Purification and characterization of phosphoenolpyruvate carboxykinase,a catabolic CO2-fixing enzyme, from Anaerobiospirillum succiniciproducens.
Phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.49) from the obligate anaerobe Anaerobiospirillum succiniciproducens was purified 18-fold. The enzyme was monomeric, with an Mr of 57,000 +/- 2,000. The enzyme was oxygen stable, had a pH optimum of 6.7-7.1, and was stable from pH 5.0 to 9.0. The enzyme displayed Michaelis-Menten kinetics for the substrate PEP and the cosubstrates bicarbonate and ADP with a Km of 0.54 mM, 17 mM and 0.42 mM, respectively. The enzyme required Mn(2+) or Co(2+) in addition to Mg(2+) to exhibit maximum activity. p-Chloromercuribenzoate inhibited activity and phosphoenolpyruvate protected the enzyme against inactivation, suggesting that an essential cysteine may be in the active site.
Podkovyrov
S M
SM
Department of Biochemistry, Michigan State University, East Lansing 48824.
Zeikus
J G
JG
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001438:Bacteroidaceae / Q000201:enzymology*; D001639:Bicarbonates / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D001639:Bicarbonates; D008670:Metals; D002245:Carbon Dioxide; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D010729:Phosphoenolpyruvate Carboxykinase (GTP)