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.     

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。     

Dynamic Behavior of Rat Phosphoenolpyruvate Carboxykinase Inhibitors: New Mechanism for Enzyme Inhibition

As an enzyme acting at the junction of gluconeogenic pathway, phosphoenolpyruvate carboxykinase (PEPCK) controls substrate flow from Krebs cycle toward glucose production. Therefore, it would be advantageous to design effective inhibitors to inactivate PEPCK in diabetes mellitus and other abnormalities caused by insulin resistance. Such inhibitors may compensate the metabolic consequences of ex-activity of PEPCK at these conditions. Understanding the mechanism by which inhibitors exert their effect on enzyme activity is of great interest for designing stronger inhibitors. In the present work, molecular dynamic simulations were used to study enzyme-inhibitor interactions. Our results indicate that inhibitors of PEPCK with their short chains interact with enzyme active site through non-covalent interactions of electrostatic and hydrogen bond nature. The data also show that inhibitors neither reach a stable state in their binding site nor make static complex with the enzyme active site. Instead, they interact with functional groups of active site residues in a dynamic fashion. In this way, oxalate and sulfoacetate carrying two negative groups of higher charge density and optimum spacing from each other, show more dynamic behavior (lower stability in their binding site) and more inhibitory effects than other inhibitors used (phosphonoformate, phosphoglycolate and 3-phosphonopropionate).     

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.     

Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells

The Saccharomyces cerevisiae cell wall provides a semipermeable barrier that can retain intracellular proteins but still permits small molecules to pass through. When S. cerevisiae cells expressing E. coli lacZ are treated with detergent to extract the cell membrane, beta-galactosidase activity in the permeabilized cells is approximately 40% of the activity of the protein in cell extract. However, the permeabilized cells can easily be collected and reused over 15 times without appreciable loss in activity. Cell wall composition and thickness can be modified using different cell strains for enzyme expression or by mutating genes involved in cell wall biosynthesis or degradation. The Sigma1278b strain cell wall is less permeable than the walls of BY4742 and W303 cells, and deleting EXG1, which encodes a 1,3-beta-glucanase, can further reduce permeability. A short Zymolyase treatment can increase cell wall permeability without rupturing the cells. Encapsulating multiple enzymes in permeabilized cells can offer kinetic advantages over the same enzymes in solution. Regeneration of ATP from AMP by adenylate kinase and pyruvate kinase encapsulated in the same cell proceeded more rapidly than regeneration using a cell extract. Combining permeabilized cells containing adenylate kinase with permeabilized cells containing pyruvate kinase can also regenerate ATP from AMP, but the kinetics of this reaction are slower than regeneration using cell extract or permeabilized cells expressing both enzymes.     

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.     

Enzyme activities during early ascosporulation in Saccharomyces cerevisiae

1. Several enzyme activities were examined during the initial sporulating phase in Saccharomyces cerevisiae. 2. Catalase activity increased obviously after transfer to sporulation medium. 3. Catalase is probably considered to play an essential role in sporulation. 4. Both activities of inorganic pyrophosphatase and glycerol-2-phosphatase decreased. 5. Conditions necessary for sporulation were suggested.     

苹果酸酶基因转化酿酒酵母的研究进展

微生物降酸是现代葡萄酒酿造工艺中重要环节之一。利用现代生物技术将粟酒裂殖酵母中的苹果酸酶基因和苹果酸通透酶基因共同转化到酿酒酵母中,构建苹果酸-酒精酵母,使之既能进行酒精发酵,又能分解苹果酸。主要对近些年粟酒裂殖酵母苹果酸酶性质、基因结构及其转化酿酒酵母的研究做了回顾与总结,并指出了有待于解决的问题。     

Effects of Metal Ions on Cattle Liver Phosphoenolpyruvate Carboxykinase Activity in Vitro

A plant glycosphingolipid, O-(β-d-mannopyranosyl)-(l → 4)-O-(β-d-glucopyranosyl)-(l → l)-(2S,3S,4R)-4-hydroxy-N-tetracosanoylsphinganine 1, and the stereoisomer, O-(α-d-mannopyranosyl)-(1 → 4)-O-(β-d-glucopyranosyl)-(l → l)-(2S,3S,4R)-4-hydroxy-N-tetracosanoylsphinganine 6, were synthesized in a stereo- and regio-controlled way.     

Phosphoenolpyruvate Carboxykinase Is Involved in the Decarboxylation of Aspartate in the Bundle Sheath of Maize

We recently showed that maize (Zea mays L.) leaves contain appreciable amounts of phosphoenolpyruvate carboxykinase (PEPCK; R.P. Walker, R.M. Acheson, L.I. Técsi, R.C. Leegood [1997] Aust J Plant Physiol 24: 459–468). In the present study, we investigated the role of PEPCK in C₄ photosynthesis in maize. PEPCK activity and protein were enriched in extracts from bundle-sheath (BS) strands compared with whole-leaf extracts. Decarboxylation of [4-¹⁴C]aspartate (Asp) by BS strands was dependent on the presence of 2-oxoglutarate and Mn²⁺, was stimulated by ATP, was inhibited by the PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent of illumination. The principal product of Asp metabolism was phosphoenolpyruvate, whereas pyruvate was a minor product. Decarboxylation of [4-¹⁴C]malate was stimulated severalfold by Asp and 3-phosphoglycerate, was only slightly reduced in the absence of Mn²⁺ or in the presence of 3-mercaptopicolinic acid, and was light dependent. Our data show that decarboxylation of Asp and malate in BS cells of maize occurs via two different pathways: Whereas malate is mainly decarboxylated by NADP-malic enzyme, decarboxylation of Asp is dependent on the activity of PEPCK.     

Regulation of the acuF Gene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous Fungus Aspergillus nidulans

Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle. Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability of A. nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S. cerevisiae. The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S. cerevisiae Hap complex and the mammalian NFY complex.     

L-Asparaginase of Saccharomyces cerevisiae: an extracellular Enzyme.

During recent studies conducted with suspensions of three strains of Saccharomyces cerevisiae, it was observed that ammonia was rapidly liberated when L-asparagine was added to the medium. Subsequent investigation has revealed that these strains of S. cerevisiae have an externally active asparaginase as well as an internally active one. The appearance of the external asparaginase is stimulated by nitrogen starvation, requires an available energy source, and is prevented by cycloheximide. The internal enzyme appears to be constitutive. The external activity is relatively insensitive to para-hydroxymercuribenzoate inhibition, whereas the internal activity is highly inhibited by this compound.     

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.