高等植物中的磷酸烯醇式丙酮酸羧激酶

简要介绍了近年来有关高等植物中磷酸烯醇式丙酮酸羧激酶（ＰＥＰＣＫ）的研究进展,并讨论了此酶的结构、功能和调节等方面的问题。     

植物磷酸烯醇式丙酮酸羧激酶(PEPCK)研究进展

磷酸烯醇式丙酮酸羧激酶(PEPCK)是一个广泛存在于开花植物中的酶, 在植物体内仅存在于特定的组织和细胞中, 其活性受自身磷酸化和一些相关代谢产物的调节。PEPCK的磷酸化在多种植物体内受光调控。ATP存在时, PEPCK催化OAA生成PEP, 而PEP是多种反应的前体物质。通过不同的代谢途径, PEPCK间接地参与贮油植物种子萌发和植物果实成熟的糖异生过程, C₄和CAM(景天科代谢)植物光合作用中的CO₂浓缩过程, 细胞内pH值平衡和植物体内氮代谢过程等, 从而调节植物的生长发育。该文综述了植物中已发现的PEPCK及其在植物生命活动过程中的自身活性调节和生理功能。     

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.     

Characterization of Phosphoenolpyruvate Carboxykinase from Panicum maximum

Phosphoenolpyruvate carboxykinase, EC 4.1.1.32 (PEPCK), was purified 43-fold from the grass Panicum maximum. Michaelis constants (Km) were determined for the exchange reaction, the carboxylation reaction, and the decarboxylation reaction. The Km values for oxaloacetate and ATP in the decarboxylation reaction were found to be lower than the Km values for the substrates used in the exchange reaction and in the carboxylation reaction. Phosphoenolpyruvate carboxylase was not detectable in the purified PEPCK preparation.     

Activating Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Combination for Improvement of Succinate Production

Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production.     

Effects of Phosphorylation on Phosphoenolpyruvate Carboxykinase from the C4 Plant Guinea Grass

In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves. To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves. The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms. In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate. The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations. Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass. Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves. There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation.     

菠萝叶片PEP羧激酶活性及草酰乙酸与腺苷酸库的昼夜变化

自然条件下生长的菠萝植株,其叶片的ＰＥＰ羧激酶的脱羧与可逆羧化活性于中午１２时达最大值。脱羧／羧化比值在上午明显高于下午和晚上。脱羧反应底物ＯＡＡ含量于夜间增高,至次日早晨６时最高,随后下降。白天的ＯＡＡ／Ｍａｌ值高于夜间。参与Ｍａｌ及ＯＡＡ形成转化的ＰＥＰＣ和ＭＤＨ活性的昼夜变化与ＰＥＰ羧激酶（ＰＥＰＣＫ）活性的图式相似。ＰＥＰ羧激酶脱羧反应的另一底物ＡＴＰ在夜间０～６时内处于低水平,白天９～１５时内则显著增高。     

Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria

Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO₂ to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PP_i)-type PEPCK (PP_i-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PP_i-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PP_i-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PP_i-PEPCK activity. The primary structure of PP_i-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PP_i-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PP_i-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply.     

Purification and Immunological Characterization of Mouse Hepatic Phosphoenolpyruvate Carboxykinase

The inhibition of glucocorticoid induction and tryptophan activation of phosphoenolpyruvate carboxyfcinase (PEPCK) by bacterial endotoxin may be explained either by decreased synthesis or by inactivation of the enzyme. To differentiate between the two possibilities, mouse hepatic PEPCK was purified using a modification of a method used for securing the enzyme from livers of other species. The techniques included high speed centrifugation of whole liver homogenates, ammonium sulfate fractionation, Sephadex G-100 filtration, DEAE-cellulose ion exchange, hydroxylapatite chromatography, and isoelectric focusing. Antibodies prepared against the purified enzyme gave a single precipitin line in gel diffusion and in Immunoelectrophoresis. The antibodies were used for enzyme titrations in whole liver homogenates of normal and endotoxin poisoned mice, as reported elsewhere.     

金属离子对地衣芽孢杆菌合成多聚γ-谷氨酸的影响

多聚γ 谷氨酸 [γ Ｐｏｌｙ(ｇｌｕｔａｍｉｃａｃｉｄ) ,γ ＰＧＡ]是由某些杆菌 (Ｂａｃｉｌｌｕｓ)合成的一种细胞外水溶性高分子氨基酸聚合物 ,是由Ｌ 谷氨酸、Ｄ 谷氨酸两种构型的单体通过γ 酰胺键聚合形成的[1 ] 。γ ＰＧＡ具有极佳的成膜性、成纤维性 ,阻氧性、可塑性、粘结性、保湿性和可生物降解等许多独特的理化和生物学特性[2 ,3] 。因此 ,γ ＰＧＡ可以被广泛用于医药制造 ,食品加工 ,蔬菜、水果、海产品防冻、保鲜 ,化妆品工业 ,烟草、皮革制造工业和植物种子保护等许多领域 ,是一种有极大开发价值和前景的多功能新型生物制…     

金属离子对蜡蚧菌几丁质酶活力的影响

以蜡蚧菌(Ll)发酵液为材料,经分离纯化获得Ll几丁质酶(EC3.2.1.14)制剂.研究了金属离子对Ll几丁质酶活力的影响.结果表明,K+、Mg2+、Zn2+、Ca2+和Fe3+对几丁质酶活性有明显的促进作用,而Na+和Cu2+完全抑制几丁质酶的活性;Mn2+在低浓度时对酶有激活作用,随着浓度的升高表现出抑制作用;Fe2+和Ba2+的浓度低于0.5 mmol/L时对酶起抑制作用,而高于该浓度时则对酶有激活作用.     

Bivalent Metal Ions Modulate Cd2+ Effects on Isolated Rat Liver Mitochondria

We have studied Cd²⁺-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd²⁺] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 M, Cd²⁺ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 M, Cd²⁺ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO₃ or NH₄NO₃ medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd²⁺ effects on mitochondria treated in sequence with 100 M of Ca²⁺, Sr²⁺, Mn²⁺ or Ba²⁺(Me²⁺) and 7.5 M RR, as well as the alterations in Cd²⁺ action on the uptake of ¹³⁷Cs⁺ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca²⁺ or RR. The evidence obtained indicate that Ca²⁺ exhibits a synergestic action on all Cd²⁺ effects examined, whereas Sr²⁺ and Mn²⁺, conversely, are antagonistic. In the presence of RR, the Cd²⁺ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd²⁺, conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd²⁺-stimulated swelling and ¹³⁷Cs⁺ (analog of K⁺) uptake in the acetate media. For the first time, we have shown that Cd²⁺-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.     

重金属对绿球藻硝酸还原酶活性的影响

研究4种重金属离子(Cu2 、Cd2 、Zn2 、Pb2 )对绿球藻硝酸还原酶(NR)活性的影响。4种重金属离子的实验浓度分别为0.01、0.1、1、10、50、100、200 mg/L;0.1、1、5、10、50、100、200 mg/L;0.1、1、5、10、50、100、200 mg/L;0.1、1、10、50、100、200、400 mg/L,BG11培养基作对照。研究结果表明:Cu2 、Cd2 分别在低浓度0.01、0.1 mg/L时,便开始抑制硝酸还原酶活性,随着Cu2 、Cd2 浓度增加,抑制作用增强,酶活性逐渐降低;当Cu2 、Cd2 ≥10 mg/L时,NR活性检测不到。当Zn2 、Pb2 浓度分别低于0.1 mg/L和50 mg/L时,NR活性随着培养基中Zn2 、Pb2 浓度的增大逐渐增强;当Zn2 、Pb2 浓度分别大于0.1 mg/L和50 mg/L时,NR活性逐渐降低。结果提示:从4种重金属离子对NR活性的影响看,绿球藻对Zn2 、Pb2 耐受力高,特别是Pb2 ,而对Cu2 、Cd2 的耐受能力要低些。     

Effect of Overexpression of Actinobacillus succinogenes Phosphoenolpyruvate Carboxykinase on Succinate Production in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.     

Site-Directed Mutagenesis in Basic Amino Acid Residues of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V _max/K _mADP. These results indicate that Arg76 is not involved in CO₂ binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207–210], does not perform an essential function for the enzyme activity.