1. The molecular weights of the G6PD subunits from the three yeast sources, bovine adrenals and rat liver were estimated by SDS-PAGE and found to be in the region of 55-59 K. An estimate of 46 K was similarly made for the G6PD subunit from the prokaryote, L. mesenteroides. 2. Two close bands or sometimes one diffuse band appeared on the polyacrylamide slab gel following electrophoresis and Coomassie blue staining for rat liver G6PD. These corresponded to the molecular weight region of about 57-59 K. So far, the evidence from peptide sequences does not reveal more than one subunit sequence. 3. G6PD from bakers' yeast was inactivated with sodium [1-. C] acetylsalicylic acid, following whichit was shown that 1.1 mole of 14 C moiety had been incorporated per enzyme subunit and that a lysine residue, essential to enzyme activity, had been modified. 4. Inactivation of bakers' yeast G6PD in the presence of high concentrations of substrate or coenzyme indicated that the acetylsalicylic acid was binding to the enzyme at a site which was directly or indirectly involved in substrate binding. The observation that high concentrations of coenzyme in the incubation mixture did not offer protection against acetylsalicylic acid inhibition was supplemented by TRNOE studies which showed no significant change in the conformation of coenzyme bound to bakers' yeast G6PD inactivated with acetylsalicylic acid compared with the active enzyme. 5. Acetylsalicylic acid did not inactivate rat liver G6PD to the same extent as the bakers' yeast enzyme. Inactivation of rat liver 14 G6PD with C-HIAB was relatively slow and resulted in the partial labelling of many cysteine residues. 6. Sequence studies on rat liver G6PD resulted in the isolation of a peptide which was homologous in sequence to the bakers' yeast G6PD tryptic peptide containing the reactive lysine residue, although no evidence for special reactivity of the corresponding lysine residue in the rat liver enzyme was found. 7. A high degree of sequence homology was noted between rat liver G6PD and human erythrocyte G6PD. It was also established that the human erythrocyte G6PD sequence published by Beutler (1983) was incorrect due to the misalignment of tryptic peptides.

No description available.

No description available.

The homotetrameric enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from Escherichia coli was used as a model for probing oligomeric structure in enzymes. Dimeric mutants of this enzyme have been found in previous work to be largely inactive, due to the trapping of a covalent adduct. Partial restoration of catalytic activity has been achieved by incubation in the presence of the substrate pyruvate to displace the adduct. It was hypothesized that the buttressing of dimeric units against one another in the wildtype tetrameric form of DHDPS provides stability in the dimer interface, necessary to maintain optimum catalytic performance and substrate specificity. We hypothesized that buttressing a dimeric DHDPS mutant against a surface would result in restoration of catalytic activity by mimicking the buttressing proposed to occur in the tetrameric structure. To test this hypothesis, dimeric DHDPS mutants were immobilised against an agarose support and the immobilised enzymes characterised. Three DHDPS mutants were prepared, the double mutant DHDPS-C20S/L167C was produced by mutagenesis and a crystal structure obtained in collaboration with Dr Renwick Dobson. Two other mutants, DHDPS-Ll67C and DHDPS-Ll97Y were also over expressed and purified. The quaternary structures of the three mutants were characterised in solution, DHDPS-Ll67C was determined to be tetrameric, DHDPS-C20S-Ll67C was found to equilibrate between tetramer and dimer and DHDPS-Ll97Y was confirmed as a dimer, consistent with previous findings. Modification experiments indicated that the sulfhydryl groups of DHDPS-C20S/L167C were available for immobilisation. Activation experiments indicated that both DHDPS-Ll67C and DHDPS-Ll97Y activated. These results were in accord with those of others in indicating that the displacement of an a-ketoglutarate adduct from the active site was responsible for the activation of mutant DHDPS enzymes. Wild-type DHDPS and the mutants were immobilised through amine and sulfhydryl groups. The free and immobilised enzymes were rigorously characterised, with thermal stability, pH optima, kinetic and lysine inhibition properties determined and compared to wild-type DHDPS. Following immobilisation, substrate affinity was found to decrease for wild-type and mutant enzymes, wild-type KmPyr = 0.26 mM free, 0.8-1.2 mM immobilised, Km(S)-ASA = 0.10 mM free, 1.5-2.5 mM immobilised. Lysine inhibition was determined to be largely unaffected by immobilisation. The largest change in K, was an increase to double that of the free enzyme. Restoration of some catalytic activity was found following the immobilisation of dimeric DHDPS-Ll97Y, the immobilised enzyme was 31 ± 12% more active than free DHDPS-Ll97Y. DHDPS-C20S/L167C was also found to immobilise as a dimer. Comparison ofthe immobilised DHDPS-C20S/L167C dimer with a derivatised free dimeric form ofthis enzyme indicated that an increase from 3% to 9% of wild-type activity had resulted from immobilisation. These results supported the hypothesis that buttressing of a dimeric mutant of DHDPS against a support surface would increase catalytic activity and that buttressing across the dimerdimer interface is essential for optimal catalytic activity in DHDPS enzymes.

No description available.

The homotetrameric enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from Escherichia coli was used as a model for probing oligomeric structure in enzymes. Dimeric mutants of this enzyme have been found in previous work to be largely inactive, due to the trapping of a covalent adduct. Partial restoration of catalytic activity has been achieved by incubation in the presence of the substrate pyruvate to displace the adduct. It was hypothesized that the buttressing of dimeric units against one another in the wildtype tetrameric form of DHDPS provides stability in the dimer interface, necessary to maintain optimum catalytic performance and substrate specificity. We hypothesized that buttressing a dimeric DHDPS mutant against a surface would result in restoration of catalytic activity by mimicking the buttressing proposed to occur in the tetrameric structure. To test this hypothesis, dimeric DHDPS mutants were immobilised against an agarose support and the immobilised enzymes characterised. Three DHDPS mutants were prepared, the double mutant DHDPS-C20S/L167C was produced by mutagenesis and a crystal structure obtained in collaboration with Dr Renwick Dobson. Two other mutants, DHDPS-Ll67C and DHDPS-Ll97Y were also over expressed and purified. The quaternary structures of the three mutants were characterised in solution, DHDPS-Ll67C was determined to be tetrameric, DHDPS-C20S-Ll67C was found to equilibrate between tetramer and dimer and DHDPS-Ll97Y was confirmed as a dimer, consistent with previous findings. Modification experiments indicated that the sulfhydryl groups of DHDPS-C20S/L167C were available for immobilisation. Activation experiments indicated that both DHDPS-Ll67C and DHDPS-Ll97Y activated. These results were in accord with those of others in indicating that the displacement of an a-ketoglutarate adduct from the active site was responsible for the activation of mutant DHDPS enzymes. Wild-type DHDPS and the mutants were immobilised through amine and sulfhydryl groups. The free and immobilised enzymes were rigorously characterised, with thermal stability, pH optima, kinetic and lysine inhibition properties determined and compared to wild-type DHDPS. Following immobilisation, substrate affinity was found to decrease for wild-type and mutant enzymes, wild-type KmPyr = 0.26 mM free, 0.8-1.2 mM immobilised, Km(S)-ASA = 0.10 mM free, 1.5-2.5 mM immobilised. Lysine inhibition was determined to be largely unaffected by immobilisation. The largest change in K, was an increase to double that of the free enzyme. Restoration of some catalytic activity was found following the immobilisation of dimeric DHDPS-Ll97Y, the immobilised enzyme was 31 ± 12% more active than free DHDPS-Ll97Y. DHDPS-C20S/L167C was also found to immobilise as a dimer. Comparison ofthe immobilised DHDPS-C20S/L167C dimer with a derivatised free dimeric form ofthis enzyme indicated that an increase from 3% to 9% of wild-type activity had resulted from immobilisation. These results supported the hypothesis that buttressing of a dimeric mutant of DHDPS against a support surface would increase catalytic activity and that buttressing across the dimerdimer interface is essential for optimal catalytic activity in DHDPS enzymes.

Nanoparticles (NPs) are the basis of nanotechnology and finding numerous applications in various fields such as health, electronics, environment, personal care products, transportation, and catalysis. To fulfill these functions, the nanoparticles must be synthesized, passivated to control their chemical reactivity, stabilized against aggregation and functionalized to achieve specific performances. The chemistry of metal nanoparticles especially that of noble metals (Gold, Platinum…) is a growing field. The nanoparticles have indeed different properties from those of the corresponding bulk material. These properties are largely influenced by several parameters; the most important are the size, shape, and the local environment of the nanoparticles. One of the most common synthetic methods for the preparation of gold nanoparticles (AuNPs) is based on stabilization by citrate. Since it was reported first by Turkevich et al. in 1951, this synthetic scheme has been widely used, studied and a substantial amount of important information regarding this system has been reported in the literature. The most popular method developed by Frens for controlling the size of the noble gold nanoparticles based on citrate was achieved by varying the concentration of sodium citrate. Despite a large number of investigations focused on utilizing Cit-AuNPs, the structural details of citrate anions adsorbed on the AuNP surface are still unknown. It is known only that citrate anions “coordinate” to the metal surface by inner sphere complexation of the carboxylate groups and there are trace amounts of AuCl4−, Cl−, and OH− on the metal surface. Moreover, it is generally accepted that the ligand shell morphology of Au nanoparticles can be partly responsible for important properties such as oxidation of carbon monoxide. The use of Au-NPs in heterogeneous catalysis started mostly with Haruta who discovered the effect of particle size on the activity for carbon monoxide oxidation at low temperature. The structure of the citrate layer on the AuNP surface may be a key factor in gaining a more detailed understanding of nanoparticle formation and stabilization. This can be affecting the catalytic activity. These thoughts invited us to systematically examine the role of sodium citrate as a stabilizer of gold nanoparticles, which is the main theme of this thesis. This research is focused on three main objectives, controlling the size of the gold nanoparticles based on citrate (and other carboxylate ligands Trisodium citrate dihydrate, Isocitric Acid, Citric acid, Trimesic acid, Succinic Acid, Phthalic acid, Disodium glutarate, Tartaric Acid, Sodium acetate, Acetic Acid and Formic Acid by varying the concentration of Gold/sodium citrate, investigating the interaction of the citrate layer on the AuNP surface, and testing the activity of the Au/TiO2 catalysts for the oxidation of carbon monoxide. This thesis will be divided into five chapters. In Chapter 1, a general literature study on the various applications and methods of synthesis of Au nanoparticles is described. Then we present the main synthetic pathways of Au nanoparticles we selected. A part of the bibliographic study was given to the use of Au nanoparticles in catalysis. In Chapter 2, we give a brief description of the different experimental procedures and characterization techniques utilized over the course of the present work. The study of the size control and the interaction between gold nanoparticles and the stabilizer (carboxylate groups) was achieved by using various characterization techniques such as UV-visible spectroscopy, Transmission Electron Microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Nuclear Magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FTIR). In Chapter 3, we discuss the synthesis and size control of Au nanoparticles by following the growth of these nanoparticles by UV-Visible spectroscopy and TEM. We then describe the effect of the concentrations and of various type of the stabilizer, and the post-synthesis treatment on gold nanoparticles size. In Chapter 4, we focus on determining the nature of the interactions at molecular level between citrate (and other carboxylate-containing ligands) and AuNP in terms of the mode of coordination at the surface, and the formal oxidation state of Au when interacting with these negatively charged carboxylate ligands (i.e., LX- in the Green formalism). We achieve this by combining very advanced 13C CP/MAS, 23Na MAS and low-temperature SSNMR, high-resolution transmission electron microscopy (HRTEM) and density functional theory (DFT) calculations. A particular emphasis will be based on SS-NMR. In Chapter 5, we study the influence of pretreatment of 1% Au/TiO2 catalysts on the resulting activity in the oxidation of carbon monoxide, the effect of the concentration and the type of the ligands on the catalytic activity. The catalysts were characterized by TPO, XRD, and TEM spectroscopy.

PrnB is a heme-containing enzyme, which catalyzes the ring rearrangement reaction of 7-chlorotryptophan to produce 3-(3-Chloro-2-nitrophenyl)pyrrole. This thesis describes the initial isolation and characterization of PrnB, the second enzyme associated with the pyrrolnitrin biosynthetic pathway in Burkholderia ambifaria. Additionally, alternative peroxidase reactivity was used to study how amino-acids bind to the substrate binding pocket of PrnB. The peroxidase activity of PrnB was measured using three different peroxidase activity assays at various pH values. The peroxidase data was compared to similar studies with the classic peroxidase, Horseradish peroxidase (HRP). Generally, PrnB showed weak peroxidase reactivity. However this weak reactivity was an experimental handhold, where tryptophan and other substrate binding events can be explored using classic inhibition steady-state kinetics. The rate of 2-aminophenol oxidation by PrnB was used as a model assay to monitor how molecules such as L-tryptophan, L-alanine, indole, L-phenylalanine, and L-tyrosine interact with the PrnB active site.

Vanadium and molybdenum oxide catalysts have been prepared on different Ti02 supports by a variety of methods. Solutions of VOC13, VO(O'Bu)3 and MoOC14 were used to graft VOX and MoOX monolayers onto the supports in a single treatment. The other methods were intended to produce more than one monolayer (i. e. aqueous impregnation and multiple treatments of VOC13 and VO(O1Bu)3 ). TPR and Raman spectroscopy showed the formation above the monolayer of a phase denoted as disordered vanadium oxide, which has the same reducibility as the monolayer species but which has a band in the Raman spectrum at 995 cm-l. Raman spectroscopy also showed the formation of a disordered molybdenum oxide phase. With supports which contained phosphorus and potassium as impurities, TPR and Raman spectoscopy indicated a potassium-containing vanadium oxide, which was difficult to reduce and which showed no band at 995 cm-1. "Paracrystalline" V205 and MoO3 are formed when the oxide content exceeds four monolayers. XPS measurements confirm the dispersion of MOX species (M = V, Mo) on the surface of the support in the monolayer region; they also show that disordered and paracrystalline oxide phases occupy a limited area of the monolayer surface, but could not distinguish between them. ESR results showed 95% of the supported vanadium in the oxidation state +5. Phosphorus and potassium impurities in (or on) the TiO2 support influence the structure and catalytic properties of the VOX monolayer phase. In the case of catalysts made on supports with low impurities, activities in butadiene oxidation and isopropanol decomposition are principally due to the monolayer species and little contribution is made by the disordered or paracrystalli ne V205, while in the catalysts made on the supports with relatively high level of impurities, the activities in both reactions increase with V205 content in the region of one to four monolayers. MoOX catalysts showed low activities and selectivities in butadiene oxidation.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 5 uppsatser.