Purification and characterization of the phosphoenolpyruvate carboxylase from the facultative chemolithotroph Thiobacillus novellus (ATCC 8093).
Phosphoenolpyruvate carboxylase (EC4.1.1.31), which catalyzes the carboxylation of phosphoenolpyruvate to produce oxaloacetate was purified 465-fold from extracts of organotrophically grown Thiobacillus 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 degrees 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 Km for PEP 0.64 mM, HCO3- 0.11 mM, and acetyl CoA a potent activator, 1.3 microM. A divalent cation best served by Mg2+ gave sigmoidal initial velocity plots. Hill plots of the data gave coefficients (nH) of 2.6. None of the metabolites tested, nucleotide triophosphates excepted, significantly affected enzyme activity. Binding studies with 14C-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.
Charles
A M
AM
Department of Biology, University of Waterloo, Ontario, Canada.
Sykora
Y
Y
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010071:Oxaloacetates / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013855:Thiobacillus / Q000201:enzymology*
D002413:Cations, Divalent; D010071:Oxaloacetates; D000105:Acetyl Coenzyme A; D010728:Phosphoenolpyruvate; D010730:Phosphoenolpyruvate Carboxylase; D008274:Magnesium