Inhibitor Of Phosphoenolpyruvate Carboxylase Research Articles

Coleataenia prionitis, a C4-like species in the Poaceae.

C4-like plants represent the penultimate stage of evolution from C3 to C4 plants. Although Coleataenia prionitis (formerly Panicum prionitis) has been described as a C4 plant, its leaf anatomy and gas exchange traits suggest that it may be a C4-like plant. Here, we reexamined the leaf structure and biochemical and physiological traits of photosynthesis in this grass. The large vascular bundles were surrounded by two layers of bundle sheath (BS): a colorless outer BS and a chloroplast-rich inner BS. Small vascular bundles, which generally had a single BS layer with various vascular structures, also occurred throughout the mesophyll together with BS cells not associated with vascular tissue. The mesophyll cells did not show a radial arrangement typical of Kranz anatomy. These features suggest that the leaf anatomy of C. prionitis is on the evolutionary pathway to a complete C4 Kranz type. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate, Pi dikinase occurred in the mesophyll and outer BS. Glycine decarboxylase was confined to the inner BS. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulated in the mesophyll and both BSs. C. prionitis had biochemical traits of NADP-malic enzyme type, whereas its gas exchange traits were close to those of C4-like intermediate plants rather than C4 plants. A gas exchange study with a PEPC inhibitor suggested that Rubisco in the mesophyll could fix atmospheric CO2. These data demonstrate that C. prionitis is not a true C4 plant but should be considered as a C4-like plant.

Molecular Cloning of Novel-Type Phosphoenolpyruvate Carboxylase Isoforms in Pitaya (Hylocereus undatus).

Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme involved in the initial CO2 fixation of crassulacean acid metabolism (CAM) photosynthesis. To understand the cultivation characteristics of a CAM plant pitaya, it is necessary to clarify the characteristics of PEPC in this species. Here, we cloned three PEPC cDNAs in pitaya, HuPPC1, HuPPC2, and HuPPC3, which encode 942, 934, and 966 amino acid residues, respectively. Phylogenetic analysis indicated that these PEPC belonged to plant-type PEPC (PTPC), although HuPPC1 and HuPPC2 have no Ser-phosphorylation motif in N-terminal region, which is a crucial regulation site in PTPC and contributes to CAM periodicity. HuPPC1 and HuPPC2 phylogenetically unique to the Cactaceae family, whereas HuPPC3 was included in a CAM clade. Two isoforms were partially purified at the protein level and were assigned as HuPPC2 and HuPPC3 using MASCOT analysis. The most distinct difference in enzymatic properties between the two was sensitivity to malate and aspartate, both of which are allosteric inhibitors of PEPC. With 2 mM malate, HuPPC3 was inhibited to 10% of the initial activity, whereas HuPPC2 activity was maintained at 70%. Aspartate inhibited HuPPC3 activity by approximately 50% at 5 mM; however, such inhibition was not observed for HuPPC2 at 10 mM. These results suggest that HuPPC3 corresponds to a general CAM-related PEPC, whereas HuPPC1 and HuPPC2 are related to carbon and/or nitrogen metabolism, with a characteristic regulation mechanism similar to those of Cactaceae plants.

Artificial miRNA inhibition of phosphoenolpyruvate carboxylase increases fatty acid production in a green microalga Chlamydomonas reinhardtii

BackgroundNutrient limitation, such as nitrogen depletion, is the most widely used method for improving microalgae fatty acid production; however, these harsh conditions also inhibit algal growth significantly and even kill cells at all. To avoid these problems, we used artificial microRNA (amiRNA) technology as a useful tool to manipulate metabolic pathways to increase fatty acid contents effectively in the green microalga Chlamydomonas reinhardtii. We down-regulated the expression of phosphoenolpyruvate carboxylase (PEPC), which catalyzes the formation of oxaloacetate from phosphoenolpyruvate and regulates carbon flux.ResultsamiRNAs against two CrPEPC genes were designed and transformed into Chlamydomonas cells and amiRNAs were induced by heat shock treatment. The transcription levels of amiRNAs increased 16–28 times, resulting in the remarkable decreases of the expression of CrPEPCs. In the end, inhibiting the expression of the CrPEPC genes dramatically increased the total fatty acid content in the transgenic algae by 28.7–48.6%, which mostly increased the content of C16–C22 fatty acids. Furthermore, the highest content was that of C18:3n3 with an average increase of 35.75%, while C20–C22 fatty acid content significantly increased by 85–160%.ConclusionsOverall our results suggest that heat shock treatment induced the expression of amiRNAs, which can effectively down-regulate the expression of CrPEPCs in C. reinhardtii, resulting in an increase of fatty acid synthesis with the most significant increase occurring for C16 to C22 fatty acids.

Expression and inhibition of the carboxylating and decarboxylating enzymes in the photosynthetic C4 pathway of marine diatoms.

There is evidence that the CO(2)-concentrating mechanism in the marine diatom Thalassiosira weissflogii operates as a type of single-cell C(4) photosynthesis. In quantitative-PCR assays, we observed 2- to 4-fold up-regulation of two phosphoenolpyruvate carboxylase (PEPC) gene transcripts in Thalassiosira pseudonana cells adapted to low pCO(2), but did not detect such regulation in Phaeodactylum tricornutum grown under similar conditions. Transcripts encoding phosphoenolpyruvate carboxykinase did not appear to be regulated by pCO(2) in either diatom. In T. pseudonana and T. weissflogii, net CO(2) fixation was blocked by 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (a specific inhibitor of PEPC), but was restored by about 50% and 80%, respectively, by addition of millimolar concentrations of KHCO(3). In T. pseudonana, T. weissflogii, and P. tricornutum, rates of net O(2) evolution were reduced by an average of 67%, 55%, and 62%, respectively, in the presence of 20 microm quercetin, also an inhibitor of PEPC. Quercetin promoted net CO(2) leakage from inhibited cells to levels in excess of the equilibrium CO(2) concentration, suggesting that a fraction of the HCO(3)(-) taken up is fated to leak back into the medium as CO(2) when PEPC activity is blocked. In parallel to these experiments, in vitro assays on crude extracts of T. pseudonana demonstrated mean inhibition of 65% of PEPC activity by quercetin. In the presence of 5 mm 3-mercaptopicolinic acid (3-MPA), a classic inhibitor of phosphoenolpyruvate carboxykinase, photosynthetic O(2) evolution was reduced by 90% in T. pseudonana. In T. weissflogii and P. tricornutum, 5 mm 3-MPA totally inhibited net CO(2) fixation and O(2) evolution. Neither quercetin nor 3-MPA had a significant inhibitory effect on photosynthetic O(2) evolution or CO(2) uptake in the marine chlorophyte isolates Chlamydomonas sp. or Dunaliella tertiolecta. Our evidence supports the idea that C(4)-based CO(2)-concentrating mechanisms are generally distributed in diatoms. This conclusion is discussed within the context of the evolutionary success of diatoms.

The role of the C4 pathway in carbon accumulation and fixation in a marine diatom.

The role of a C(4) pathway in photosynthetic carbon fixation by marine diatoms is presently debated. Previous labeling studies have shown the transfer of photosynthetically fixed carbon through a C(4) pathway and recent genomic data provide evidence for the existence of key enzymes involved in C(4) metabolism. Nonetheless, the importance of the C(4) pathway in photosynthesis has been questioned and this pathway is seen as redundant to the known CO(2) concentrating mechanism of diatoms. Here we show that the inhibition of phosphoenolpyruvate carboxylase (PEPCase) by 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate resulted in a more than 90% decrease in whole cell photosynthesis in Thalassiosira weissflogii cells acclimated to low CO(2) (10 microm), but had little effect on photosynthesis in the C(3) marine Chlorophyte, Chlamydomonas sp. In 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate-treated T. weissflogii cells, elevated CO(2) (150 microm) or low O(2) (80-180 microm) restored photosynthesis to the control rate linking PEPCase inhibition with CO(2) supply in this diatom. In C(4) organic carbon-inorganic carbon competition experiments, the (12)C-labeled C(4) products of PEPCase, oxaloacetic acid and its reduced form malic acid suppressed the fixation of (14)C-labeled inorganic carbon by 40% to 50%, but had no effect on O(2) evolution in photosynthesizing diatoms. Oxaloacetic acid-dependent O(2) evolution in T. weissflogii was twice as high in cells acclimated to 10 microm rather than 22 microm CO(2), indicating that the use of C(4) compounds for photosynthesis is regulated over the range of CO(2) concentrations observed in marine surface waters. Short-term (14)C uptake (silicone oil centrifugation) and CO(2) release (membrane inlet mass spectrometry) experiments that employed a protein denaturing cell extraction solution containing the PEPCKase inhibitor mercaptopicolinic acid revealed that much of the carbon taken up by diatoms during photosynthesis is stored as organic carbon before being fixed in the Calvin cycle, as expected if the C(4) pathway functions as a CO(2) concentrating mechanism. Together these results demonstrate that the C(4) pathway is important in carbon accumulation and photosynthetic carbon fixation in diatoms at low (atmospheric) CO(2).

Influence of the nitrogen source and metabolites on the Vmax of phosphoenolpyruvate carboxylase from the unicellular green alga Dunaliella parva CCAP 19/9 (Volvocales, Chlorophyceae)

The activity of phosphoenolpyruvate carboxylase (PEPC) was measured in cell-free extracts of the salt-tolerant unicellular green alga Dunaliella parva Lerche CCAP 19/9. For cells grown in batch cultures with 5 mM NaNO3 as the sole source of nitrogen, the optimum pH for PEPC activity was 8 and the reaction was saturated by 1 mM phosphoenolpyruvate (PEP). The K0.5 for PEP was 150 μM and the Vmax was 0.18 ± 0.04 μmol h−1 mg−1 soluble protein. The effect of key metabolites on PEPC activity was determined under optimum assay conditions. Glycerol, the main osmoticum of Dunaliella, had little or no effect on PEPC activity, but dihydroxyacetone phosphate, pyruvate, α-ketoglutarate, orthophosphate, glutamine, glutamate and aspartate all stimulated activity. Surprisingly, the largest stimulation was exerted by aspartate, generally an inhibitor of PEPC, which enhanced PEPC activity up to 30-fold. Oxaloacetate slightly inhibited PEPC activity, but the most effective inhibitor was malate, which caused an 80–90% decrease in the carboxylation, even at low concentration. The response of PEPC activity to replacement of NaNO3 with NH4Cl was tested in continuous culture. The activity of PEPC increased when NH4+ was added to the medium and, even if it decreased somewhat during acclimation to the new nitrogen source, it stayed at levels that were appreciably higher than those observed in NO3−-grown cells. These results are discussed with regard to the role that PEPC may play under different nitrogen growth regimes.

Effects of an inhibitor of phosphoenolpyruvate carboxylase on photosynthesis of the terrestrial forms of amphibious Eleocharis species.

The leafless amphibious sedge Eleocharis vivipara develops culms with C(4) traits and Kranz anatomy under terrestrial conditions, but develops culms with C(3) traits and non-Kranz anatomy under submerged conditions. The culms of the terrestrial form have high C(4) enzyme activities, while those of the submerged form have decreased C(4) enzyme activities. The culms accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the mesophyll cells (MC) and the bundle sheath cells. The Rubisco in the MC may be responsible for the operation of the C(3) pathway in the submerged form. To verify the presence of the C(3) cycle in the MC, we examined the effects of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl) -propenoate (DCDP), an inhibitor of phosphoenolpyruvate carboxylase (PEPC), on photosynthesis in culms of the terrestrial forms of E. vivipara and related amphibious species, E. baldwinii and E. retroflexa ssp. chaetaria. When 1 mM DCDP was fed via the transpiration stream to excised leaves, photosynthesis was inhibited completely in Fimbristylis dichotoma (C(4) control), but by only 20% in potato (C(3) control). In the terrestrial Eleocharis plants, the degree of inhibition of photosynthesis by DCDP was intermediate between those of the C(4) and C(3) plants, at 58-81%. These results suggest that photosynthesis under DCDP treatment in the terrestrial Eleocharis plants is due mainly to fixation of atmospheric CO(2) by Rubisco and probably the C(3) cycle in the MC. These features are reminiscent of those in C(4)-like plants. Differential effects of DCDP on photosynthesis of the 3 Eleocharis species are discussed in relation to differences in the degree of Rubisco accumulation and C(3) activity in the MC.

The interaction of shikimic acid and protein phosphorylation with pep carboxylase from the C 4 dicot Amaranthus viridis

The effect of shikimic acid was studied on the purified dephospho and phosphorylated (in vitro) forms of PEPC from Amaranthus viridis. Kinetic analysis showed that the inhibitory effect of shikimic acid was dependent on the phosphorylation state of the enzyme. Shikimic acid also decreased 32P incorporation from Mg[γ- 32P]ATP into the dark-form of C 4 PEPC. Shikimic acid mimics the behavior of l-malate, in that it decreases both the enzyme's activity and phosphorylatability. A possible role for shikimic acid in the regulation of PEPC activity in plants is suggested. Shikimic acid has been described as a potent competitive inhibitor of the activity of C 4 phospho- enolpyruvate carboxylase (PEPC) from Amaranthus viridis. In the present study, the effects of shikimic acid were examined further with the dephospho (dark-form) and in vitro phosphorylated forms of homogeneous PEPC from A. viridis. Kinetic analysis showed that the inhibitory effect of shikimic acid was dependent on the phosphorylation state of the enzyme. Thus, the I 50 value of shikimic acid for dark-form PEPC was six times lower than that for the phosphorylated enzyme (12 vs 71 μM, respectively). When Glc6P, an activator of C 4 PEPC, was present in the assay medium, the I 50 value increased 2- and 3-times with the phospho and dephospho PEPC-forms, respectively. Shikimic acid also markedly decreased 32P incorporation from Mg[γ- 32P]ATP into the dark-formsof C 4 PEPC, but not casein, catalyzed by protein kinase A. In this way, shikimic acid mimics the behaviour of l-malate, a well-known inhibitor of PEPC, in that it decreases both the enzyme's activity and phosphorylatability. Based on these data, a possible role for shikimic acid in the regulation of PEPC activity in plants is suggested.

Regulation and Localization of Key Enzymes during the Induction of Kranz-Less, C4-Type Photosynthesis in Hydrilla verticillata.

Kranz-less, C4-type photosynthesis was induced in the submersed monocot Hydrilla verticillata (L.f.) Royle. During a 12-d induction period the CO2 compensation point and O2 inhibition of photosynthesis declined linearly. Phosphoenolpyruvate carboxylase (PEPC) activity increased 16-fold, with the major increase occurring within 3 d. Asparagine and alanine aminotransferases were also induced rapidly. Pyruvate orthophosphate dikinase (PPDK) and NADP-malic enzyme (ME) activities increased 10-fold but slowly over 15 d. Total ribulose-1,5-bisphosphate carboxylase/oxygenase activity did not increase, and its activation declined from 82 to 50%. Western blots for PEPC, PPDK, and NADP-ME indicated that increased protein levels were involved in their induction. The H. verticillata NADP-ME polypeptide was larger (90 kD) than the maize C4 enzyme (62 kD). PEPC and PPDK exhibited up-regulation in the light. Subcellular fractionation of C4-type leaves showed that PEPC was cytosolic, whereas PPDK and NADP-ME were located in the chloroplasts. The O2 inhibition of photosynthesis was doubled when C4-type but not C3-type leaves were exposed to diethyl oxalacetate, a PEPC inhibitor. The data are consistent with a C4-cycle concentrating CO2 in H. verticillata chloroplasts and indicate that Kranz anatomy is not obligatory for C4-type photosynthesis. H. verticillata predates modern terrestrial C4 monocots; therefore, this inducible CO2-concentrating mechanism may represent an ancient form of C4 photosynthesis.

Both rubisco and phosphoenolpyruvate carboxylase are beneficial for stomatal function in epidermal strips of Commelina benghalensis

The relative importance of the Calvin cycle and β-carboxylation, through phosphoenolpyruvate carboxylase (PEPC), pathways was assessed in relation to stomatal opening in epidermal strips of Commelina benghalensis, using d,l-glyceraldehyde (an inhibitor of the Calvin cycle) and 3,3-dichloro-2-dihydroxyphosphinoyl-methyl-2-propenoate (DCDP, an inhibitor of PEPC) or metabolites related to the Calvin cycle or PEPC. Stomatal opening was markedly inhibited by both DCDP and d,l-glyceraldehyde. In the absence of these inhibitors, exogenous metabolites such as PEP or ribose-5-P (R-5-P) had little or no effect on stomatal opening. On the other hand, in the presence of DCDP or d,l-glyceraldehyde, metabolites related to not only PEPC (PEP or malate) but also the Calvin cycle (R-5-P or PGA) relieved the inhibition of stomatal opening. We suggest that both Calvin cycle and β-carboxylation activity are beneficial for stomatal opening, particularly when either of these pathways is restricted.

Regulation of Phosphoenolpyruvate Carboxylase in Zea mays by Protein Phosphorylation and Metabolites and Their Roles in Photosynthesis

The effects of metabolites, protein phosphorylation and malate inhibition on phosphoenolpyruvate carboxylase (PEPC) activity were investigated at pH 7.0 in partially purified enzyme from maize leaves. Glycine, glucose 6-phosphate or alanine stimulated the activity two- to three-fold. Glycine and glucose 6-phosphate increased the affinity for PEP by factors of eight and four respectively. These metabolites changed the response of the enzyme activity to pH. Activity increased between pH 6.8 and 8.0 by 10-fold in the absence and 26% in the presence of these metabolites. In vitro phosphorylation of PEPC increased the activity two-fold in the absence but not in the presence of these metabolites. Malate was a strong inhibitor of PEPC, the KI value being 0.25-0.5 mM. Protein phosphorylation and the above metabolites increased the Ki value by factors of three and 12 respectively, but they synergistically increased the Ki 50-fold, thus providing maximal protection against malate inhibition. In the crude extracts from light- and dark-adapted leaves in the presence of a physiological concentration of malate (20 mM), PEPC activity comparable to the photosynthetic rate was obtained only from the light-adapted leaves in the presence of metabolites indicating that both light-induced protein phosphorylation and metabolic activators were essential for PEPC activation during photosynthesis. We propose that both these factors act synergistically to modulate PEPC during photosynthesis in maize.

Phosphoenol acetylphosphonates: Substrate analogues as inhibitors of phosphoenolpyruvate enzymes

Phosphoenol acetylphosphonate (1) and its bis methyl ester (2) are structurally and electrostatically defined analogues of phosphoenolpyruvate (PEP) in which the COO − moiety of PEP is replaced by a P(O)O −(OR) group. It is expected that such materials will bind to electrostatic receptors but the stereoelectronic properties of the phosphonate group will align the molecule at an angle that differs from that of the carboxylate substrate. ▪ The ability of these analogues to bind in place of PEP was evaluated with three enzymes that utilize PEP as a substrate in distinct reactions. Both 1 and 2 inhibit enolase and PEP carboxylase but they do not bind to pyruvate kinase. The inhibition of enolase at pH 6.8 is competitive with respect to 2-phosphoglycerate (the substrate in the reverse of the reaction that utilizes PEP) with K i = 2.2 ± 0.4 m m for 1 and 27 ± 4 m m for 2. The inhibition of enolase by 1 follows a titration curve consistent with the trianionic form of 1 being the active inhibitory form of the analogue. The specificity for the trianionic form shows that the compound is not an analogue of the aci-carboxylate reaction intermediate, which is a tetraanion. Neither 1 nor 2 are substrates for any of the enzymes. Inhibition of PEP carboxylase is pH dependent: at pH 7.8, 1 is a competitive inhibitor of the enzyme with respect to PEP ( K i = 2.2 ± 0.6 m m) while at pH 6.8 it is an activator; 2 has no effect on this enzyme. The diversity of the effects of the phosphonate analogues is consistent with the existence of distinct classes of binding sites for PEP. The variation in binding patterns suggests that the relationship of the points of contact of the substrate with the enzyme and with other substrates differs markedly in each of the enzymes studied.

Regulation of phospho enolpyruvate carboxylase from Crassula argentea: Effect of incubation with ligands and dilution on oligomeric state, activity, and allosteric properties

The relationship between the aggregation state and allosteric properties of purified phospho enolpyruvate carboxylase from Crassula argentea was examined using both kinetic and physical techniques. Analysis by native polyacrylamide gel electrophoresis showed that dilution induced a dissociation of the active tetramer to a less active dimer. Kinetic assays showed that inhibition of phospho enolpyruvate carboxylase by 5 m m malate measured at a saturating phospho enolpyruvate concentration rose to nearly 80% with increasing preassay dilution while the activity in the absence of malate remained constant. Kinetic bursts were observed when enzyme-initiated assays were measured at a subsaturating phospho enolpyruvate concentration. At saturating phospho enolpyruvate concentrations, however, increasing lags developed in response to increasing the preassay dilution of the enzyme. Further, dynamic laser-light scattering measurements showed that preincubation of the dilute enzyme with phospho enolpyruvate stabilized the tetramer while the presence of malate induced dimer formation. These observations confirm and extend earlier work with the extracted active malate insensitive night and less active, malate-sensitive day forms of the enzyme (Wu and Wedding [1985] Plant Physiol. 77, 667–675). Activity measured at subsaturating phospho enolpyruvate concentrations dropped with increasing preassay dilution of enzyme, while activation by 3.2 m m glucose 6-phosphate, assayed at a low phospho enolpyruvate concentration (0.044 m m), increased with dilution to nearly 400%. In this case activation results from a decrease in the control rate as the activity measured in the presence of glucose 6-phosphate was nearly constant, similar in effect to saturating phospho enolpyruvate in the assay. Glucose 6-phosphate induced tetramer formation of the dilute enzyme as measured by light-scattering similar to the effects induced by PEP. In addition, when diluted (dimeric) PEPC was preincubated with PEP or glucose 6-phosphate the enzyme became less sensitive to malate inhibition, while the active-site directed ligand 2-phosphoglycolate had no effect on malate inhibition. These results indicate that both the substrate PEP and the activator glucose 6-phosphate stabilize the active tetramer via binding and interaction at an activator site separate from the active site.

Metabolite Regulation of Partially Purified Soybean Nodule Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was purified 40-fold from soybean (Glycine max L. Merr.) nodules to a specific activity of 5.2 units per milligram per protein and an estimated purity of 28%. Native and subunit molecular masses were determined to be 440 and 100 kilodaltons, respectively, indicating that the enzyme is a homotetramer. The response of enzyme activity to phosphoenolpyruvate (PEP) concentration and to various effectors was influenced by assay pH and glycerol addition to the assay. At pH 7 in the absence of glycerol, the K(m) (PEP) was about twofold greater than at pH 7 in the presence of glycerol or at pH 8. At pH 7 or pH 8 the K(m) (MgPEP) was found to be significantly lower than the respective K(m) (PEP) values. Glucose-6-phosphate, fructose-6-phosphate, glucose-1-phosphate, and dihydroxyacetone phosphate activated PEPC at pH 7 in the absence of glycerol, but had no effect under the other assay conditions. Malate, aspartate, glutamate, citrate, and 2-oxoglutarate were potent inhibitors of PEPC at pH 7 in the absence of glycerol, but their effectiveness was decreased by raising the pH to 8 and/or by adding glycerol. In contrast, 3-phosphoglycerate and 2-phosphoglycerate were less effective inhibitors at pH 7 in the absence of glycerol than under the other assay conditions. Inorganic phosphate (up to 20 millimolar) was an activator at pH 7 in the absence of glycerol but an inhibitor under the other assay conditions. The possible significance of metabolite regulation of PEPC is discussed in relation to the proposed functions of this enzyme in legume nodule metabolism.

Partial characterization of guard-cell phospho enolpyruvate carboxylase: Kinetic datum collection in real time from single-cell activities

Maximum velocity and K m(PEP·Mg) of phospho enolpyruvate carboxylase (PEPC) from stomatal guard cells of Vicia faba L. were determined as a function of pH, presence of malate, and physiological state of guard cells. The biochemical rationale for these measurements is that (a) massive proton extrusion from guard cells, the primary event that drives stomatal movements, has been speculated to alkalinize the cell; (b) guard-cell malate concentration increases severalfold on stomatal opening, and malate, generally an inhibitor of PEPC's, affects the oligomeric state of some PEPC's; and (c) the apparent in vivo activity of guard-cell PEPC is greatly enhanced during stomatal opening, compared with that of other physiological states of these cells. As there are precedents for cell-specific expression of particular forms of PEPC and for labile reversible, posttranslational modifications (which are manifested kinetically as distinct physiological-state isoforms), individual assays were initiated on the addition of a single stomatal complex directly to a microdroplet of assay cocktail. The stomatal complexes (each of which comprises a pair of guard cells having a mass of 6 × 10 −9 g) were dissected from lyophilized leaf tissue that had been freeze-quenched either before, during, or after a treatment to open stomata. V max at pH 7.0 was not significantly different from that at pH 8.5. Neither V max nor K m(PEP·Mg) was distinguished on the basis of the physiological state of the tissue from which the enzyme was extracted. However, K m(PEP·Mg) was >4 × lower at pH 8.5 than at pH 7.0. Malate inhibition was competitive at both pH's, but inhibition was >3 × greater at the lower pH. These data indicate that the combined effects of pH and malate over the range studied can produce changes in enzyme velocity of approximately 24-fold. Thus, the results are consistent with an interpretation that guard-cell PEPC is regulated by the cytoplasmic chemical environment and not by alternations between physiological-state isoforms.

Are‐examination of NaCI effects on phosphoenolpyruvate carboxylase at high (physiological) enzyme concentrations

Inhibition of phosphoenolpyruvate carboxylase (EC 4.1.1 31) from the C4‐halophyte Salsola soda L. by NaCI is compeutive to phosphoenolpyruvate (PEP). Physiological (betaine, glycerol) and synthetic (polyethylene glycol) osmotica and the allosteric activator glucose‐6‐phosphate (G6P) increase the apparent affinity of the enzyme for PEP and also alleviate the inhibition by NaCl. Physiological osmotica that either increase the Km(PEP) (proline) or are neutral (sorbitol), do not protect the enzyme against NaCI attack. In the absence of cosolutes and, G6P, the enzyme is self‐protected when its concentration in the assay medium is increased to more physiological values. In addition, the amount of betaine needed for complete protection is inversely related to native protein concentration in the assay. Exogenous protein (bovine serum albumin or bovine skin gelatin) have no effect on either Km(PEP), or extent of NaCl inhibition. These results can be better explained with the exclusion volume theory and the inferred assumption that both cosolutes and high protein concentration strengthen intrinsic aggregation properties of enzymes. It is suggested that the extremely high phosphoenolpyruvate carboxylase concentration in the cytoplasm and the accumulation of compatible solutes in response to water stress fully protect the enzyme in vivo against the chaotropic effects of NaCI.

Kinetic Properties of Phosphoenolpyruvate Carboxylase in Peperomia camptotricha

Kinetic characteristics of phosphoenolpyruvate carboxylase (PEPC) from the epiphytic C(3) or C(4): CAM intermediate plant, Peperomia camptotricha, were investigated. Few day versus night differences in V(max),K(m(PEP))), or malate inhibition were observed, even in extracts from water-stressed plants which characteristically perform CAM, regardless of efforts to stabilize day/night forms. The PEPC extracted from plants during the light period remained stable, without much of an increase or decrease in activity for at least 22 hours at 0 to 4 degrees C. Extracts from mature, fully developed leaves had slightly greater PEPC activity than from very young, developing leaves. Generally, however, the kinetic properties of PEPC extracted from mature leaves of plants grown under short day (SD), long day (LD), or 1-week water-stress conditions, as well as from young, developing leaves, were similar. The PEPC inhibitor, l-malate, decreased the V(max) and increased the K(m(PEP)) for all treatments. Under specific conditions, malate did not inhibit PEPC rates in the dark extracts as much as the light. The PEPC activator, glucose-6-phosphate (G-6-P), lowered the K(m(PEP)) for all treatments. At saturating PEP concentrations, PEPC activity was independent of pH in the range of 7.5 to 9.0. At subsaturating PEP concentrations, the pH optimum was 7.8. The rates of PEPC activity were lower in the light period extracts than the dark, at pH 7.1, but day/night PEPC was equally active at pH 7.8. At pH 7.5 and a subsaturating PEP concentration, G-6-P significantly activated PEPC. At pH 8, however, only slight activation by G-6-P was observed. The lower pH of 7.5 combined with l-malate addition, greatly inhibited PEPC, particularly in extracts from young, developing leaves which were completely inhibited at an l-malate concentration of 1 millimolar. However, malate did not further inhibit PEPC activity in mature leaves when assayed at pH 7.1. The fairly constant day/night kinetic and regulatory properties of PEPC from P. camptotricha are unlike those of PEPC from CAM or C(4) species studied, and are consistent with the photosynthetic metabolism of this plant.

Inhibition of phosphoenolpyruvate carboxylase from maize by 2-phosphoglycollate

The phosphoenolpyruvate carboxylase from maize leaf was strongly inhibited by 2-phosphoglycollate. The pH of the reaction did not influence the extent of inhibition by 2-phosphoglycollate. The kinetic analysis of the inhibition data by Lineweaver-Burk method showed that 2-phosphoglycollate inhibition was competitive with respect to phosphoenolpyruvate. The secondary plot of the data showed nonlinearity indicating that there may be two 2-phosphoglycollate binding sites with Ki values of 0.4 mM and 0.16 mM. The biphasic nature of the inhibition was also evident when the data were plotted using the method of Dixon. 2-phosphoglycollate inhibition was uncompetitive with respect to Mg(2+) suggestting that it binds only to enzyme-Mg(2+) complex.

Activity of maize leaf phosphoenolpyruvate carboxylase in relation to tautomerization and nonenzymatic decarboxylation of oxaloacetate

The keto form of oxaloacetate (OAA), a product of phosphoenolpyruvate carboxylase (PEPC) activity, can undergo various nonenzymatic conversions which make conventional methods of assaying the enzyme difficult, because the products may either act as inhibitors or go undetected. In studies with PEPC isolated from leaves of maize, an assay coupled with reduction of OAA to malate was compared with product analysis using high-performance liquid chromatography and an assay based on P i release. The results show that activity of the enzyme in the assay coupled to malate dehydrogenase is underestimated, to varying extents, depending on magnesium concentration, buffer, and pH. In the assay coupled to malate dehydrogenase, inaccuracies occur due to conversion of the keto form of OAA to the enol form, which is not utilized as a substrate, and due to loss of OAA by decarboxylation to pyruvate. The assay based on P i formation is considered to give the true rate of catalysis. With this assay the pH optimum is 7.8, compared to 8.3–8.5 for the assay coupled to malate dehydrogenase. The metal enol complex of oxaloacetate (M-OAA enol) is an inhibitor of PEPC and conditions which are favorable for forming this tautomer, high pH with divalent metal ions or high concentrations of Tris buffer at a pH below its p K a value, limit catalysis. Glycine stimulates enzyme activity, and it may have its effect by preventing the formation of the hydrated M-OAA enol complex and maintaining more of the OAA in the keto form. This interpretation is consistent with glycine stimulation of malate synthesis in the assay of PEPC coupled to malate dehydrogenase, with glycine stimulation of the decarboxylation of OAA, and with a reduction in the level of the M-OAA enol complex in the presence of glycine.

Active-site-directed inhibition of phosphoenolpyruvate carboxylase from maize leaves by bromopyruvate

Bromopyruvate is a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase with respect to phosphoenolpyruvate ( K i : 2.3 m m at pH 8). Relatively low concentrations of this compound completely and irreversibly inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The haloacid combines first with the carboxylase to give a reversible enzyme-bromopyruvate complex and then alkylates the enzyme. The maximum inactivation rate constant was 0.27 min −1 at pH 7.2 and 30 °C and the concentration of bromopyruvate giving half-maximum rate of inactivation was 1.8 m m. The inactivation was prevented by the substrate phosphoenolpyruvate, in the absence or presence of MgCl 2, and by the competitive inhibitor P-glycolate. Malate afforded protection at pH 7 but not at pH 8. MgCl 2 enhanced the inactivation when it was carried out at pH 7; its effect was mainly due to a decrease in the dissociation constant of the complex between bromopyruvate and the enzyme from 2 to 1.4 m m. This behavior was not observed at pH 8. Analysis of the inactivation at different pH suggests that a group of p K a near 7.5 is important for the binding of the reagent to the carboxylase. Determination of the number of sulfhydryl groups of the native and modified enzyme with [ 3H]- N-ethylmaleimide suggests that the inactivation correlates with the modification of thiol groups in the enzyme. The substrate prevented the modification of these groups. The results suggest that the alkylating reagent modifies cysteinyl residues at the phosphoenolpyruvate binding site of the carboxylase.