Kinetic Analysis of the Non-Phosphorylated, in Vitro Phosphorylated, and Phosphorylation-Site-Mutant (Asp8) Forms of Intact Recombinant C4 Phosphoenolpyruvate Carboxylase from Sorghum (original) (raw)
Related papers
1995
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 4.1.1.3 1) 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 1mM 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-pyruv...
Photosynthesis Research, 1995
A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C4-phosphoenolpyruvate carboxylase (PEPC) was synthesized, purified and used to raise an antiserum in rabbits. Affinity-purified IgGs prevented PEPC phosphorylation in a reconstituted in vitro assay and reacted with both the phosphorylated and dephosphorylated forms of either native or denatured PEPC in immunoblotting experiments. Saturation of dephospho-PEPC with these specific IgGs resulted in a marked alteration of its functional and regulatory properties that mimicked phosphorylation of Ser-8. A series of recombinant C4 PEPCs mutated in the N-terminal phosphorylation domain and a C3-1ike PEPC isozyme from Sorghum behaved similarly to their C4 counterpart with respect to these phosphorylation-site antibodies.
The Journal of biological chemistry, 1992
The properties of the dephospho and in vitro phosphorylated forms of recombinant sorghum phosphoenolpyruvate carboxylase have been compared with those of the authentic dark (dephospho) and light (phospho) leaf enzyme forms and two mutant enzymes in which the phosphorylatable serine residue (Ser8) has been changed by site-directed mutagenesis to Cys (S8C) or Asp (S8D). Kinetic analysis of the purified recombinant, mutant, and leaf enzyme forms at pH 8.0 indicated virtually identical Vmax, apparent Km (phosphoenolpyruvate), and half-maximal activation (glucose 6-P) values of about 44 units/mg, 1.1 mM, and 0.23 mM, respectively. In contrast, the Ser8, S8C, and dark leaf enzymes were about 3-fold more sensitive to inhibition by L-malate at pH 7.3 than the Ser8-P, S8D, and light leaf enzyme forms. These comparative results indicate that: (i) Ser8 is an important determinant in the regulation of sorghum phosphoenolpyruvate carboxylase activity by negative (L-malate), but not positive (glu...
European Journal …, 1995
Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8→Asp) forms of purified recombinant sorghum C 4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.3 1) 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 K i for the negative allosteric effector l-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent K a for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1mM 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 C 4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and K m (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by l-malate and, most especially, K a (Glc6P) and K i (l-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C 4-photosynthesis enzyme.
PLANT PHYSIOLOGY, 1991
Reversible seryl-phosphorylation contributes to the light/dark regulation of C4-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malatesensitivity of the target enzyme has been recently identified as Ser-15 in 32P-phosphorylated/activated dark-form maize PEPC
1991
Reversible seryl-phosphorylation contributes to the light/dark regulation of C4-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malatesensitivity of the target enzyme has been recently identified as Ser-15 in 32P-phosphorylated/activated dark-form maize PEPC (J-A Jiao, R Chollet [1990] Arch Biochem Biophys 283: 300-305). In order to ascertain whether this N-terminal seryl residue is, indeed, the in vivo regulatory phosphorylation site, [32P]phosphopeptides were isolated and purified from in vivo 32P-labeled maize and sorghum leaf PEPC and subjected to automated Edman degradation analysis. The results show that purified light-form maize PEPC contains 14-fold more 32P-radioactivity than the corresponding dark-form enzyme on an equal protein basis and, more notably, only a single N-terminal serine residu...
Planta
Main conclusion A synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect. Abstract Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC pro...
The regulatory role of residues 226–232 in phosphoenolpyruvate carboxylase from maize
Photosynthesis Research, 2006
The regulatory properties of maize phosphoenolpyruvate carboxylase were significantly altered by site-directed mutagenesis of residues 226 through 232. This conserved sequence element, RTDEIRR, is part of a surface loop at the dimer interface. Mutation of individual residues in this sequence caused various kinetic changes, including desensitization of the enzyme to key allosteric effectors or alteration of the K0.5 PEP for the substrate phosphoenolpyruvate. R231A, and especially R232Q, displayed decreased apparent affinity for the activator glucose-6-phosphate. Apparent affinity for the activator glycine was reduced in D228N and R232Q, while the maximum activation caused by glycine was greatly reduced in R226Q and E229A. R226Q and E229A also showed significantly lower sensitivity to the inhibitors malate and aspartate. E229A exhibited a low K0.5 PEP, while the K0.5 PEP of R232Q was significantly higher than that of wild type. Thus these seven residues are critical determinants of the enzyme’s kinetic responses to activators, inhibitors and substrate. The present results support an earlier suggestion that Arg 231 contributes to the binding site of the allosteric activator glucose-6-phosphate, and are consistent with other proposals that the substrate phosphoenolpyruvate allosterically activates the enzyme by binding at or near the glucose-6-phosphate site. The results also suggest that the glycine binding site may be contiguous with the glucose-6-phosphate binding site. Glu 229, which extends from this interface region through the interior of the protein and emerges near the aspartate binding site, may provide a physical link for propagating conformational changes between the allosteric activator and inhibitor binding regions.