Properties of phosphoenolpyruvate carboxylase in rapidly prepared,desalted leaf extracts of the Crassulacean acid metabolism plant Mesembryanthemum crystallinum L.

Properties of phosphoenolpyruvate (PEP) carboxylase, obtained from leaves of Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism (CAM), were determined at frequent time points during a 12-h light/12-h dark cycle. Leaf extracts were rapidly desalted and PEP carboxylase activity as a function of PEP concentration, malate concentration, and pH was measured within 2 min after homogenization of the tissue. Maximum velocity of PEP carboxylase was similar in the light and dark at pH 7.5 and pH 8.0. However, PEP carboxylase had as much as a 12-fold lower K _m for PEP and as much as a 20-fold higher K _i for malate during the dark than during the light periods, the magnitude of these differences being dependent on the assay pH. Assuming that enzyme properties immediately after isolation reflect the approximate state of the enzyme in vivo, these differences in enzyme properties reduce the potential for CO₂ fixation via PEP carboxylase in the light. A small decrease in cytoplasmic pH in the light would greatly magnify the above differences in day/night properties of PEP carboxylase, because the sensitivity of PEP carboxylase to inhibition by malate increased with decreasing pH. Properties of PEP carboxylase were also studied in plants exposed to short-term perturbations of the normal 12-h light/12-h dark cycle (e.g., prolonged light period, prolonged dark period). Under all light/dark regimes, there was a close correlation between change in properties of PEP carboxylase and changes of the tissue from acidification to deacidification, and vice versa. Changes in properties of PEP carboxylase were not merely light/dark phenomena because they were also observed in plants exposed to continuous light or dark. the data indicate that, during CAM, PEP carboxylase exists in two stages which differ in their capacity for net malate synthesis. The physiologically-active state is distinguished by a low K _m for PEP and a high K _i for malate and favors malate synthesis. The physiologically-inactive state has a high K _m for PEP and a low K _i for malate and exists during periods of deacidification and other periods lacking synthesis of malic acid.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPC PEP carboxylase - RuBP ribulose 1,5-bisphosphate - RH relative humidity     

The influence of temperature on malic Acid metabolism in grape berries: I. Enzyme responses

Phosphoenolpyruvate (PEP) carboxylase activity in immature `Carignane' grape berries (Vitis vinifera L.) had a temperature optimum of about 38 C, whereas malic enzyme activity rose with increasing temperature between 10 and 46 C. In vitro temperature inactivation rates for the PEP carboxylase were markedly greater than for the malic enzyme activity. From the simultaneous action of malic acid-producing enzymes (PEP carboxylase and malic dehydrogenase) and malic acid-degradating enzyme (malic enzyme) systems at different temperatures, the greatest tendency for malic acid accumulation in immature grape berries was at 20 to 25 C. Time-course measurements of enzymic activity from heated, intact berries revealed greater in vivo temperature stability for the malic enzyme activity than for the PEP carboxylase activity.     

Activity and quantity of ribulose bisphosphate carboxylase-and phosphoenolpyruvate carboxylase-protein in two Crassulacean acid metabolism plants in relation to leaf age,nitrogen nutrition,and point in time during a day/night cycle

Activity of ribulose 1,5-bisphosphate (RuBP) carboxylase in leaf extracts of the constitutive Crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers. decreased with increasing leaf age, whereas the activity of phosphoenolpyruvate (PEP) carboxylase increased. Changes in enzyme activities were associated with changes in the amount of enzyme proteins as determined by immunochemical analysis, sucrose density gradient centrifugation, and SDS gel electrophoresis of leaf extracts. Young developing leaves of plants which received high amounts of NO ₃ ^- during growth contained about 30% of the total soluble protein in the form of RuBP carboxylase; this value declined to about 17% in mature leaves. The level of PEP carboxylase in young leaves of plants at high NO ₃ ^- was an estimated 1% of the total soluble protein and increased to approximately 10% in mature leaves, which showed maximum capacity for dark CO₂ fixation. The growth of plants at low levels of NO ₃ ^- decreased the content of soluble protein per unit leaf area as well as the extractable activity and the percentage contribution of both RUBP carboxylase and PEP carboxylase to total soluble leaf protein. There was no definite change in the ratio of RuBP carboxylase to PEP carboxylase activity with a varying supply of NO ₃ ^- during growth. It has been suggested (e.g., Planta 144, 143–151, 1978) that a rhythmic pattern of synthesis and degradation of PEP carboxylase protein is involved in the regulation of -carboxylation during a day/night cycle in CAM. No such changes in the quantity of PEP carboxylase protein were observed in the leaves of Kalanchoe pinnata (Lam.) Pers. or in the leaves of the inducible CAM plant Mesembryanthemum crystallinum L.Abbreviations CAM Crassulacean acid metabolism - RuBP ribulose 1,5-bisphosphate - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

Anaplerotic metabolism of Aspergillus nidulans and its effect on biomass synthesis in carbon limited chemostats

Anaplerotic fixation of carbon dioxide by the fungus Aspergillus nidulans when grown under carbon-limited conditions was mediated by pyruvate carboxylase and a phosphoenol pyruvate (PEP)-metabolising enzyme which has been tentatively designated as PEP carboxylase. The activities of both enzymes were growth rate dependent and measurements of H¹⁴CO₃ incorporation by growing mycelium indicated that they were responsible for almost all the assimilated carbon dioxide. In carbon-limited chemostats, the maximum rate of bicarbonate assimilation occurred at a dilution rate of 0.11 h^–1, equivalent to 1/2 _max. The affinity of the pyruvate carboxylase for bicarbonate was twice that of the PEP carboxylase under the conditons of growth used. The effect of changing the bicarbonate concentration in carbon-limited chemostats was substantial: increasing the HCO ₃ ^– concentration over the range 0.7–2.8 mM enhanced biomass synthesis by 22%. Over-shoots in bicarbonate assimilation and carboxylase activity occurred when steady state chemostat cultures were subjected to a step down in dilution rate.     

Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L.: Enzyme studies

The activities of several enzymes, including ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z. mays. Mature leaf tissue had a RuDP-carboxylase activity of 296.7 mol CO₂ g^-1 fresh weight h^-1 and a PEP-carboxylase activity of 660.6 mol CO₂ g^-1 fresh weight h^-1. In young corn leaves the activity of the two enzymes was 11 and 29%, respectively, of the mature leaves. In senescent leaf tissue, RuDP carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of NADP malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and NAD malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and RuDP carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C₄ acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total ¹⁴C remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in ¹²CO₂. The activity of RuDP carboxylase during leaf ontogeny in Z. mays parallels the development of the activity of this enzyme in C₃ plants.Abbreviations RuDP ribulose-1,5-diphosphate - PEP phosphoenol pyruvate - PGA 3-phosphoglycerate     

Carboanhydrase-Aktivität und Enzyme des Glykolatweges in der Blaualge Anacystis nidulans

Summary The blue-green alga Anacystis nidulans (strain L 1402-1) was grown in air (0.03 vol. % CO₂) and in 3.0 vol. % CO₂ at +35° C. Levels of carbonic anhydrase were 3-fold higher in air-grown cells than in CO₂-grown algae. CO₂ content during growth has no effect on activity of RuDP carboxylase. Activities of PEP carboxylase, malic enzyme and catalase were higher in CO₂-grown Anacystis cells. In air-grown cells higher activities of malate dehydrogenase, glycolate dehydrogenase, serine-pyruvate aminotransferase and aspartate--ketoglutarate aminotransferase were found. Levels of these enzymes are relatively low compared to those in green algae and higher plants.     

The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression

Summary The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal c. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5 and 3 flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103154) which shows 34% similarity with the respective E. coli enzyme. Present address: Institut für Biotechnologie 1 der Kernforschungsanlage, Postfach 1913, D-5170 Jülich, Federal Republic of Germany     

Purification and some properties of extracellular invertase B from Zymomonas mobilis

Zymomonas mobilis is a Gram-negative ethanologen that can ferment glucose, fructose, and sucrose. Three enzymes that hydrolyze sucrose were found in a zymogram of electrophoretically separated proteins of Z. mobilis CP4. Two were invertase,, Inv A and Inv B; the latter was studied. Inv B is extracellular and accounts for at least 60% of the saccharolytic activity found in the culture broth of Z. mobilis CP4. The enzyme was purified 51-fold in 17% yield from culture broth of Z. mobilis grown on sucrose. It is a -fructosidase, monomeric with a molecular mass of 47 kDa and pI of 4.3. Its K _m for sucrose is 86 mm, and it has high catalytic activity (V _max = 1800 mol product/min per milligram protein). The purification and some properties of Inv B are presented. Correspondence to: D. E. Eveleigh     

Interactions of Phosphoenolpyruvate Carboxylase and Pyruvic Kinase in Developing Soybean Seeds

Developing soybean seeds contain phosphoenolpyruvate (PEP) carboxylase,pyruvic kinase, malate dehydrogenase, aspartate aminotransferase,alanine aminotransferase and malic enzyme activities. PEP carboxylasemay be important in competing with pyruvic kinase and directinga portion of glycolytic carbon towards oxaloacetate synthesis.The oxaloacetate can then be converted to aspartate and malate.Malic enzyme produces pyruvate and NADPH from malate, and thismay be an important additional source of reducing power forlipid biosynthesis. In the presence of high levels of PEP carboxylaseit is possible to demonstrate PEP formation by pyruvic kinase.PEP carboxylase and pyruvic kinase independently compete forPEP in a mixed system. Soybean seed extracts readily convertedradioactive PEP into alanine and aspartate when supplementedwith ADP, Mg²⁺, K+, HCO₃– and glutamate. Under varyingconditions of pH, metal ions, PEP, enzyme concentration andtime both alanine and aspartate were always produced. Possiblythe final products of glycolysis should be considered as pyruvateand oxaloacetate in plants. (Received April 22, 1981; Accepted June 26, 1981)     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Biochemical Specialization of Photosynthetic Cell Layers and Carbon Flow Paths in Suaeda monoica

Suaeda monoica Frossk. ex J. F. Gmel is a C₄ plant with three different photosynthesizing cell layers. The outer chlorenchymatous layer shows a high activity of phosphoenolpyruvate (PEP) carboxylase but none of ribulose bisphosphate (RuBP) carboxylase. The electrophoretic protein band of RuBP carboxylase was missing in this layer. The second chlorenchymatous cells layer shows a very high activity of RuBP carboxylase and NAD malic enzyme and only traces of activity of PEP carboxylase. The third photosynthesizing cell type is comprised of the water tissue. It has moderate activities of RuBP carboxylase and PEP carboxylase. A model for carbon flow in Suaeda monoica leaves is proposed.     

Dawn signal as a rhythmical timer for the seasonal adaptive variation of CAM: a model

Abstract Photoperiodism, known to control the level of CAM in different types of CAM plants (e.g. Kalanchoe blossfeldiana, Bryophyllum daigremontianum), would act as a reliable timer for seasonal coherent adaptation. Two different endogenous rhythms (malic enzyme activity, PEP carboxylase capacity) appear to be separately coupled to dawn and dusk, respectively, thus achieving time-compartmentation of CAM; this feature suggests involvement of an ‘internal coincidence’ type of clock mechanism. Persistence in continuous darkness of the rhythm of malic enzyme activity (measured by label incorporation into pyruvate or by CO₂ output) establishes its endogenous character and shows that light is not necessary for malate decarboxylation. The role of the dawn signal would be to entrain the CAM system, i.e. to phase the endogenous rhythm of malic enzyme activity correctly to local time. The possibility of an effect of the phase of the PEP carboxylase rhythm on the phase of the decarboxylation rhythm is ruled out by the presence of the intermediary malate storage step. Phase responses to red and to far-red show that phytochrome is involved in rephasing the rhythm of malic enzyme activity. The relative position of dusk affords a ‘measurement’ of the season by the CAM system entrained by dawn. According to the dusk-dawn interval, the level of PEP carboxylase capacity (amount of active enzyme) is modified, resulting in changes of CAM level (high activity under short days).     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Rapid triggering of malate accumulation in the C3/CAM intermediate plant Sedum telephium: relationship with water status and phosphoenolpyruvate carboxylase

Sedum telephium is a C₃/CAM intermediate plant in which expressionof CAM is caused by water deficit. The timing of the C₃-CAMswitch and its relationship with water status and phosphoenolpyruvate(PEP) carboxylase activity have been investigated. Water deficitwas provided by application of polyethylene glycol (PEG) solutionsso that roots were exposed to water potentials from 0 to –2.0 MPa below that of the nutrient solution. The response ofthe plants was measured during the first dark period after PEGaddition and 7 d later. Malic acid accumulation was triggeredduring the first dark period at root water potentials of –0.3MPa or less. This corresponded with very small decreases inleaf water potential and relative water content. The capacityof PEP carboxylase was not altered at any water potential duringthe first dark period. After 7 d the capacity of PEP carboxylaseprogressively increased as water potential declined to –0.4MPa. At this, and more negative, water potentials it was 5-foldhigher than in well-watered leaves. Malic acid fluctuationsincreased with decreasing PEG water potential below a thresholdof –0.1 MPa. Malic acid levels at the end of the lightperiod were progressively lower as water potential decreased.NAD- and NADP-malic enzyme activity were not affected by lowwater potential. Leaves detached from well-watered plants in the middle of thelight period and kept hydrated did not accumulate malic acidduring the following dark period. Allowing the leaves to lose10% of their water content induced malic acid accumulation duringthe same time. Conversely, leaves detached from long-term droughtedplants (which had malate fluctuations and a PEP carboxylasecapacity 5-fold higher than well-watered plants) accumulatedmalate during the night if maintained at the same low hydrationstate (82%RWC), whereas malic acid accumulation was promptlyreduced if they were rehydrated. Malic acid accumulation couldtherefore be rapidly altered by changing the hydration stateof the leaves. The short-term rehydration treatments did notalter PEP carboxylase capacity. However, alteration of leafhydration affected the apparent K_m (PEP) of PEP carboxylaseextracted 1 h before the end of the dark period. The K_m wasincreased by rehydration and decreased by dehydration. Sensitivityto feedback inhibition by malate was not affected by hydrationstate and was high for PEP carboxylase from well-watered leavesand lower for PEP carboxylase from long-term droughted leaves. Taken together, the responses of intact plants and detachedleaves show that malic acid accumulation can be triggered veryrapidly by small water deficits in the leaves. The extent ofnight-time malic acid accumulation is independent of PEP carboxylasecapacity. However, a change in the hydration state of the leavescan rapidly alter the affinity of PEP carboxylase for PEP. Theregulation of malic acid accumulation in relation to the drought-inducedtriggering of CAM is discussed. Key words: Crassulacean acid metabolism, water stress, Sedum telephium, phosphoenolpyruvate carboxylase (PEP carboxylase), malic enzyme     

Regulation of phospho(enol)-pyruvate-and oxaloacetate-converting enzymes in Corynebacterium glutamicum

The presence and properties of the enzymes involved in the synthesis and conversion of phospho(enol)pyruvate (PEP) and oxaloacetate (OAA), the precursors for aspartate-derived amino acids, were investigated in three different Corynebacterium strains. This study revealed the presence of both PEP carboxykinase 0.29 mol·min^–1·mg^–1 of protein [units (U)·mg^–1] and PEP synthetase (0.13 U·mg^–1) in C. 2 glutamicum as well as pyruvate kinase (1.4 U·mg^–1) and PEP carboxylase (0.16 U·mg^–1). With the exception of PEP carboxykinase these activities were also present in glucose-grown C. flavum and C. lactofermentum. Pyruvate carboxylase activity was not detected in all three species cultivated on glucose or lactate. At least five enzyme activities that utilize OAA as a substrate were detected in crude extracts of C. glutamicum: citrate synthase (2 U·mg^–1), malate dehydrogenase (2.5 U·mg^–1), glutamate: OAA transaminase (1 U·mg^–1), OAA-decarboxylating activity (0.89 U·mg^–1) and the previously mentioned PEP carboxykinase (0.29 U·mg^–1). The partially purified OAA-decarboxylase activity of C. glutamicum was completely dependent on the presence of inosine diphosphate and Mn²⁺, had a Michaelis constant (K _m) of 2.0mm for OAA and was inhibited by ADP and coenzyme A (CoA). Examination of the kinetic properties showed that adenine nucleotides and CoA derivatives have reciprocal but reinforcing effects on the enzymes catalyzing the interconversion of pyruvate, PEP and OAA in C. glutamicum. A model for the regulation of the carbon flow based on these findings is presented.Correspondence to: M. S. M. Jetten     

Increases in phosphoenolpyruvate carboxylase activity in iron-deficient sugar beet roots: Analysis of spatial localization and post-translational modification

Root tips of Fe-deficient and Fe-sufficient sugar beet plants grown in hydroponics have been used to study the changes in the amount and activity of the cytosolic enzyme phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31). Phosphoenolpyruvate carboxylase activity in extracts of the yellow Fe-deficient root tips was, at pH 7.3, 30-fold higher (when expressed on a FW basis) and 7.1-fold higher (when expressed on a protein basis) than that found in the extracts of Fe-sufficient root tips. The amount of phosphoenolpyruvate carboxylase protein determined by immuno-blotting was, on a protein basis, 35-fold larger in the yellow zone of Fe-deficient root tips than in the Fe-sufficient root tips. The inhibition of the phosphoenolpyruvate carboxylase activity by 500 m malate was 41 and 58% in the extracts Fe-deficient and Fe-sufficient roots. The possibility that post-translational regulation of phosphoenolpyruvate carboxylase may occur mediated through phosphorylation, was studied by immunological detection of phosphoserine residues in root tip extracts.     

Phosphoenolpyruvate carboxylase from Acetobacter aceti

In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K _m values for PEP and NaHCO₃ were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K _m for Mn²⁺, Co²⁺ and Mg²⁺ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg²⁺. Mn²⁺ and Co²⁺ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA (K _a 0.18 mM) or propionyl CoA (K _a 0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid     

Heat inactivation of leaf phosphoenolpyruvate carboxylase: Protection by aspartate and malate in C4 plants

The activity of phosphoenolpyruvate (PEP) carboxylase EC 4.1.1.31 in leaf extracts of Eleusine indica L. Gaertn., a C₄ plant, exhibited a temperature optimum of 35–37° C with a complete loss of activity at 50° C. However, the enzyme was protected effectively from heat inactivation up to 55° C by L-aspartate. Activation energies (Ea) for the enzyme in the presence of aspartate were 2.5 times lower than that of the control enzyme. Arrhenius plots of PEP carboxylase activity (±aspartate) showed a break in the slope around 17–20° C with a 3-fold increase in the Ea below the break. The discontinuity in the slopes was abolished by treating the enzyme extracts with Triton X-100, suggesting that PEP carboxylase in C₄ plants is associated with lipid and may be a membrane bound enzyme. Depending upon the species, the major C₄ acid formed during photosynthesis (malate or aspartate) was found to be more protective than the minor C₄ acid against the heat inactivation of their PEP carboxylase. Oxaloacetate, the reaction product, was less effective compared to malate or aspartate. Several allosteric inhibitors of PEP carboxylase were found to be moderately to highly effective in protecting the C₄ enzyme while its activators showed no significant effect. PEP carboxylase from C₃ species was not protected from thermal inactivation by the C₄ acids. The physiological significance of these results is discussed in relation to the high temperature tolerance of C₄ plants.Abbreviations CAM crassulaccan acid metabolism - Chl chlorophyll - Ea activation energy - PEP phosphoenolypyruvate Journal Series Paper, New Jersey Agricultural Experiment Station     

Interspecific variation in assimilation of 14CO2 into C4 acids by leaves of C3, C4 and C3−C4 intermediate Flaveria species near the CO2 compensation concentration

The assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of C₃, C₄ and C₃–C₄ intermediate Flaveria species was investigated near the CO₂ compensation concentration ^* in order to determine the potential role of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in reducing photorespiration in the intermediates. Relative to air concentrations of CO₂, the proportion of CO₂ fixed by PEP carboxylase at ^* increased in all six C₃–C₄ intermediate species examined. However, F. floridana J.R. Johnston and F. ramosissima Klatt were shown to be markedly less responsive to reduced external CO₂, with only about a 1.6-fold enhancement of CO₂ assimilation by PEP carboxylase, as compared to a 3.0- to 3.7-fold increase for the other C₃–C₄ species examined, namely, F. linearis Lag., F. anomala B.L. Robinson, F. chloraefolia A. Gray and F. pubescens Rydb. The C₃ species F. pringlei Gandoger and F. cronquistii A.M. Powell exhibited a 1.5- and 2.9-fold increase in labeled malate and aspartate, respectively, at ^*. Assimilation of CO₂ by PEP carboxylase in the C₄ species F. trinervia (Spreng.) C. Mohr, F. australasica Hook., and the C₄-like species F. brownii A.M. Powell was relatively insensitive to subatmospheric levels of CO₂. The interspecific variation among the intermediate Flaverias may signify that F. floridana and F. ramosissima possess a more C₄-like compartmentation of PEP carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) between the mesophyll and bundle-sheath cells. Chasing recently labeled malate and aspartate with ¹²CO₂ for 5 min at ^* resulted in an apparent turnover of 25% and 30% of the radiocarbon in these C₄ acids for F. ramosissima and F. floridana, respectively. No substantial turnover was detected for F. linearis, F. anomala, F. chloraefolia or F. pubescens. With the exception of F. floridana and F. ramosissima, it is unlikely that enhanced CO₂ fixation by PEP carboxylase at the CO₂ compensation concentration is a major mechanism for reducing photorespiration in the intermediate Flaveria species. Moreover, these findings support previous related ¹⁴CO₂-labeling studies at air-levels of CO₂ which indicated that F. floridana and F. ramosissima were more C₄-like intermediate species. This is further substantiated by the demonstration that F. floridana PEP carboxylase, like the enzyme in C₄ plants, undergoes a substantial activation (2.2-fold) upon illuminating dark-adapted green leaves. In contrast, light activation was not observed for the enzyme in F. linearis or F. chloraefolia.Abbreviations and symbols PEP phosphoenolpyruvate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CO₂ compensation concentration - ^* a subatmospheric level of CO₂ approximating Published as Paper No. 8832, Journal Series, Nebraska Agricultural Research Division     

Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization

Pyruvate decarboxylase (EC 4.1.1.1) from the ethanol producing bacterium Zymomonas mobilis was purified to homogeneity. This enzyme is an acidic protein with an isoelectric point of 4.87 and has an apparent molecular weight of 200,000±10,000. The enzyme showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 56,500±4,000 which indicated that the enzyme consists of four probably identical subunits. The dissociation of the cofactors Mg²⁺ and thiamine pyrophosphate at pH 8.9 resulted in a total loss of enzyme activity which could be restored to 99.5% at pH 6.0 in the presence of both cofactors. For the apoenzyme the apparent K _m values for Mg²⁺ and thiamine pyrophosphate were determined to be 24 M and 1.28 M. The apparent K _m value for the substrate pyruvate was 0.4 mM. Antiserum prepared against this purified pyruvate decarboxylase failed to crossreact with cell extracts of the reportedly pyruvate decarboxylase positive bacteria Sarcina ventriculi, Erwinia amylovora, or Gluconobacter oxydans, or with cell extracts of Saccharomyces cerevisiae.Abbreviations Tris-buffer 0,01 M tris-HCl buffer, containing 1 mM MgCl₂ 0.1 mM EDTA, 1.0 mM thiamine pyrophosphate, 2 mM mercaptopropanediol, pH 7.0