Using sunlight to drive chemical reactions has long been one of the goals in developing sustainable processes. Previous research has focused on solar fuel production in the form of H₂, but this thesis demonstrates that solar-to-chemicals processes can be constructed to produce more complex compounds, using hybrid systems composed of enzymes and inorganic materials. Tetrachloroethene reductive dehalogenase (PceA), an enzyme that catalyzes the conversion of tetrachloroethene (PCE) to trichloroethene (TCE) and subsequently to cis-dichloroethene (cDCE), was shown to accept electrons from both graphite and TiO₂ electrodes. Irradiation by UV light onto PceA-adsorbed TiO₂ particles led to the selective production of TCE and cDCE, which was not possible without PceA as a catalyst. Ferredoxin-NADP⁺ reductase (FNR) is a key enzyme in photosynthesis, as it receives energetic electrons from Photosystem I and produces NADPH as an energy carrier for downstream 'Dark' reactions involving CO₂ assimilation. This thesis presents the discovery of FNR activity on indium tin oxide (ITO) electrodes which led to direct electrochemical investigation of the properties of FNR, both in the absence and presence of its substrate, NADP⁺. The FNR-adsorbed electrode, termed 'the electrochemical leaf', rapidly interconverts NADP⁺/NADPH, and this was coupled to a downstream NADPH-dependent enzyme, thus demonstrating a new approach to cofactor regeneration for enzyme-catalyzed organic synthesis. The NADP⁺ reduction by FNR was also driven by light using a photoanode made of visible-light responsive semiconductors.

Magister Scientiae - MSc / Human angiotensin-converting enzyme (ACE) is a key enzyme in the regulation of blood pressure via the renin-angiotensin and kallikrein-kinin systems. A number of orally active drugs have been developed over the years that target somatic ACE, for the treatment of hypertension, myocardial infarction and congestive heart failure. Protein structural information about ACE is an important key for the understanding of the mechanism and substrate-specificity of the enzyme. However, this information has only begun to be elucidated in the past year, with the solution of crystal structures of human testis ACE (tACE), and homologues Drosophila AnCE and human ACE2. tACE is identical to the C-terminal domain of somatic ACE, which consists of two homologous domains, each having a slightly different substrate-specificity. This thesis describes the purification, crystallisation and X-ray crystal structure-determination of a glycosylation-deficient mutant of tACE, tACEG1,3, to 2.9 Å. The structure of tACE-G1,3 aligns closely with that of native tACE, indicating that the mutations did not alter the conformation. The ability to achieve minimal glycosylation of tACE for crystallisation purposes via mutation, rather than using expensive glycosidase inhibitors, iii should prove advantageous for further structural studies, such as the study of the binding of novel inhibitors. In all of the tACE structures thus far observed, the active site is closed off from the external medium in a deep cleft, so that it is unclear how a large substrate molecule could gain access. However, a hinge motion that opens this cleft has been observed in the structures of ACE2. Temperature factor and sequence comparison between tACE, tACE-G1,3, AnCE and ACE2 suggests the functional conservation of three flexible loop regions, as well as the sequence conservation of three constrained regions, involved in the hinge. Normal mode analysis reveals the intrinsic flexibility of tACE, and further suggests that a putative open form of tACE would behave similarly to the open form of ACE2. Based on these indications, a conservation of the ACE2 hinge-bending mechanism is proposed. Temperature factor analysis also reveals that subdomain II, containing bound chloride ions, is more structurally rigid than subdomain I, in all structures considered. Based on these results, lines of investigation are suggested that should yield insight into the mechanisms of action of ACE and its association with various substrates and inhibitors, ideally aiding in the development of novel drugs for the treatment of cardiac disease. / South Africa

The aim of this work was to investigate the problems involved in production and separation of a yeast microsomal enzyme, cytochrome P450. Immobilisation of the microsomal preparation, and utilisation of the immobilised system for the removal of polycyclic aromatic hydrocarbons, particularly the carcinogen benzo(a)pyrene [B(a)P], from aqueous systems was also investigated. High recoveries of the microsomal enzyme were obtained using rapid, low-speed centrifugation (5 minutes, 3000xg) by prior precipitation with a non-ionic polymer, polyethylene glycol (PEG). PEG was found to effectively replace centrifugal acceleration. A semi-empirical mathematical model of the process based on the size distribution of the agglomerates formed and the proportion of the agglomerates sedimented by centrfugation was developed. The effect of PEG was consistent with its increasing the mean effective diameter of protein agglomerates in proportion to PEG concentration to an exponent of 0.619 and with its increasing the spread of the size distribution in proportion to the mean effective agglomerate diameter. Binding spectra studies established that B(a)P binds to the active site of yeast cytochrome P450 and is unaffected by PEG. The B(a)P assay by fluorescence following hexane extraction was also unaffected by PEG. The stability of the microsomal cytochrome P450 preparation at different temperatures was investigated. The enzyme half-life was 66 minutes at 37° and was also unaffected by PEG. The enzyme preparation was satisfactorily immoblilsed by encapsulation in calcium alginate gel and enzyme distribution profiles were determined in sections of alginate stained with coomasie blue by a novel technique using a scanning optical densitometer, Diffusivity coefficients of B(a)P and NADP in alginate gel beads were determined as 7.5 and 2.5 x 10[-10] m[2]/S, respectively by the Tanaka method, fitting solute depletion profiles to Crank's theoretical model. The microsomal enzyme preparation immobilised in alginate beads was used to remove B(a)P from aqueous solution. B(a)P removal was shown to be by a non-specific affinity absorption mechanism and the removal profile was found to correspond well to a theoretical model of a diffusion-reaction system, for an affinity ligand system. The activaiton energy for deactivation of microsomal P450 was measured as 33.56kJ/mole.

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Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-30Bitstream added on 2014-06-13T20:10:33Z : No. of bitstreams: 1 santos_lfj_me_jabo.pdf: 849764 bytes, checksum: f44b7996a2cb16332037d11523efd658 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Todos os seres vivos necessitam de um suprimento energético, que deve ser obtido de fontes externas. As substâncias simples da dieta dos animas, como a água, os sais minerais e as vitaminas (exceto a vitamina B12), podem ser absorvidas ao longo do trato digestório sem sofrer transformações físicas ou químicas. Entretanto, macromoléculas tais como carboidratos, proteínas e lipídeos, têm de ser convertidas em moléculas pequenas e menos complexas para serem absorvidas. A digestão química dos nutrientes é realizada por hidrólise enzimática, que consiste na cisão de uma ligação química e inserção de uma molécula de água, sendo este processo catalisado pela ação das enzimas digestivas. O objetivo deste estudo foi o de se analisar a variação nas atividades de cinco hidrolases do sistema digestório de rã-touro (Lithobates catesbeianus) durante o desenvolvimento larval, para fornecer subsídios a nutrição animal. Os girinos foram mantidos em aquários, à 27ºC e separados por estádios de desenvolvimento. Amostras de estômago, pâncreas e intestino foram retiradas de 100 animais de cada estádio, congeladas em nitrogênio liquido e armazenadas a -70ºC para posterior homogeneização e determinação da atividade enzimática. A atividade das enzimas tripsina, -amilase e lipase pancreáticas (44,30 U.mg-1, 3,17 U.mg-1, 2,99 U.mg-1 respectivamente), e da maltase intestinal (26,99 U.mg-1), foram detectadas desde o início da fase de alimentação (estádio 26) enquanto que a atividade de catepsina-E gástrica (13,82 U.mg-1), foi estudada a partir do estádio 28, devido a dificuldade de se obter de estômagos antes deste estádio. Durante a pré-metamorfose a catepsina-E não apresentou grande variação em sua atividade, sendo observado aumento ao final do período da prómetamorfose. A atividade das demais... / All living beings require energy supply, wich must be obtained from external sources. Simple substances in the diet of the animals, such as water, minerals and vitamins (except vitamin B12) can be absorbed along the digestive tract without undergoing chemical or physical changes. However, macromolecules such as carbohydrates, proteins and lipids, must be converted into smaller and less complex molecules to be absorbed. The chemical digestion of nutrients is accomplished through enzymatic hydrolysis, which means to break a chemical bond and inserti a water molecule. This process is catalysed by the action of digestive enzymes. The aim of this study was to analyze the activity variation of five hydrolases of the bullfrog’s (Lithobates catesbeianus) digestive system during larval development, to provide subsidies to animal nutrition. The tadpoles were kept in aquaria, at 27 °C and separated by stages of development. Samples of stomach, pancreas and intestine were taken from 100 animals of each stage, frozen in liquid nitrogen and stored at -70ºC for later homogenization and enzyme activity determination. The activity of trypsin, -amylase and pancreatic lipase (44.30 U.mg-1, 3.17 U.mg-1, 2.99 U.mg-1 respectively), and intestinal maltase (26.99 U.mg-1) were detected since the start of feeding (stage 26) while the gastric cathepsin E (13.82 U.mg-1) activity was studied since stage 28, due to difficulty in obtaining stomachs before this stage. During pre-metamorphosis cathepsin-E did not show great variation in its activity, but an increase was observed at the end of the period of prometamorphosis. The other enzymes activity increased significantly in periods that precede metamorphosis, with the highest activities in stage 38 (261.97 U.mg-1 for trypsin, 28.19 U.mg-1 for -amylase and... (Complete abstract click electronic access below)

Orientador: Maria Isabel Rodrigues / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-06T16:00:21Z (GMT). No. of bitstreams: 1 Mendes_GuilhermeLopes_M.pdf: 1683620 bytes, checksum: 61fa8e0621ddee5d8253c384bb74286f (MD5) Previous issue date: 2006 / Mestrado / Mestre em Engenharia de Alimentos

Glycogen phosphorylase (GPb) regulates the degradation of glycogen to glucose-1-phosphate and catalyses the first step of the reaction. Many studies have provided insights into the essentials of the catalytic mechanism. Previous time resolved crystallographic work using heptenitol has revealed a putative phosphate binding site at the active site of phosphorylase. Using nojirimycin tetrazole, a transition state analogue, complexed with phosphate and both T and R state GPb crystals, this work has conclusively located the phosphate binding site and the concomitant active site conformational changes. This has confirmed the previous heptenitol results. Using R state crystals complexed with tetrazole and phosphate, data were collected to 2.5Å resolution, higher than for the original 2.8Å resolution native structure, and used to position water molecules in the R state model. Further to this, direct observation of the phosphate ion orientation was made possible using flash-frozen T state crystals to collect 1.7Å resolution data at 100K. As part of this cryocrystallographic work the relationship of cryoprotectant concentration with crystal mosaicity was established, aiding the systematic search for flash-freezing cryoprotectant conditions for all protein crystals. Collection of a new native T state data set to 1.5Å resolution was made possible using the flash-freezing technique. Refinement has produced a new higher precision native model (R factor currently 22.8%) containing additional N terminal residues (14-18) and 330 new water molecules. A molecule of glycerol, the cryoprotectant, was located at the active site. This study represents a considerable improvement over the 1.9Å resolution room temperature native data, and is also the first time such high resolution data have been collected from such a large enzyme. In a further analysis of the phosphate binding properties, a link between structure, atomic charges and the ability of a ligand to bind phosphate at the phosphorylase active site was established. In order of phosphate binding ability, the nojirimycin tetrazole, nitroglucal, glucal and glucose complexes with T state GPb and phosphate were structurally analysed. As the charge difference between the pyranose ring oxygen (or equivalent atom) and anomeric atom becomes more negative (charges estimated using MOPAC) the tendency to bind phosphate decreases. The ligand must possess a half-chair conformation as this is essential to bind phosphate; glucose, having the most negative charge difference and a full chair conformation, does not bind phosphate significantly in the crystal. A novel surface binding site for nitroglucal (covalently linked to His 73) was also located during this work. As part of an ongoing search for potential drugs for diabetes, two gluco-hydantoin inhibitors of GPb were investigated. One proved to be the best inhibitor to date, and the inhibition was rationalised using the structural results from this work. A new improved inhibitor has been proposed on the basis of these results.

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