Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

Abstract Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis-Menten kinetics for the substrates PEP, IDP, KHCO₃, OAA and ITP with a K _m of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.     

Conservation from rat to human of cytosolic phosphoenolpyruvate carboxykinase and the control of its gene expression

Structural conservation of cytosolic phosphoenolpyruvate carboxykinase protein and mRNA sequence was found in all species examined from rodents to human. The mitochondrial isoenzyme, in all species tested, represents a distinct protein. Moreover, irrespective of the ratio of cytosolic to mitochondrial isoenzyme, cytosolic phosphoenolpyruvate carboxykinase activity in the human as in the rat is controlled at the level of gene expression and through the same multiple hormonal stimulation. This evolutionary conservation of the cytosolic phosphoenolpyruvate carboxykinase structure and mode of regulation supports the enzymes' physiological importance in mammals.     

Phosphoenolpyruvate carboxykinase from Fasciola hepatica

Behm C. A. and Bryant C. 1982. Phosphoenolpyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology12: 271–278. The kinetic properties of a partially purified preparation of phosphoenolpyruvate carboxykinase (PEPCK) from F. hepatica were examined. The pH optimum for the carboxylation reaction is 5.8–6.2. The enzyme is more active with Mn²⁺ than Mg²⁺ and the Mn²⁺ saturation curve was sigmoid. Apparent K_m values for the substrates GDP, IDP, PEP and HCO₃^? were determined and found to be in the same range as those reported for other helminths except that the enzyme is less sensitive to low PEP concentrations. GTP and ATP at 0.5 and 1.0 mM inhibit the enzyme; the GTP inhibition was greater in the presence of Mg²⁺ than Mn²⁺ and was competitive with GDP. It was concluded that the activity of PEPCK from F. hepatica is controlled by the concentration of reactants and the ambient pH, that the accumulation of GTP is a sensitive mechanism for inhibiting the carboxylation reaction and that PEPCK activity in the cytosol is likely to be favoured over that of pyruvate kinase except when pH is high and PEP concentration low.     

PEPCK不同启动子调控报告基因表达效率的比较

目的构建两种不同的磷酸烯醇式丙酮酸羧激酶（PEPCK）基因启动子调控报告基因表达质粒,比较该启动子在各种细胞系中的表达效率。方法采用PCR克隆大鼠PEPCK启动子1323 bp和560 bp两条片段,将两个PEPCK启动子片段插入pGL4.26-Luc2报告质粒中,将重组质粒转染到肝脏细胞系和脂肪细胞系,以非脂肪或肝脏细胞系做对照,比较PEPCK驱动荧光素酶表达效率。结果通过酶切鉴定及基因测序,证明pGL4.26-PEPCK-Luc2荧光素酶表达质粒构建成功,且能够在体外表达荧光素酶。结论两种同片段的PEPCK启动子在各细胞系中的表达效率差异无显著性。     

A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples

A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels.     

Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site

We report crystal structures of the human enzyme phosphoenolpyruvate carboxykinase (PEPCK) with and without bound substrates. These structures are the first to be determined for a GTP-dependent PEPCK, and provide the first view of a novel GTP-binding site unique to the GTP-dependent PEPCK family. Three phenylalanine residues form the walls of the guanine-binding pocket on the enzyme's surface and, most surprisingly, one of the phenylalanine side-chains contributes to the enzyme's specificity for GTP. PEPCK catalyzes the rate-limiting step in the metabolic pathway that produces glucose from lactate and other precursors derived from the citric acid cycle. Because the gluconeogenic pathway contributes to the fasting hyperglycemia of type II diabetes, inhibitors of PEPCK may be useful in the treatment of diabetes.     

产琥珀酸重组大肠杆菌的发酵性能研究

研究了重组大肠杆菌JM001(△ppc)/pTrc99a-pck发酵产琥珀酸的性能,结果表明厌氧条件下其耗糖能力和产酸能力分别为对照菌株JM001的4.2倍和15.3倍。进一步优化发酵条件表明：采用接入菌泥的发酵方式比按照10%接种量转接厌氧发酵的效果要好,琥珀酸的对葡萄糖的质量收率提高了约10%,且副产物乙酸的量进一步降低。初始葡萄糖浓度高于60g/L时会对菌株的生长和产酸产生抑制,且浓度越高,抑制作用越明显。7L发酵罐放大实验中,整个厌氧发酵阶段葡萄糖的消耗速率为0.42g/(L.h),琥珀酸对葡萄糖的质量收率为67.75%,琥珀酸的生产强度为0.28g/(L.h)。     

Expression and manipulation of phosphoenolpyruvate carboxykinase 1 identifies a role for malate metabolism in stomatal closure

Malate, along with potassium and chloride ions, is an important solute for maintaining turgor pressure during stomatal opening. Although malate is exported from guard cells during stomatal closure, there is controversy as to whether malate is also metabolised. We provide evidence that phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in malate metabolism and gluconeogenesis, is necessary for full stomatal closure in the dark. Analysis of the Arabidopsis PCK1 gene promoter indicated that this PEPCK isoform is specifically expressed in guard cells and trichomes of the leaf. Spatially distinct promoter elements were found to be required for post-germinative, vascular expression and guard cell/trichome expression of PCK1. We show that pck1 mutant plants have reduced drought tolerance, and show increased stomatal conductance and wider stomatal apertures compared with the wild type. During light-dark transients the PEPCK mutant plants show both increased overall stomatal conductance and less responsiveness of the stomata to darkness than the wild type, indicating that stomata get 'jammed' in the open position. These results show that malate metabolism is important during dark-induced stomatal closure and that PEPCK is involved in this process.     

The Relation Between Activity of Phosphoenolpyruvate Carboxykinase and Photosynthesis in Aloe vera Leaves

The chlorophyllous layer of leaf of a PEP-CK type CAM plant Aloe vera was stripped tiff from the colorIess water storage tissue and used to stuly the interrelation between the activity of decarboxylating enzyme phosphoenolpyruvate carboxykinase (PEPCK) and photosynthesis. Oxaloacetate, malate+ADP, and NaHCO3 were found to stimulate photosynthetic oxygen evolution. During the period from 6:00 to 18:00 of the day time, a diurnal fluctuation was observed in both PEPCK activity and the rate of oxygen evolution. The maximum of photosynthesis appeared at 10-12:00, but the maximum PEPCK activity appeared at 14:00. The PEPCK activity and photosynthetic rate in leaf discs increased with temperature from 10 to 35℃, then decreased at 45℃. Similar decline of both parameters was found in the leaf discs stressed by different concentration of PEG-6000 solution for 4.5 h. At light intensity of 900 mol m-2 s-1 and 25℃, the PEPCK activity and photosynthetic rate of leaf discs rised with the illumination time, then a slight inhibition followed at the time of 30 min (Pn) or 40 min (PEPCK). The strong response of PEPCK activity to high light intensity in leaf discs, and a progressive increase of PEPCK activity in direct illumination of crude enzyme extractm the range of 0-55 min, indicated that light s likely to be an activator for PEPCK. Leaf discs were infiltrated with 3-(3,4-dichlorophenyl)-l, 1-dimethylurea, DL-glyceraldehyde and 2,4-dimitrophenol resulted in the partial inhibition of light-ependent photosynthesis and decarboxylation of C4 acid. The activity of PEPCK was also stimulated by Mg2+ or Mg2++ATP infiltrated into the leaf discs in the dark. The evidence presented here suggested that PEPCK activity of CAM plants showed a close interrelation with photosynthesis. Both of them were regulated by the environmental changes. The activity of PEPCK might be coupled to electron trsnsport and photophosphorylatiou.     

pH and carbon supply control the expression of phosphoenolpyruvate carboxylase kinase genes in Arabidopsis thaliana

Phosphoenolpyruvate carboxylase (PEPC) is thought to play many roles in C(3) plants including the provision of biosynthetic precursors and control of pH during N assimilation. Its activity is controlled via phosphorylation catalysed by PEPC kinases, which are encoded by PPCK genes. We examined PPCK expression in response to changes in the supply of N or C, and to changes in intracellular pH, using cultured Arabidopsis cells and seedlings. The results show that expression of both PPCK1 and PPCK2 is increased by C availability, but does not respond to N availability. Expression of the two PPCK genes and the phosphorylation state of PEPC are increased in response to increasing intracellular pH. Elevated pH also reduces the repression of PPCK gene expression by P(i). Expression of phosphoenolpyruvate carboxykinase (PEPCK), which catalyses the decarboxylation of oxaloacetate, is decreased in response to increasing intracellular pH. pH homeostasis may be mediated at least partly by reciprocal changes in the expression of PPCK genes and PEPCK.     

Mapping of the PCK1 gene encoding phosphoenolpyruvate carboxykinase on chromosome XI of Saccharomyces cerevisiae

The gene PCK1 encoding phosphoenolpyruvate carboxykinase of Saccharomyces cerevisiae has been mapped on the right arm of chromosome XI, 12.7 centimorgans proximal to MAL4 and 20.1 centimorgans distal to MET1. In order to map the gene, hybridization of PCK1 DNA with separated yeast chromosomes and tetrad analysis of diploids with adequate markers were carried out.     

Gluconeogenesis and P-enolpyruvate carboxykinase in liver and kidney of long-term fasted quails

The activity of cytoplasmic and mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) in kidney and liver, and in vivo gluconeogenic activity, were determined during different phases of prolonged fasting in quails. The fasting-induced changes in the activity of kidney cytoplasmic PEPCK were positively correlated with the changes in gluconeogenesis. Both activities increased at the initial phase (I) of fasting to levels 65% to 100% higher than fed values, and decreased during the protein-sparing period (phase II), although remaining higher than in fed birds. At the catabolic final phase (III) both kidney cytoplasmic PEPCK activity and gluconeogenesis increased markedly, attaining levels 115% to 150% higher than fed values. The activity of liver cytoplasmic PEPCK, present in appreciable amounts in quails, did not change during phases I and II of fasting, but increased to levels 60% higher than fed values at the final phase (III). Plasma glucose levels at phase III did not differ significantly from those at phases I and II. In both kidney and liver the activity of the mitochondrial PEPCK was not significantly affected by fasting. The data suggest that the kidney cytoplasmic PEPCK is the main enzyme responsible for gluconeogenesis adjustments during food deprivation in quails, and that this function is complemented at the final phase by enzyme present in liver cytosol. Accepted: 14 April 2000     

Endotoxin-induced alterations in hepatic glucose-6-phosphatase activity and gene expression

The mechanisms responsible for the glycemic changes associated with endotoxic shock are not fully understood, but are known to involve the ability of the liver to produce glucose. The purpose of the present study was to determine whether endotoxin (LPS) influences the expression and activity of glucose-6-phosphatase (Glu-6-Pase) during the early hyperglycemic phase and the later hypoglycemic phase. Rats were injected with a relatively large dose of LPS (20 mg/kg) or saline (control), and sacrificed at 1 or 5 h post-injection. Both the plasma glucose concentration and glucose production were elevated 1 h post-LPS (2-fold) and both decreased at 5 h postinjection (50%). Compared to time-matched control values, hepatic glucose-6-phosphate and fructose-6-phosphate levels were significantly decreased at both 1 and 5 h. Hepatic Glu-6-Pase activity and mRNA levels were moderately increased, 1 h after injection of LPS. At 5 h, an 88% decrease in mRNA abundance for Glu-6-Pase was associated with a 30% decrease in activity of this enzyme. Plasma insulin concentrations were not different 1 h after LPS and were elevated 2-fold from control values at 5 h. Circulating levels of glucagon and corticosterone were elevated at both time points following LPS. Our data indicate that the LPS-induced hypoglycemia and reduction in hepatic glucose production were accompanied by a depression in Glu-6-Pase activity and gene expression.     

Reactivity of cysteinyl, arginyl, and lysyl residues ofEscherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents

Calcium-activated phosphoenolpyruvate carboxykinase fromEscheria coli is not inactivated by a number of sulfhydryl-directed reagents [5,5-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N-(5-sulfo-l-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn²⁺, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.Abbreviations used: DTNB, 5,5-dithiobis(2-nitrobenzoate); Hepes, N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid; 1,5-IAEDANS, N-(iodoacetyl)-N-(5-sulfo-1-naphthyl) ethylenediamine; EPE, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PG, 1-pyrenylglyoxal; PLP, pyridoxal 5-phosphate.