Bibliography: leaves 148-157 / viii, 157, [126] leaves : ill ; 28 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1985

The synthesis of a new thiamine analogue, 2'-hydroxythiamine, is reported. Kinetic studies with thiamine pyrophosphate analogues and apopyruvate decarboxylase (EC 4.1.1.1) from brewer's yeast, gave the values of 2.3 X 10^-5 M as the K_m for thiamine pyrophosphate and 2.0 X 10^-5 M as the K_m for 2'-ethylthiamine pyrophosphate. The V_max for the latter was 14% that of thiamine pyrophosphate. Inhibitor constants, K_i, were determined for the following competitive inhibitors of thiamine pyrophosphate with the apoenzyme. All values are given for the pyrophosphate esters: tetrahydrothiamine, 0.65 X 10^-5 M; oxythiamine, 2.0 X 10^-5 M; 2'-n-butylthiamine, 4.5 X 10^-5 M; 2'-methoxythiamine, 7.0 X 10^-5 M; pyrithiamine, 7.8 X 10^-5; thiochrome, 15. X 10^-5 M; 2'-desmethylthiamine, 22. X 10^-5 M; 2'-hydroxythiamine, 38. X 10^-5 M. None of the inhibitors exhibited coenzyme activity. A hydrophobic interaction of the 2'-methyl group of thiamine pyrophosphate with the apoenzyme is suggested from these studies. The formation of a fluorescent complex at pH 6.7 between apopyruvate decarboxylase and thiochrome pyrophosphate was detected and found to be dependent upon Mg(II) ions. A similar complex between thiochrome and the apoenzyme could not be detected, demonstrating the importance of the pyrophosphate function in binding to the protein. The shift in the fluorescence emission spectrum of thiochrome pyrophosphate toward lower wavelengths upon complex formation with the apoenzyme, coincided with the behavior of thiochrome in solvents of decreasing dielectric constant. This latter observation suggests involvement of the thiochrome pyrophosphate with a hydrophobic region of the enzyme. A study of the pH dependency of the enzyme-coenzyme complex indicated considerable recombination of apoenzyme and coenzyme at alkaline pH, where dissociation of the coenzyme usually takes place. A rationale for the interpretation of the pH-behavior of the enzyme-coenzyme complex is offered. An amino acid analysis of a highly purified sample of pyruvate decarboxylase, considered to be essentially homogeneous, is reported. Assuming a molecular weight of 175,000 for the enzyme, a total of 1317 amino acid residues were calculated, of which 52.1% fall into the non-polar category. the half-cystine content was calculated as 10.3%, and the proline content, as 4.6%. The specific volume was calculated as 0.737 ml per g. A single low-angle X-ray diffraction study gave a value of 35.5 ± 1.5 A for the radius of gyration of pyruvate decarboxylase. Assuming a spherical shape, a diameter of 91.6 ± 4.0 A was calculated.

A purification of pyruvic carboxylase from dried brewer's yeast is reported. The procedure consists of an initial extraction of one part yeast with three parts of a 5% glycerol-water mixture for two hours at room temperature, an acetone fractionation at -5° between the concentration (v/v) limits of 50% and 85%, an ammonium sulfate fractionation at 0° between the saturation limits of 0.50 and 0.60 and precipitation of inert material with 0.1 M lead acetate. After precipitation of the enzyme with ammonium sulfate, gel filtration on a Sephadex column at a pH of 6.8 is carried out. A mixture of equal amounts of Sephadex types G-100 and G-200 were used. The best purification obtained from the Sephadex column exhibited a single peak in the analytical ultracentrifuge. This represents a more highly purified preparation than the best purification previously reported (Holzer, H. and Beaucamp, K., Biochim. Biophys. Acta, 26, 225 (1961) ). Paper electrophoresis revealed a small amount of a second component. Therefore, homogeneity cannot, as yet be claimed for the preparation. A molecular weight of 250,000 to 300,000, based on the sedimentation coefficient (S_20o, W°) of 8.78 X 10^-13 cm.^2 sec.^-1, which was obtained by means of the ultracentrifuge, is estimated. This estimate is from two to three times previously reported molecular weight values. A conversion of 138.3 micromoles of pyruvate to acetaldehyde per minute per milligram protein has been calculated. A value of 35,000 moles of pyruvate converted to acetaldehyde per mole of pyruvic carboxylase per minute at 30° can be considered an approximate turnover number. Ultrasonic disintegration liberated 1.95 mg. of pyruvic carboxylase per g. of dried brewer's yeast. This yeast can therefore be estimated to contain the enzyme in the amount of 0.2% by weight. Preincubation of pyruvic carboxylase with thiamine pyrophosphate before assaying for activity caused a significant increase in the reaction rate, even with the initial extract. Both thiamine pyrophosphate and Mg(II) were present in the reaction mixture of the assay. It is suggested that dissociation of thiamine pyrophosphate (and probably some Mg(II) also) occurs throughout the purification procedure, which is carried out in the slightly acid pH range. A simple, rapid procedure for the preparation of apocarboxylase by gel filtration with Sephadex G-75 at a pH of 8.0 is also described.

No description available.

Many diseases correlate with abnormal glucose metabolism in cells and organisms. For instance, the human M2 isoform of the glycolytic enzyme pyruvate kinase (M2PYK) plays an important role in metabolic reprogramming of tumour cells whereby aerobic glycolysis or the ‘Warburg effect’ supports cell proliferation by accumulating necessary biomass. By contrast, gluconeogenesis may play an important role, as observed in certain types of trypanosomatid parasites (e.g. the amastigote form of Leishmania major) where anabolism is essential for infectious properties. Hence, these glucose metabolising enzymes are important potential drug targets for cancer and trypanosomiasis. However, many aspects of their regulatory mechanisms are still poorly understood. This thesis describes biochemical and structural studies on M2PYK and on L. major fructose-1,6-bisphosphatase (LmFBPase), providing insights into allosteric mechanisms and structure-based drug design for both enzymes. Human PYKs and LmFBPase were expressed and purified from Escherichia coli, and their kinetics were fully characterised. It was shown that certain amino acids regulate the activity of M2PYK allosterically, but in opposite ways, with some being inhibitors and others activators. X-ray crystallographic structures and biophysical data of M2PYK complexes with alanine, phenylalanine, serine or tryptophan reveal an R-/T-state oscillating model of M2PYK involving a 11° rotation of each subunit. In addition, M2PYK was demonstrated to be a redox-sensitive enzyme. Reducing reagents were shown to help maintain the tetramer and prevent its dissociation, and thereby to activate M2PYK, whereas oxidation and nitrosylation reagents functioned in the opposite sense. Nitrosylation assays showed that the main nitrosylated residue is Cys326 of M2PYK, which is located on the tetramer interface. Dynamics and modulator effects of PYKs were further studied by hydrogen–deuterium exchange by mass spectrometry. These observations highlight the important effects of amino acids on M2PYK regulation. M1PYK by contrast, was demonstrated to be a constitutively fully active pyruvate kinase, with minor effects from modulators. The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is a potential drug target against leishmaniasis. Here we present biochemical and structural studies for LmFBPase, by characterising its activity in a metal-dependent reaction, as well as its inhibition by AMP. The crystal structure of LmFBPase is a homotetramer, composed of monomers with alternating α/β/α/β/α ‘club sandwich’ topologies. In comparison with previously revealed LmFBPase structures, the AMP-complexed structure shows a rotated form of the tetramer. Comparisons of the structures reveal an ‘unlock-androtate’ allosteric mechanism in which AMP binding causes a series of structural changes culminating in an incomplete and non-productive active site. The structure of the effector site of LmFBPase shows a different conformation from human FBPases, thereby offering a potential specific target for Leishmania.

Bioethanol, produced from organic waste as a second-generation biofuel, is an important renewable energy source. Here, recalcitrant carbohydrate sources, such as municipal and agricultural waste, and plants grown on land not suitable for food crops, are exploited. The thermophilic, Gram-positive bacterium Geobacillus thermoglucosidasius is naturally very flexible in its growth substrates and produces a variety of fermentation products, including lactate, formate, acetate and ethanol. TMO Renewables Ltd. used metabolic engineering to enhance ethanol production, creating the production strain TM242 (NCIMB 11955 ∆ldh, ∆pfl, pdhup). Ethanol yield has been increased to 82% of the theoretical maximum on glucose and up to 92% of the theoretical maximum on cellobiose. However, this strain still produces acetate, presumably derived from the overproduction of acetyl-CoA through the upregulated pdh gene encoding the pyruvate dehydrogenase complex. An alternative to the mixed fermentation pathway found in G. thermoglucosidasius is to introduce a homoethanologenic pathway. Yeast and a very limited range of mesophilic bacteria use the homoethanol fermentation pathway, which employs pyruvate decarboxylase (PDC) in conjunction with alcohol dehydrogenase (ADH), to convert pyruvate to ethanol. Despite extensive screening, no PDC has yet been identified in a thermophilic organism. Using the thermophile G. thermoglucosidasius as a host platform, we endeavoured to develop a thermophilic version of the homoethanol pathway for use in Geobacillus spp. This Thesis reports the in vitro characterization and crystal structure of one of the most thermostable bacterial PDCs from the mesophile Zymobacter palmae (ZpPDC) and describes strategies to improve expression of active PDC at high growth temperatures. This includes codon harmonization and the successful development of a PET (producer of ethanol) operon. Furthermore, ancestral sequence reconstruction was explored as an alternative engineering approach, but did not yield a PDC more thermostable than ZpPDC. In vitro ZpPDC is most active at 65°C with a denaturation temperature of 70°C, when sourced from a recombinant mesophilic host. Codon harmonization improved detectable PDC activity in G. thermoglucosidasius cultures grown up to 65°C by up to 42%. Pairing this PDC with G. thermoglucosidasius ADH6 produced a PET functional up to 65°C with ethanol yields of 87% of the theoretical maximum on glucose. This increase in yield at temperatures of up to 15°C higher than previously reported for any PDC expressed.

Graduation date: 1999

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on hepatic pyruvate carboxylase (PC) gene expression were investigated in C57BL/6J Ah[superscipt b/b male mice. A dose-dependent reduction of PC levels and activity occurred in animals given a single intraperitoneal dose of TCDD in a corn oil carrier. The dose ranged from 1 to 75 ug/kg body weight and the analysis performed 8 days postinjection. At the maximum TCDD level investigated, a 10-fold reduction in PC activity occurred. At doses beyond those required to initiate a reduction in PC, a lactate dehydrogenase isozyme patterns shift is observed. This is accompanied by increases in blood lactic acid levels. Northern blot analysis on RNA extracts from hepatic tissues indicated that at 8 days post TCDD treatment, a dose-dependent reduction of hepatic mRNA levels occurs. The aryl hydrocarbon receptor (AhR) is believed to mediate all responses to TCDD. Liganded AhR and the aryl hydrocarbon receptor nuclear translocator (ARNT) protein form a heterodimeric transcription factor which interacts with dioxin response elements (DREs). These are found in enhancer/promoter regions of many genes that respond transcriptionally to TCDD exposure. Cloning and sequencing a region approximately 1.4 kb upstream of the PC translational start site revealed an untranslated leader sequence of 124 nucleotides starting with adenosine. Primary structural analysis of the upstream region revealed an 1nr element in place of a TATA element. Additional transcription factor elements were identified including: Spl, GCF, UBP-1, GRE, CREB, NF-1, HNF-4, TFII-I and E-boxes; DRE elements were notably lacking. A tandem series of 10 evenly spaced E-boxes, which bind ARNT homodimers, are each juxtaposed to a TFII-I element, possibly forming composite elements. Tertiary structure analysis revealed the positioning of nine composite elements displayed as a trio of phased elements. Transient transfections into Hepa lc1c7 cells, using a luciferase reporter gene under the transcriptional control of the PC upstream region, unlike the animal studies, produced an induction in activity in the presence of 10 nM TCDD. Co-transfections with an ARNT encoding plasmid reduced induction indicating overexpression of ARNT protein partially overrides the TCDD-induced increase in activity. These results in relationship to whole animal experiments are discussed. / Graduation date: 1997

No description available.

Glycolytic enzymes have been indicated as potential drug targets in trypanosomatid parasites such as Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania spp. Pyruvate kinase (PYK) catalyses the final reaction in the glycolytic pathway to produce ATP and pyruvate from ADP and phosphoenolpyruvate (PEP), and has been validated by RNAi experiments as a suitable drug target in T. brucei. This thesis describes biochemical and structural studies of PYKs from T. cruzi (TcPYK) and T. brucei (TbPYK), providing not only a foundation but also new clues for PYK-specific inhibitor screening and structure-based drug design. Soluble TcPYK and TbPYK (81% sequence identity) have been expressed and purified from E. coli, and their kinetics have been fully characterised. X-ray crystal structures of apoenzyme TcPYK (apo TcPYK), and of TbPYK in complex with fructose 2,6-bisphosphate (F26BP) (TbPYK/F26BP/Mg) have been determined, and each possesses a tetrameric architecture composed of four identical protein chains. Each chain contains four domains which are A-domain, B-domain, C-domain and N-terminal domain. The active site is located in the cleft between the A- and B-domains, while the F26BP-bound effector site is within the C-domain. The conformational transition between inactive T-state and active R-state for both enzymes requires a concerted 8o rigid-body rotation of each of the four AC-cores (Aand C-domains) in the tetramer. During the T- to R-state transition induced by F26BP binding, the side chain of Arg311 is re-orientated to stabilise the short Aα6′ helix at the active site, and the flexible loop at the effector site is stabilised by F26BP. In this active conformation additional salt bridges form across the C-C interface to lock the enzyme in a more stable R-state. TbPYK/F26BP/Mg is the first ‘effector only’ PYK structure and identifies a third Mg2+ binding site (Mg-3) which is distinct from the two canonical Mg2+ binding sites. The substrate PEP was soaked into crystals of TbPYK/F26BP/Mg resulting in an ‘in crystallo’ 23° B-domain rotation forming a partially closed active site. This is accompanied by active site side-chain reorientations, and the movement of Mg2+ from its ‘priming’ position Mg-3 to its canonical position Mg-1. It is plausible that Mg2+ is retained in its ‘priming’ position after product release to act as a co-activator with F26BP to maintain the enzyme in its R-state conformation, as long as F26BP is present. The inherent oxaloacetate decarboxylase activity of PYK was reported over 30 years ago and has been further characterised by 1H NMR studies in this thesis. In addition, a series of TbPYK structures in complex with product (pyruvate), with analogues of the decarboxylase substrate oxaloacetate (D-malate and α-ketoglutarate), or with the competitive inhibitor oxalate have been determined by crystal soaking, and indicate that both decarboxylase activity and kinase activity share a common active site. A proposed mechanism explains the conserved decarboxylase activity of PYK where the active-site Mg2+ and Lys239 in TbPYK (which is conserved between species) play essential roles in the decarboxylation reaction. Three strategies for designing novel inhibitors against trypanosomatid PYKs have been proposed in this thesis. (1) Develop selective modulators to increase the binding affinity of inhibitors. As an example, F16BP has been shown to regulate the inhibitory effect of PEP analogues (oxalate, D-malate, α-ketoglutarate, malonate and L-tartrate) on TbPYK activity. (2) Develop allosteric inhibitors in order to lock trypanosomatid PYKs in an inactive state where the enzyme has low affinity for substrate binding. (3) A third strategy is to combine multiple modulators and inhibitors to increase the inhibition efficiency and selectivity.