caa a22 4500 469093153 CHVBK 20180323133023.0 cr unu---uuuuu 170328e19921001xx s 000 0 eng 10.1007/BF00582575 doi (NATIONALLICENCE)springer-10.1007/BF00582575 Purification and characterization of the phosphoenolpyruvate carboxylase from the facultative chemolithotroph Thiobacillus novellus (ATCC 8093) [Elektronische Daten] [A. Charles, Yvona Sykora] 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 Mr 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 Mr 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 μM. 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 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. Kluwer Academic Publishers, 1992 CO2 fixation nationallicence carboxylase nationallicence enzymology nationallicence enzyme regulation nationallicence carboxylation nationallicence chemolithotrophy nationallicence thiobacilli nationallicence Charles A. Department of Biology, University of Waterloo, N2L 3G1, Waterloo, Ontario, Canada aut Sykora Yvona Department of Biology, University of Waterloo, N2L 3G1, Waterloo, Ontario, Canada aut Antonie van Leeuwenhoek Kluwer Academic Publishers 62/3(1992-10-01), 155-165 0003-6072 62:3<155 1992 62 10482 https://doi.org/10.1007/BF00582575 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00582575 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Charles A. Department of Biology, University of Waterloo, N2L 3G1, Waterloo, Ontario, Canada aut NATIONALLICENCE 700 1- Sykora Yvona Department of Biology, University of Waterloo, N2L 3G1, Waterloo, Ontario, Canada aut NATIONALLICENCE 773 0- Antonie van Leeuwenhoek Kluwer Academic Publishers 62/3(1992-10-01), 155-165 0003-6072 62:3<155 1992 62 10482 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer