Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Escherichia coli phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxylase (transphorsphorylating) EC 4.1.1.49) catalyzes the decarboxylation and subsequent phosphorylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of Mg2+ATP and synergistic catalysis has been observed in the presence of Ca2+ or Mn2+. Structural analyses have shown that active site residues Arg333, Ser250 and Tyr207 are coordinated differently in E. coli PCK structures complexed with Mg2+ATP-oxalate, Mg2+ATP-Mn2+-pyruvate and Mg2+ATP-Ca2+-pyruvate; hence, we hypothesize that the function of Arg333, Ser250 and Tyr207, depends on the absence or presence of Ca2+ or Mn2+ during catalysis by E. coli phosphoenolpyruvate carboxykinase (PCK). In order to verify this hypothesis, site directed mutagenesis of the pckA gene was used to convert Arg333 to Gln, Ser250 to Ala and Tyr207 to Phe, while 14CO2 exchange assay and x-ray crystallography were used to determine the effects of these mutations on catalysis by E. coli PCK in the presence of OAA and Mg2+ATP with Ca2+ or Mn2+ metal ions. Kinetic analysis showed that the Tyr207Phe mutation decrease kcat by 1.7 fold, while Ser250Ala and Arg333Gln reduced kcat by 10.8 and 4,555 fold respectively in the presence of Mg2+ATP and OAA. In the presence of Mg2+ATP, OAA and Ca2+, Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 11,688, 44 and 2 fold respectively. In the presence of Mg2+ATP, OAA and Mn2+ Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 2,880, 4 and 5.5 fold respectively. The crystal structure of Ser250Ala complexed with Mg2+ATP-Mn2+-pyruvate, showed that in the presence of Mn2+, Ser250Ala mutation reduced the angle between the γ-phosphate of ATP and residue 250 by 6.2 Å and increased the distance between the hydroxyl group of Tyr207 and the CH2 group of pyruvate by 0.5 Å. As a result we conclude that Arg333 is important for oxaloacetate decarboxylation and phosphorylation. During catalysis in the presence of Mg2+ATP with or without Ca2+ or Mn2+, Ser250 functions to maintain one γ-phosphate oxygen of ATP in an eclipsed conformation, while Tyr207 functions to drive oxaloacetate decarboxylation during catalysis in the presence of Mn2+ ion. Kinetic and structural studies of E. coli PCK have previously been used to show that Asp269 is involved in metal coordination, while Lys254 and Arg65 are important for Mg2+ATP and OAA binding to E. coli PCK respectively. In this study the E. coli PCK Asp269Asn-Mg2+ATP-Ca2+-pyruvate crystal structure showed that the Asp269Asn mutation reduced the number of ligands coordinating Ca2+ from seven to three, while no electron density was observed for Mg2+ATP and OAA in Lys254Ser and Arg65Gln crystal structures respectively.

PEPC [PEP(phosphoenolpyruvate) carboxylase] is an essential and tightly controlled enzyme located at the core of plant C-metabolism. It fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and N-assimilation. In plants, a small multigene family encodes several closely related plant-type PEPC (PTPC) isozymes along with a distantly related bacterial-type PEPC (BTPC) isozyme. The PTPCs are well studied ~110-kDa subunits that typically exist as a homotetramer (Class-1 PEPC). By contrast, little is known about the larger ~118-kDa BTPC isozyme except that it occurs in developing castor (Ricinus communis) endosperm in tight association with PTPC subunits as a ~900-kDa hetero-octameric complex (Class-2 PEPC) that is greatly desensitized to metabolic effectors compared to Class-1 PEPC. This thesis elucidates the physiological purpose of the BTPC subunits by examining their structure/function relationship within Class-2 PEPC and identifying mechanisms of post-translational control. Recombinant expression and purification of the castor bean BTPC revealed unusual physical and kinetic properties including a remarkable insensitivity to metabolic effectors and a dependence upon PTPC subunits for structural stability. The first purification of a non-proteolyzed plant Class-2 PEPC complex was performed, and the kinetic analysis determined that the BTPC and PTPC subunits have complimentary catalytic properties. The BTPC subunits’ high Km(PEP) and desensitization to metabolic effectors may function as a metabolic overflow mechanism for sustaining flux from PEP to malate when PTPC subunits become feedback inhibited. An anti-PTPC co-immunopurification strategy was utilized to highly enrich non-proteolyzed BTPC from developing castor endosperm for downstream immunological and mass spectrometric analysis. BTPC was in vivo phosphorylated at multiple novel sites, identified by mass spectrometry as Thr4 or 5, Ser425 and Ser451. Phosphosite-specific antibodies towards Ser425 and Ser451 confirmed the existence of these sites in vivo and comparisons of Ser425 phosphorylation patterns established that the castor BTPC and PTPC phosphorytation sites are regulated independently. Phosphomimetic mutants of Ser425 caused BTPC inhibition by increasing its Km(PEP) and sensitivity to feedback inhibition. These results establish a novel mechanism of PEPC control whose implications within plant carbon metabolism are discussed. / Thesis (Ph.D, Biology) -- Queen's University, 2011-09-04 16:46:22.024

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Phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step of gluconeogenesis, and the gene which encodes this enzyme (pckA) is transcriptionally regulated. High pckA expression is observed in succinate-grown cells, while little expression is observed in glucose-grown cells. pckA regulatory mutants have previously been isolated (Osteras et al. 1997) and pckR, a gene encoding a Lacl-GaIR DNA-binding transcriptional regulator, has been implicated in the regulation of pckA transcription. Here we shew that pckR insertion mutations result in a dramatic decrease in pckA expression even in succinate-grown cells. We demonstrate that the previously identified rpk-9 mutation is tightly linked to pckR. The rpk-9 mutation results in constitutive pckA expression, and we show that plasmids carrying the pckR gene complement the rpk-9 mutation in glucose-grown cells. A putative Lacl-GaIR operator binding site has been identified in the pckA promoter, however no evidence of an interaction between this site and the pckR gene product could be found. / Thesis / Master of Science (MS)

Our laboratory is interested in studying the junction of glycolysis and the tricarboxylic acid (TCA) cycle, specifically the enzymes phosphoenolpyruvate carboxykinase, pyruvate carboxylase and phosphoenolpyruvate carboxylase. All produce oxaloacetate (OAA) for the cell. OAA production is critical for cell carbon synthesis in the methanogenic archaea. Therefore OAA-generating enzymes are essential for the survival of methanogens. In part of this study we investigated archaeal-type phosphoenolpyruvate carboxylase (PpcA), a new type of phosphoenolpyruvate carboxylase, which is widespread in the archaea and is found in three bacterial species. The form of phosphoenolpyruvate carboxylase (Ppc) that is prevalent in bacteria and plants is not found in the archaea. Due to complications expressing PpcA in the soluble form and difficulty purifying this enzyme from methanogens, an in-depth investigation of this enzyme's biochemical properties has yet to occur. In this study we demonstrate the successful expression of a PpcA homolog in the soluble fraction of Escherichia coli. We purified the recombinant protein to homogeneity. This development provides the means to study the enzyme's biochemical properties and manipulate the primary sequence in order to identify residues critical to the enzyme's function. We also show that this PpcA homolog does have the postulated activity and investigate its biochemical properties. The data show that PpcA has unique properties in regard to the enzyme's substrate and its regulation by metabolites. Our data also reveal that PpcA is a membrane associated protein, unlike Ppc, which is a soluble protein. We also show that pyruvate carboxylase (Pyc) can be expressed recombinantly in Pseudomonas aeruginosa at levels sufficient for structure-function studies. This is a major step forward in the expression in Pyc because it cannot be expressed at high levels in Escherichia coli. These are important developments in studying the enzymes that connect glycolysis and the TCA cycle. / Master of Science

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Plants exhibit a flexible array of morphological, physiological and biochemical adaptations during phosphorous limitation. Legumes are vulnerable to P deficiency, because it affects their ability to fix atmospheric nitrogen (N2). In particular, legumes from nutrient-poor ecosystems, such as the Fynbos in the Cape Floristic Region (CFR) evolved on P deficient soils and may therefore display unique adaptations to soil P stress. In general, very few studies on legumes have focussed on the belowground structures of nodules as a plant organ. Moreover, even less is known about the P stressed responses in nodules from legumes in nutrient-poor ecosystems. The aim of this research was to investigate the metabolic flexibility of organic acid and amino acid metabolism in the nodulated root system of the Fynbos legume Virgilia divaricata, during low P stress. Virgilia divaricata, which grows in the Cape Floristic Region, was used in this study to enhance our knowledge regarding the vital role that the cytosolic enzyme, phosphoenol pyruvate carboxylase (PEPC) plays in phosphoenol pyruvate (PEP) metabolism, in roots and nodules of this legume during phosphate stress. V. divaricata was grown under glasshouse conditions (20 - 25°C) in sterilized quartz sand for 2-3 months whilst being inoculated with the nitrogen fixation bacteria, Burkholderia phytofirmans, which was isolated from V. divaricata nodules grown in fynbos soil. Two phosphate treatments, 5 μM and 500 μM, were applied simulating low-phosphate and high phosphate conditions respectively using a modified Long Ashton Nutrient Solution to simulate a low nutrient ecosystem such as the Cape Floristic Region. Roots and nodules were then analysed for growth kinetics, nutrient acquisition and distribution, enzyme activity and genetic responses. It was shown that during phosphate deficiency, V. divaricata nodules experienced less Pi stress than roots, due to increased metabolic phosphate conservation reactions during organic acid synthesis via an increased PEPC activity. The increased PEPC activity resulted in an increase in downstream metabolic products such as organic acids, (malic acid and citric acid), and amino acids (glutamate, aspartate and asparagine). Although the biological nitrogen fixation (BNF) declined, the high efficiency of BNF may be underpinned by these altered phosphate conservation pathways and enhanced resource allocation during growth particularly under low phosphate (LP) conditions. Therefore, it can be concluded that the efficiency of the nodules via an increased allocation of resources and P acquiring mechanisms in V. divaricata may be the key to the plant’s ability to adapt to poor P environments and thus sustaining its reliance on BNF. From the data obtained as well as previous findings, it has been established that the phosphate conservation mechanisms in roots and nodules, involve the non-adenylate requiring PEPC-bypass route. 13C Nuclear magnetic resonance (NMR) gave us a better understanding regarding the incorporation rates of the PEPCderived C into malate, α-ketoglutarate and asparagine. It therefore is suggested that V. divaricata nodules may use their large PEPC-derived malate pool to prevent large declines in BNF under low phosphate conditions. The nodules of V. divaricata were able to offset an excessive drop in BNF, despite a decline in inorganic phophosphate (Pi) levels. It therefore appears that nodules have evolved to acquire different mechanisms than roots to adapt to phosphate deficiency in order to maintain their function. This was achieved via increased regulation of nodule PEPC and its downstream products. This implies that compared to roots under low P, nodules alter the metabolism of PEPC derived C, in order to maintain nodule respiration and amino acid synthesis. This trait could be observed in the synthesis of larger 13C malate pools of nodules compared to roots, from PEPC, which was underpinned by their different regulation mechanisms of enzyme activity, of the same protein isoform. Since malate is a potent inhibitor of PEPC activity, roots appear to have invested in more PEPC protein compared to nodules. In contrast, nodules with lower PEPC protein, achieved greater enzyme activity than roots, possibly due to higher phosphorylation in order to reduce the malate effect. The subsequent metabolism of this PEPCderived malate, caused roots and nodules to synthesise asparagine via different pathways. These findings imply that roots and nodules under P stress, synthesise their major export amino acid, asparagine, via different routes. This research has generated new knowledge regarding the physiological impact of the organic and amino acid metabolism, derived from PEPC-C in the roots and nodules of legumes growing in nutrient poor ecosystems. It has demonstrated for the first time that the nodules of legume from a nutrient-poor ecosystem rely on improved resource allocation, Pi distribution, and PEPC-derived organic acids to maintain the efficient functioning of N assimilation under P stress. This may be a consequence of having evolved in a nutrient-poor ecosystem, so that nodule-bacteroid respiration and N metabolism can be maintained in P-poor soils such as the Fynbos. / AFRIKAANSE OPSOMMING: Tydens fosfaat stremming maak plante gebruik van buigsame kombinasies van morfologiese, fisiologiese en biochemiese aanpassings. Peulplante is sensitief vir fosfaat tekorte omdat dit die vermoë om atmosferiese stikstof te kan fikseer, grootliks beïnvloed. Peulplante vanuit ekosisteme met mineraal-arme gronde, soos Fynbos binne die Kaapse Blommeryk, het ontwikkel in grond met lae fosfaatvlakke en mag dus unieke aanpassings tot fosfaat tekorte toon. Oor die algemeen is daar baie min peulplant studies wat fokus op die ondergrondse strukture van wortelknoppies as ‘n plant orgaan. Nog minder inligting is beskikbaar oor wortelknoppies, van peulplante, vanuit mineraalarme ekosisteme, se reaksie teenoor ‘n fosfaat tekort. Die doel van hierdie navorsing was om die metaboliese buigsaamheid van organiese- en aminosuur metabolisme in die (nodulated) wortelknoppie-wortelstelsel van die Fynbos peulplant Virgilia divaricata, tydens fosfaat tekort te ondersoek. Virgilia divaricata wat voorkom in die Kaapse Blommeryk, was gebruik in hierdie studie om die huidige kennis te verbeter van die essensiële rol wat die sitisoliese ensiem, fosfo-enol piruvaat karboksilase (PEPC) in fosfo-enol piruvaat metabolisme tydens ‘n fosfaat tekort speel binne die wortels en wortelknoppies van hierdie peulplant. V. divaricata was gegroei onder glashuis toestande (20 - 25°C) in gesteriliseerde kwartssand vir 2-3 maande. Die plante was geïnokuleer met die stikstoffikserende bakterie, Burkholderia phytofirmans, wat geïsoleer is vanaf V. divaricata wortelknoppies wat in Fynbos grond gegroei is. Twee fosfaatbehandelings, 5μM and 500μM, was toegedien om lae en hoë fosfaat toestande, onderskeidelik, na te boots deur gebruik te maak van ‘n aangepasde Long Ashton voedingstofmengsel om ‘n ekosisteem, soos die Kaapse Blommeryk, met lae voedingstofvlakke na te boots. Die wortels en knoppies was geanaliseer ten opsigte van die groeikinetika, opname en verspreiding van voedingstowwe, ensiemaktiwiteit en genetiese aanpassings. Dis is bewys dat tydens fosfaat tekort V. divaricata wortelknoppies minder fosfaat stres ervaar as die wortels, as gevolg van die verhoogde metaboliese fosfaat bewaringsreaksies tydens organise suur sintese via die styging in PEPC aktiwiteit. Die styging in PEPC aktiwiteit lei tot ‘n verhoging in stroomaf metaboliese produkte soos organiese- (appel- en sitroënsuur) en aminosure (glutamaat, aspartaat en asparagien). Alhoewel biologiese stikstoffiksering verlaag het, kan die hoë doeltreffendheid daarvan ondersteun word deur díe aangepasde fosfaat bewarings weë asook verhoogde hulpbron toekenning tydens groei onder lae fosfaat omstandighede. Dit kan dus afgelei word dat die doeltreffendheid van die wortelknoppies via die verhoging in belegging van hulpbronne en fosfaat opname meganismes in V. divaricata moontlik die sleutel is tot die plant se vermoë om aan te pas tot omgewings met lae fosfaatvlakke en sodoende die afhanklikheid van biologiese stikstofbinding te kan onderhou. Data in hierdie as ook vorige studies, wys dat die fosfaat bewaringsmeganismes in wortels en wortelknoppies die PEPC-ompad roete, wat nie adenilaat benodig nie, gebruik. 13C NMR het meer lig gewerp aangaande die vaslegging van koolstof vanaf PEPC na malaat, α-ketoglutaraat en asparagien. Dit word voorgestel dat V. divaricata knoppies ‘n groot hoeveelheid malaat, afkomstig van PEPC-werking, gebruik om groot dalings in biologiese stikstofbinding tydens fosfaat tekort, te verhoed. Die wortelknoppies van V. divaricata kon ‘n oormatige verlaging in biologiese stikstofbinding voorkom ten spyte van die verlaging in fosfaatvlakke. Dit wil voorkom dat wortelknoppies ander meganismes as die wortels ontwikkel het om aan te pas tot fosfaat tekort en sodoende dus hul funksie behou. Dit word bereik deur ‘n verhoging in die regulering van PEPC en die stroomaf produkte in die wortelknoppies. Dit blyk dat wortelknoppies tydens fosfaat te kort, in vergelyking met wortels, die metabolisme van die koolstof vanaf PEPC verander om sodoende respirasie en aminosuursintese te onderhou. Dit wil voorkom dat hierdie meganismes verskil van die van wortel meganismes. Hierdie eienskap kan toegeskryf word aan die produksie van ‘n groter hoeveelheid van 13C malaat vanaf PEPC in die wortelknoppies teenoor die wortels, wat ondersteun word die verskillende reguleringsmeganismes van ensiemaktiwiteit van dieselfde proteïen isoform. Malaat is ‘n kragtige inhibeerder van PEPC-aktiwiteit, dus blyk dit dat die wortels belê in meer PEPC proteïene as die wortelknoppies. In teenstelling, toon die wortelknoppies met laer PEPC proteïene, ‘n hoër ensiem aktiwiteit as die wortels. Dit kan wees as gevolg van hoër fosforilasie om die effek van malaat te verlaag. Die metabolisme van die malaat vanaf PEPC het die sintese van asparagien in die wortels en wortelknoppies via verskillende roetes tot gevolg gehad. Dit impliseer dat tydens ‘n tekort aan fosfaat, wortels en wortelknoppies hul hoof uitvoer aminosuur, asparagien, deur verskillende roetes sintetiseer. Hierdie studie het nuwe kennis aangaande die fisiologiese impak van organiese- en aminosuur metabolisme met koolstof vanaf PEPC in die wortels en wortelknoppies van peulplante wat voorkom in ekosisteme met lae voedingstofvlakke, voortgebring. Vir die eerste keer is dit bewys dat die wortelknoppies vanaf peulplante wat voorkom in mineraal-arme ekosisteme, staatmaak op verbeterde hulpbron beleggings, fosfaat verspreiding en organiese sure vanaf PEPC om die doeltreffendheid van funksionele stikstofassimilasie tydens fosfaat tekort, te onderhou. Dit mag die gevolg wees van, om in ‘n voedingstof arme ekosisteem te ontwikkel sodat die wortelknoppiebakteroïed respirasie en stikstofmetabolisme onderhou kan word in fosfaat arme grond soos die Fynbos.

In cultured rat hepatocytes the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) is known to be induced by glucagon via an elevation of cAMP. Prostaglandin E₂ has been shown to antagonize the glucagon-activated cAMP formation, glycogen phosphorylase activity and glucose output in hepatocytes. It was the purpose of the current investigation to study the potential of PGE₂ to inhibit the glucagon-induced expression of PCK on the level of mRNA and enzyme activity. PCK mRNA and enzyme activity were increased by 0.1 nM glucagon to a maximum after 2 h and 4 h, respectively. This increase was completely inhibited if 10 μM PGE2 was added concomitantly with glucagon. This inhibition by PGE₂ of glucagon-induced PCK activity was abolished by pertussis toxin treatment. When added at the maximum of PCK mRNA at 2 h, PGE₂ accelerated the decay of mRNA and reduced enzyme activity. This effect was not reversed by pertussis toxin treatment. Since in liver PGE₂ is derived from Kupffer cells, which play a key role in the local inflammatory response, the present data imply that during inflammation PGE₂ may reduce the hepatic gluconeogenic capacity via a Gᵢ-linked signal chain.

Phosphofructokinase (PFK) from the lactic acid bacterium Lactobacillus delbrueckii subspecies bulgaricus (LbPFK) is a non-allosteric PFK with weak binding affinity for both the allosteric ligands phospho(enol)pyruvate (PEP) and magnesium adenosine diphosphate (MgADP). PEP and MgADP bind to the same allosteric binding site but exhibit opposite effects, PEP acting as an inhibitor and MgADP an activator. In 2005, Parichatttanakul, et al. solved the first crystal structure of LbPFK to 1.87 A resolution and allowed for a structural comparison of LbPFK to the allosteric forms of PFK from E. coli (EcPFK) and Bacillus stearothermophilus (BsPFK). Two additional structures of LbPFK have been determined with the first having phosphates bound at the four active sites and four allosteric sites solved to 2.20 A resolution. The second structure solved to 1.83 A resolution contains phosphates at all eight sites with the addition of the substrate fructose-6-phosphate (F6P) in the active sites. These structures are similar to the published sulfate-bound LbPFK structure. Overall, the secondary, tertiary and quaternary structure is conserved with the exception of the residues in the allosteric site. E55, H59, S211, D214, H215 and G216, as well as the long cassettes of residues 52-61 (PFKs1) and 206-218 (PFKs2) were mutated to the corresponding residue/residues in Thermus thermophilus PFK (TtPFK). PFKs1 and PFKs1 were also combined to form PFKs1s2. The single mutations along with PFKs1 and PFKs2 showed no enhancement in PEP binding, but PFKs1s2 enhanced PEP binding 10-fold with no change in MgADP binding compared to LbPFK. D12, located along the active site interface 15 A away from the allosteric site, was mutated to an alanine and exhibited enhanced binding 9-fold for both PEP and MgADP to the allosteric binding site. A crystal structure of D12A was solved to 2.30 A resolution with sulfate bound to all eight binding sites, and showed no major changes in secondary, tertiary or quaternary structure when compared to the sulfate-bound wild-type LbPFK structure. Combining D12A with PFKs1s2 (PFKs1s2/D12A) further enhanced PEP binding with a 21-fold tighter binding compared to LbPFK with MgADP binding being similar to D12A. PEP inhibition was also quantitated in PFKs1s2/D12A with a Q_ay = 0.007 plus/minus 0.0008. Coupling between PEP and F6P in PFKs1s2D12A is 2-fold stronger than the coupling measured in EcPFK and 7-fold stronger than the coupling measured in BsPFK. The coupling measured in PFKs1s2D12A is the first measured in any of the LbPFK variants.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [176]-179).