title: Kinetic analysis of the non-phosphorylated, in vitro phosphorylated, and phosphorylation-site-mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate carboxylase from sorghum.
abstract: Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8-->Asp) forms of purified recombinant sorghum C4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent Ki for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent Ka for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and Km (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, Ka (Glc6P) and Ki (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C4-photosynthesis enzyme.
authors.affiliation: Department of Biochemistry, University of Nebraska-Lincoln.
authors.forename: S M
authors.initials: SM
authors.lastname: Duff
authors.forename: C S
authors.initials: CS
authors.lastname: Andreo
authors.forename: V
authors.initials: V
authors.lastname: Pacquit
authors.forename: L
authors.initials: L
authors.lastname: Lepiniec
authors.forename: G
authors.initials: G
authors.lastname: Sarath
authors.forename: S A
authors.initials: SA
authors.lastname: Condon
authors.forename: J
authors.initials: J
authors.lastname: Vidal
authors.forename: P
authors.initials: P
authors.lastname: Gadal
authors.forename: R
authors.initials: R
authors.lastname: Chollet
mesh_terms: D002523:Edible Grain / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010729:Phosphoenolpyruvate Carboxykinase (GTP)