Purification and characterization of the phosphoenolpyruvate carboxylase from the facultative chemolithotrophThiobacillus novellus (ATCC 8093)

Abstract

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), which catalyzes the carboxylation of phosphoenolpyruvate to produce oxaloacetate was purified 465-fold from extracts of organotrophically grownThiobacillus novellus. Nondenaturing polyacrylamide gel electrophoresis (PAGE) of the purified enzyme revealed the presence of two bands after staining with Buffalo Black. Gels stained with Fast Violet B after incubation with PEP, HCO₃ ^-, Mg²⁺ and acetyl CoA also showed two bands of activity with the faster moving the more active of the two. Sodium dodecylsulfate (SDS)-PAGE of the enzyme heated at 100°C for 5 min revealed the presence of three intensely stained bands of M_r 95 K, 51 K, and 28 K. However, electrophoresis of the enzyme heated for 2 min showed a single band of about 100 K, indicating that the preparation was likely homogeneous. The 51 K and 28 K subunits are thus products of the 95 K subunit. Gel filtration studies of the native enzyme yielded a M_r of 360 K. Therefore, the enzyme is a tetramer. The optimum pH in Tris buffer was 8.0, with K_m for PEP 0.64 mM, HCO₃ ^- 0.11 mM, and acetyl CoA a potent activator, 1.3 μM. A divalent cation best served by Mg²⁺ gave sigmoidal initial velocity plots. Hill plots of the data gave coefficients (n_H) of 2.6. None of the metabolites tested, nucleotide triophosphates excepted, significantly affected enzyme activity. Binding studies with¹⁴C-labelled PEP revealed the binding of about 20 moles PEP per mole (360,000 g) of PEPC. Initial velocity studies suggest that the reaction is catalyzed by a random Bi Bi mechanism. Despite the lack of inhibition by certain metabolites, the enzyme's function is probably anaplerotic.