PHOSPHO ENOL PYRUVATE CARBOXYLASE: A Ubiquitous, Highly Regulated Enzyme in Plants (original) (raw)
Related papers
PHOSPHOENOLPYRUVATE CARBOXYLASE: A Ubiquitous, Highly Regulated Enzyme in Plants
Annual review of plant physiology and plant molecular biology, 1996
Since plant phosphoenolpyruvate carboxylase (PEPC) was last reviewed in the Annual Review of Plant Physiology over a decade ago (O'Leary 1982), significant advances have been made in our knowledge of this oligomeric, cytosolic enzyme. This review highlights this exciting progress in plant PEPC research by focusing on the three major areas of recent investigation: the enzymology of the protein; its posttranslational regulation by reversible protein phosphorylation and opposing metabolite effectors; and the structure, expression, and molecular evolution of the nuclear PEPC genes. It is hoped that the next ten years will be equally enlightening, especially with respect to the three-dimensional structure of the plant enzyme, the molecular analysis of its highly regulated protein-Ser/ Thr kinase, and the elucidation of its associated signal-transduction pathways in various plant cell types.
Phospho\u3ci\u3eenol\u3c/i\u3epyruvate Carboxylase: A Ubiquitous, Highly Regulated Enzyme in Plants
1996
Since plant phosphoenolpyruvate carboxylase (PEPC) was last reviewed in the Annual Review of Plant Physiology over a decade ago (O’Leary 1982), significant advances have been made in our knowledge of this oligomeric, cytosolic enzyme. This review highlights this exciting progress in plant PEPC research by focusing on the three major areas of recent investigation: the enzymology of the protein; its posttranslational regulation by reversible protein phosphorylation and opposing metabolite effectors; and the structure, expression, and molecular evolution of the nuclear PEPC genes. It is hoped that the next ten years will be equally enlightening, especially with respect to the three-dimensional structure of the plant enzyme, the molecular analysis of its highly regulated protein-Ser/Thr kinase, and the elucidation of its associated signal-transduction pathways in various plant cell types
FEBS Letters, 1997
In order to mimic regulatory phosphorylation of the Ser-15 of maize C 4-form phosphoewo/pyruvate carboxylase (PEPC), we replaced Ser-15 and Lys-12 with Asp (S15D) and Asn (K12N), respectively, by site-directed mutagenesis. Although both mutant enzymes were catalytically as active as the wild-type PEPC, they showed much less sensitivity to malate, an allosteric inhibitor, similarly to the phosphorylated wild-type PEPC. A maize protein kinase of 30 kDa which is known to be specific to PEPC (PEPC-PK), phosphorylated K12N as well as the wildtype PEPC but not S15D. The phosphorylation of K12N further diminished the sensitivity to malate. Thus, a positive charge of the conserved Lys-12 is not required for the recognition by PEPC-PK but contributes to the intrinsic sensitivity to malate inhibition.
In vitro phosphorylation of purified tobacco-leaf phosphoenolpyruvate carboxylase
FEBS Letters, 1993
C,-leaf phosphoenolpyruvate (PEP) carboxylase (PEPC) was purified about l,OOO-fold from tobacco and displayed a final specific activity of 35 ~mol/minlmg protein, an apparent K,,, (total PEP) of 95 mM (both at pH 8.0, 30°C). and an 1,&L-malate) value of 0.14 mM at pH 7.3, 0.2 mM PEP. The rapid, 5-step protocol involved polyethylene glycol fractionation and sequential FPLC on hydroxylapatite, phenyl-Sepharose. Mono Q and Superose 12. The electrophoretically pure protem and purified &leaf PEPC were phosphorylated in vitro in a reconstituted system with PEPC-kinase isolated from illummated tobacco and maize leaves. These reciprocal phosphorylation experiments (i) indicate that Ser" of tobacco PEPC is the likely target residue, sttuated in the plant-invariant Cm/Asp-LyslArg-X-X-Ser phosphorylation motif near the N-terminus, and (ii) lend support to the recent hypothesis that C-leaf PEPC is subject to&gulatory phosphorylation in vivo.
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...
Phosphoenolpyruvate carboxylase: structure, regulation and evolution
Plant Science, 1994
Plant phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) is encoded by a small multigene family in which the expression of each member is controlled individually by exogenous (light, environmental) and/or endogenous (hormonal and developmental) stimuli. The involvement of putative trans-acting factors and consensus cis-elements of promoters in the specific transcriptional regulation of the PEPC genes is discussed. At the post-translational level, the regulatory strategy of the plant enzyme is mainly to offset the negative effect of the feedback inhibitor, L-malate, the end-product of the oxaloacetate reduction. All plant PEPC-forms are under positive and negative allosteric control by metabolite effectors and possess a consensus phosphorylation site containing a target serine residue near their Nterminus (e.g. Ser8 in C4 PEPC from sorghum). In C 4 and Crassulacean acid metabolism (CAM) plants, a complex signal-transduction chain activates ~a Ca2+-independent protein-serine kinase responsible for regulatory phosphorylation of PEPC. A more thorough understanding of the functional and regulatory properties of the bacterial and C 4 enzymes has emerged by exploiting recombinant proteins and site-directed mutagenesis. In these newly opened areas, PEPC offers one of the best characterized paradigms of plant signaling. Finally, some emerging ideas on the evolution and phylogenetic relationships of the various PEPC isoforms are presented.
\u3ci\u3eIn Vitro\u3c/i\u3e Phosphorylation of Maize Leaf Phosphoenolpyruvate Carboxylase
1986
Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with 32P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ -32P]ATP or [β-32P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acidstable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase f...