. Hill coefficients for PEP were 1.6 in the absence of any effectors, 1.0 in the presence of acetyl-CoA, and 2.3 in the presence of aspartate. As to the mutant enzyme, only the inhibitor constant for aspartate was increased, being 0.18 mM, but other constants, coefficients, as described above, and specific activity were almost the same as those of the wild-type enzyme.
D000494:Allosteric Regulation; D001224:Aspartic Acid / Q000378:metabolism; D001951:Brevibacterium / Q000201:enzymology*; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D001224:Aspartic Acid; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase