title: The anaplerotic phosphoenolpyruvate carboxylase of the tricarboxylic acid cycle deficient Acholeplasma laidlawii B-PG9.
abstract: Phosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEP-C) was purified approximately 770-fold from the mollicute Acholeplasma laidlawii B-PG9. The partially purified PEP-C required phosphoenolpyruvate (PEP) and MnCl2 at pH 7.4 or MgCl2 at pH 8.6 for optimal activity. The product is oxaloacetate as detected by a malate dehydrogenase indicator system. The KmA (PEP variable) was 0.66 mM and the KmB (bicarbonate variable) was 1.02 mM. At low bicarbonate concentrations (0.5 mM), PEP-C activity was stimulated approximately 240% by fructose 1,6-bisphosphate. Aspartate was a non-competitive inhibitor of PEP-C activity. The KiA (PEP variable) for aspartate was 0.69 mM and the KiB (bicarbonate variable) was 0.99 mM. Malate, citrate, isocitrate, 2-oxoglutarate, acetyl-CoA, CMP, CDP, GDP, GTP, ADP and ATP had no effect on the PEP-C reaction. The Hill interaction coefficient was 0.98-1.11. The molecular mass by sucrose density gradient analysis was 353 kDa; by gel filtration chromatography it was 384 kDa. The Stokes radius was about 7.4 nm. PEP-C activity and its inhibition by aspartate in Acholeplasma laidlawii B-PG-9 extracts may reflect an involvement of this enzyme in the interdependent regulation of protein, lipid and nucleic acid precursor metabolism of this TCA-cycle-deficient and cytochrome-less mollicute.
authors.affiliation: Department of Medical Microbiology and Immunology, Ohio State University, Columbus 43210.
authors.forename: J T
authors.initials: JT
authors.lastname: Manolukas
authors.forename: M V
authors.initials: MV
authors.lastname: Williams
authors.forename: J D
authors.initials: JD
authors.lastname: Pollack
mesh_terms: D000128:Acholeplasma laidlawii / Q000201:enzymology*; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D002952:Citric Acid Cycle; D007700:Kinetics; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification* / Q000378:metabolism; D013045:Species Specificity
chemical_list: D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase