title: A kinetic study of the effects of phosphate and organic phosphates on the activity of phosphoenolpyruvate carboxylase from Crassula argentea.
abstract: The effects of phosphate and several phosphate-containing compounds on the activity of purified phosphoenolpyruvate carboxylase (PEPC) from the crassulacean acid metabolism plant, Crassula argentea, were investigated. When assayed at subsaturating phosphoenolpyruvate (PEP) concentrations, low concentrations of most of the compounds tested were found to stimulate PEPC activity. This activation, variable in extent, was found in all cases to be competitive with glucose 6-phosphate (Glc-6-P) stimulation, suggesting that these effectors bind to the Glc-6-P site. At higher concentrations, depending upon the effector molecule studied, deactivation, inhibition, or no response was observed. More detailed studies were performed with Glc-6-P, AMP, phosphoglycolate, and phosphate. AMP had previously been shown to be a specific ligand for the Glc-6-P site. The main effect of Glc-6-P and AMP on the kinetic parameters was to decrease the apparent Km and increase Vmax/Km. AMP also caused a decrease in the Vmax of the reaction. In contrast, phosphoglycolate acted essentially as a competitive inhibitor increasing the apparent Km for PEP and decreasing Vmax/Km. Inorganic phosphate had a biphasic effect on the kinetic parameters, resulting in a transient decrease in Km followed by an increase of the apparent Km for PEP with increasing concentration of phosphate. The Vmax also was decreased with increasing phosphate concentrations. Further, the enzyme appeared to respond to the complex of phosphate with magnesium. In the presence of a saturating concentration of AMP, no activation but rather inhibition was observed with increasing phosphate concentration. This is consistent with the binding of phosphate to two separate sites--the Glc-6-P activation site and an inhibitory site, a phenomenon that may be occurring with other phosphate containing compounds. High concentrations of phosphate with magnesium were found to protect enzyme activity when PEPC, previously shown to contain an essential arginine at the active site, was incubated with the specific arginyl reagent 2,3-butanedione, consistent with the binding of phosphate at the active site. Data were successfully fitted to a rapid equilibrium model allowing for binding of the phosphate-magnesium complex to both the activation site and the active site which accounts for the activation/deactivation observed at low substrate concentrations. Effects on the Vmax of the reaction are also addressed. Factors controlling the differential affinity of various effectors to the active site or activation site appear to include charge distribution, size, and other steric factors.
authors.affiliation: Department of Biochemistry, University of California, Riverside 92521.
authors.forename: C R
authors.initials: CR
authors.lastname: Meyer
authors.forename: P
authors.initials: P
authors.lastname: Rustin
authors.forename: R T
authors.initials: RT
authors.lastname: Wedding
mesh_terms: D000249:Adenosine Monophosphate / Q000494:pharmacology; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology; D006016:Glycolates / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D017616:Magnesium Compounds; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology*; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*
chemical_list: D005958:Glucosephosphates; D006016:Glycolates; D017616:Magnesium Compounds; D009943:Organophosphorus Compounds; D010710:Phosphates; D000249:Adenosine Monophosphate; C030781:magnesium phosphate; D019298:Glucose-6-Phosphate; D010728:Phosphoenolpyruvate; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase; C013193:phosphoglycolate; D008274:Magnesium