Purification and Some Properties of Phosphoenolpyruvate Carboxylase from Brevibacterium flavum and Its Aspartate-Overproducing Mutant

Abstract

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.

Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum

“…The enzyme activity of the purified PEPC was assayed by a coupling reaction catalyzed by malate dehydrogenase at 25°C as previously described (15)(16)(17). The standard reaction mixture contained 100 mM Tris-HCl (pH 7.5), 10 mM MnSO 4 , 10 mM NaHCO 3 , 2 mM PEP, 0.1 mM NADH, and 1.5 IU of malate dehydrogenase.…”

Higher plant phosphoenolpyruvate carboxylase

“…A study concerning the enzyme from Brevibacterium ffavum, reported that the mechanism is a rapid equilibrium random Bi Bi with a dead end complex enzyme-bicarbonate-Pi [55]. In view of the large differences in kinetic properties between bacterial and plant PEP carboxylase, a detailed study with the plant enzyme is required.…”

Metabolic Consequences of Altered PhosphoenolpyruvateCarboxykinase Activity in Corynebacterium glutamicum Reveal Anaplerotic Regulation Mechanisms in Vivo

“…For the amino acid producer Corynebacterium glutamicum, it was originally believed that phosphoenolpyruvate (PEP) carboxylase was the only anaplerotic enzyme for growth on glucose (Ozaki and Shiio, 1969;Mori and Shiio, 1985a); however, later also pyruvate carboxylase activity was demonstrated by Peters-Wendisch et al (1997).…”