Biochemistry I. Unit 3

Unit 3. Enzymes. Catalysis and enzyme kinetics

Studies in organobismuth chemistry

Perturbations in the bismuth market resulted in Mining and Chemical Products Ltd., seeking further outlets in the market. Together with Manchem Ltd. they were anxious to evaluate the possibility of using bismuth compounds as a replacement for lead/calcium soaps in paint driers. A range of new organobismuth compounds were synthesised of the type RBiX2 and R3BiX2 (X= halogen, OOCR, dithiocarbamate). A variety of synthetic techniques were explored, including the use of mathematical reactions, phase-transfer catalysis and microwave energy. The preparation of a range of trivalent and pentavalent organobismuth carboxylates is reported and their infra-red , 13C, lH nmr spectra. The compounds were evaluated as paint driers and in cases found to enhance paint drying to a greater degree than the standard driers, to which they were being compared. The drying times of paint films containing the organobismuth compounds are reported, together with a comparison of the drying times with the addition of bismuth tris-diethyldithiocarbamate, which may promote the cross-linking reaction that occur in paint films during the drying process. Examples are reported to illustrate the great reductions in reaction times possible when using microwave energy. Reactions such as metallation of aromatic rings, ligand redistribution and synthesis were carried out in PTFE containers in a conventional domestic microwave oven. An X-ray diffraction study of (phenylazophenyl-C,N')mercury(II) chloride has shown it to be dimeric via long Hg-Cl bridging interactions of 3.367A. Its crystal structure is reported, together with its 13C nmr spectra and mass spectrum. The Lewis acidity of compounds of the type RBiX2 was investigated. The donor group being anchored to the organo group (R). The dithiocarbamates bis- (diethyldithiobarbamato)phenylbismuth(Ill) and [2-2-pyridyl)phenylbismuth(III) were synthesised, and their crystal structures, 14N, 13C nmr ar1d infra-red spectra are reported. Both compounds are pseudo-pentagonal bipyramidal in geometry, with two long Bi-S bonds and two short Bi-S bonds. The reaction of RBiBr2 (R= 2-(pyridyl) with various ligands is reported. The infra-red evidence suggesting that the coordination of extra ligands is accompanied by a reduction of the strength of the Bi-interaction.

The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.

Elaboração de pós de ferrita de cobalto, CoFe2O4, nanoestruturados e hierarquizados: influência da morfologia sobre a detecção e oxidação catalítica de gases poluentes

This thesis is part of research on new materials for catalysis and gas sensors more active, sensitive, selective. The aim of this thesis was to develop and characterize cobalt ferrite in different morphologies, in order to study their influence on the electrical response and the catalytic activity, and to hierarchize these grains for greater diffusivity of gas in the material. The powders were produced via hydrothermal and solvothermal, and were characterized by thermogravimetric analysis, X-ray diffraction, scanning electron microscopy, transmission electron microscopy (electron diffraction, highresolution simulations), and energy dispersive spectroscopy. The catalytic and electrical properties were tested in the presence of CO and NO2 gases, the latter in different concentrations (1-100 ppm) and at different temperatures (room temperature to 350 ° C). Nanooctahedra with an average size of 20 nm were obtained by hydrothermal route. It has been determined that the shape of the grains is mainly linked to the nature of the precipitating agent and the presence of OH ions in the reaction medium. By solvothermal method CoFe2O4 spherical powders were prepared with grain size of 8 and 20 nm. CoFe2O4 powders exhibit a strong response to small amounts of NO2 (10 ppm to 200 ° C). The nanooctahedra have greater sensitivity than the spherical grains of the same size, and have smaller response time and shorter recovery times. These results were confirmed by modeling the kinetics of response and recovery of the sensor. Initial tests of catalytic activity in the oxidation of CO between temperatures of 100 °C and 350 °C show that the size effect is predominant in relation the effect of the form with respect to the conversion of the reaction. The morphology of the grains influence the rate of reaction. A higher reaction rate is obtained in the presence of nanooctahedra. In order to improve the detection and catalytic properties of the material, we have developed a methodology for hierarchizing grains which involves the use of carbonbased templates.

Características estruturais e fotocatalíticas do compósito oxihidróxido de nióbio/vermiculita

The preparation of nanostructured materials using natural clays as support, has been studied in literature under the same are found in nature and consequently, have a low price. Generally, clays serve as supports for metal oxides by increasing the number of active sites present on the surface and can be applied for various purposes such as adsorption, catalysis and photocatalysis. Some of the materials that are currently highlighted are niobium compounds, in particular, its oxides, by its characteristics such as high acidity, rigidity, water insolubility, oxidative and photocatalytic properties. In this scenario, the study aimed preparing a composite material oxyhydroxide niobium (NbO2OH) / sodium vermiculite clay and evaluate its effectiveness with respect to the natural clay (V0) and NbO2OH. The composite was prepared by precipitation-deposition method and then characterized by X-ray diffraction, infrared spectroscopy (XRD), energy dispersive X-ray (EDS), thermal analysis (TG/DTG), scanning electron microscopy (SEM), N2 adsorption-desorption and investigation of distribution of load. The application of the material NbO2OH/V0 was divided in two steps: first through oxidation and adsorption methods, and second through photocatalytic activity using solar irradiation. Studies of adsorption, oxidation and photocatalytic oxidation monitored the percentage of color removal from the dye methylene blue (MB) by UV-Vis spectroscopy. The XRD showed a decrease in reflection d (001) clay after modification; the FTIR indicated the presence of both the clay when the oxyhydroxide niobium to present bands in 1003 cm-1 related to Si-O stretching bands and 800 cm-1 to the Nb-O stretching. The presence of niobium was also confirmed by EDS indicated that 17 % by mass amount of the metal. Thermal analysis showed thermal stability of the composite at 217 °C and micrographs showed that there was a decrease in particle size. The investigation of the surface charge of NbO2OH/V0 found that the material exhibits a heterogeneous surface with average low and high negative charges. Adsorption tests showed that the composite NbO2OH/V0 higher adsorption capacity to remove 56 % of AM, while the material removed from V0 only 13 % showed no NbO2OH and adsorptive capacity due to the formation of H-aggregates. The percent removal of dye color for the oxidation tests showed little difference from the adsorption, being 18 and 66 % removal of dye color for V0 and NbO2OH/V0 respectively. The NbO2OH/V0 material shows excellent photocatalytic activity managing to remove just 95,5 % in 180 minutes of the color of MB compared to 41,4 % and 82,2 % of V0 the NbO2OH, proving the formation of a new composite with distinct properties of its precursors.

On Provable Algorithms for Determination of Continuous Protein Interdomain Motions from Residual Dipolar Couplings

Dynamics of biomolecules over various spatial and time scales are essential for biological functions such as molecular recognition, catalysis and signaling. However, reconstruction of biomolecular dynamics from experimental observables requires the determination of a conformational probability distribution. Unfortunately, these distributions cannot be fully constrained by the limited information from experiments, making the problem an ill-posed one in the terminology of Hadamard. The ill-posed nature of the problem comes from the fact that it has no unique solution. Multiple or even an infinite number of solutions may exist. To avoid the ill-posed nature, the problem needs to be regularized by making assumptions, which inevitably introduce biases into the result.

Here, I present two continuous probability density function approaches to solve an important inverse problem called the RDC trigonometric moment problem. By focusing on interdomain orientations we reduced the problem to determination of a distribution on the 3D rotational space from residual dipolar couplings (RDCs). We derived an analytical equation that relates alignment tensors of adjacent domains, which serves as the foundation of the two methods. In the first approach, the ill-posed nature of the problem was avoided by introducing a continuous distribution model, which enjoys a smoothness assumption. To find the optimal solution for the distribution, we also designed an efficient branch-and-bound algorithm that exploits the mathematical structure of the analytical solutions. The algorithm is guaranteed to find the distribution that best satisfies the analytical relationship. We observed good performance of the method when tested under various levels of experimental noise and when applied to two protein systems. The second approach avoids the use of any model by employing maximum entropy principles. This 'model-free' approach delivers the least biased result which presents our state of knowledge. In this approach, the solution is an exponential function of Lagrange multipliers. To determine the multipliers, a convex objective function is constructed. Consequently, the maximum entropy solution can be found easily by gradient descent methods. Both algorithms can be applied to biomolecular RDC data in general, including data from RNA and DNA molecules.

New biomass derived carbon catalysts for biomass valorization

Due to diminishing petroleum reserves, unsteady market situation and the environmental concerns associated with utilization of fossil resources, the utilization of renewables for production of energy and chemicals (biorefining) has gained considerable attention. Biomass is the only sustainable source of organic compounds that has been proposed as petroleum equivalent for the production of fuels, chemicals and materials. In fact, it would not be wrong to say that the only viable answer to sustainably convene our future energy and material requirements remain with a bio-based economy with biomass based industries and products. This has prompted biomass valorization (biorefining) to become an important area of industrial research. While many disciplines of science are involved in the realization of this effort, catalysis and knowledge of chemical technology are considered to be particularly important to eventually render this dream to come true. Traditionally, the catalyst research for biomass conversion has been focused primarily on commercially available catalysts like zeolites, silica and various metals (Pt, Pd, Au, Ni) supported on zeolites, silica etc. Nevertheless, the main drawbacks of these catalysts are coupled with high material cost, low activity, limited reusability etc. – all facts that render them less attractive in industrial scale applications (poor activity for the price). Thus, there is a particular need to develop active, robust and cost efficient catalytic systems capable of converting complex biomass molecules. Saccharification, esterification, transesterification and acetylation are important chemical processes in the valorization chain of biomasses (and several biomass components) for production of platform chemicals, transportation fuels, food additives and materials. In the current work, various novel acidic carbons were synthesized from wastes generated from biodiesel and allied industries, and employed as catalysts in the aforementioned reactions. The structure and surface properties of the novel materials were investigated by XRD, XPS, elemental analysis, SEM, TEM, TPD and N2-physisorption techniques. The agro-industrial waste derived sulfonic acid functionalized novel carbons exhibit excellent catalytic activity in the aforementioned reactions and easily outperformed liquid H2SO4 and conventional solid acids (zeolites, ion-exchange resins etc). The experimental results indicated strong influence of catalyst pore-structure (pore size, pore-volume), concentration of –SO3H groups and surface properties in terms of the activity and selectivity of these catalysts. Here, a large pore catalyst with high –SO3H density exhibited the highest esterification and transesterification activity, and was successfully employed in biodiesel production from fatty acids and low grade acidic oils. Also, a catalyst decay model was proposed upon biodiesel production and could explain that the catalyst loses its activity mainly due to active site blocking by adsorption of impurities and by-products. The large pore sulfonated catalyst also exhibited good catalytic performance in the selective synthesis of triacetin via acetylation of glycerol with acetic anhydride and out-performed the best zeolite H-Y with respect to reusability. It also demonstrated equally good activity in acetylation of cellulose to soluble cellulose acetates, with the possibility to control cellulose acetate yield and quality (degree of substitution, DS) by a simple adjustment of reaction time and acetic anhydride concentration. In contrast, the small pore and highly functionalized catalysts obtained by hydrothermal method and from protein rich waste (Jatropha de-oiled waste cake, DOWC), were active and selective in the esterification of glycerol with fatty acids to monoglycerides and saccharification of cellulosic materials, respectively. The operational stability and reusability of the catalyst was found to depend on the stability of –SO3H function (leaching) as well as active site blocking due to adsorption of impurities during the reaction. Thus, our results corroborate the potential of DOWC derived sulfated mesoporous active carbons as efficient integrated solid acid catalysts for valorization of biomass to platform chemicals, biofuel, bio-additive, surfactants and celluloseesters.

Conversão da lactose e síntese de galacto-oligossacarídeos: uma abordagem experimental e teórica

O presente trabalho avaliou, na etapa experimental, um processo simultâneo de catálise e fermentação láctica visando obter um iogurte com potenciais características nutracêuticas e, na sua etapa teórica, estabeleceu uma interlocução entre a vivência experimentalista e a teoria da cinética enzimática, no que se refere à conversão da lactose e à síntese de galactooligossacarídeos (GOS). Na abordagem experimental, para um substrato específico, avaliouse biocatálise conduzida simultaneamente à fermentação, defasando a adição da enzima em relação ao início do processo fermentativo. A fermentação foi realizada a partir de cultura láctica liofilizada comercial contendo dois micro-organismos probióticos, Bifidobacterium animalis e Lactobacillus acidophilus, associados aos micro-organismos característicos do iogurte, Lactobacillus bulgaricus e Streptococcus thermophilus. Foi utilizado um preparado enzimático contendo -galactosidases obtidas de duas origens distintas: Kluyveromyces lactis e Aspergillus niger. Foram avaliados os efeitos da concentração da enzima e do tempo de adição da enzima em um planejamento experimental 2 2 . As respostas foram às concentrações, ao final do processo, de lactose, de GOS, de glicose e de galactose e a hidrólise dos galactooligossacarídeos ao longo do tempo. No que se refere à abordagem teórica, o presente trabalho considerou modelos matemáticos de hidrólise de dissacarídeos e conversão da lactose, em que a inibição foi representada a partir do incremento da concentração dos produtos da reação. No que se refere à conversão da lactose e síntese de GOS, o presente trabalho buscou estabelecer um modelo matemático em que a inibição ocorreu por efeito do incremento das concentrações de glicose e de galactose, comparando-o com os modelos conhecidos na literatura. Verificou-se que o desempenho do modelo obtido no presente trabalho foi robusto em relação às premissas estabelecidas. Na comparação com resultados experimentais de conversão enzimática, o modelo mostrou-se capaz de minimizar o erro e de ajustar-se aos dados experimentais.

Recovery of gold(0) nanoparticles from aqueous solutions using effluents from a bioremediation process

The use of biological processes with the aim of the recovery of gold from low-concentration solutions derived from leaching of secondary sources is gaining increasing importance owing to the scarcity of the primary resources and the economic and environmental advantages usually presented by these methods. Thus, the addition in batch and continuous processes of different solutions containing biogenic sulphide, which was generated by the activity of sulphate-reducing bacteria (SRB), to gold(III) solutions was investigated for that purpose. In the batch experiments, AuS nanoparticles with sizes of between 6 and 14 nm were obtained (corresponding to 100% removal of Au(III) from solution) if the biogenic sulphide was generated in a typical nutrient medium for SRB, whereas Au(0) nanoparticles with sizes of below 8 nm were obtained (corresponding to 62% removal of Au(III)) if effluent from a SRB bioremediation process for treating acid mine drainage (AMD) was used instead. These results stimulated the development of a continuous process of addition, in which two sulphide-rich effluents, which resulted from a SRB bioremediation process for treating two types of AMD (from a uranium mine and a polysulphide mine), were tested. In both cases, Au(0) nanoparticles with sizes of between 6 and 15 nm were mainly obtained, and the percentage removal of Au(III) from solution ranged from 76% to 100%. The processes described allow the simultaneous treatment of AMD and recovery of metallic gold nanoparticles, which are a product with a wide range of applications (e.g., in medicine, optical devices and catalysis) and high economic value. The synthesis process described in this work can be considered as novel, because it is the first time, to our knowledge, that the use of effluent from a SRB bioremediation process has been reported for the recovery of gold(III) as gold(0) nanoparticles.

Catalysts study of adipic acid synthesis from 1,6-hexanediol

Sustainable chemicals currently have a very limited market share due to current low production but biomass is expected to become one of the major renewable energy and fine chemicals sources in the coming years. Bearing in mind the compromise of all nations to climatic change remediation, the industries will need to use efficient catalysts and green processes to meet the requirements of emissions and efficiency. This project is expected to develop new catalysts to convert 1,6-hexanediol to adipic acid through a green approach based on the “nano-catalysis” and “green chemistry” concepts. Supported Au and Pd nanoparticles were used to study one-pot reaction of HDO oxidation to AA using O2 as a final oxidant and H2O as a solvent. Catalytic results showed that under low pressure O2 atmosphere and low temperature (< 120°C) AuNPs supported on basic-supports are more active than acid and amphoteric oxides. The effect of basic oxide (MgO) addition to MgF2 was studied. The study showed that doping of MgF2 with MgO increased significantly the activity of the catalyst. The best results were obtained with the Au/0.4MgF2-0.6MgO sample, which gave the selectivity to AA of 33% at HDO conversion of 62%.

Electrocatalysis of ethanol oxidation on Pt monolayers deposited on carbon-supported Ru and Rh nanoparticles

Pt monolayers deposited on carbon- supported Ru and Rh nanoparticles were investigated as electrocatalysts for ethanol oxidation. Electronic features of the Pt monolayers were studied by in situ XANES (X-ray absorption near-edge structure). The electrochemical activity was investigated by cyclic voltammetry and cronoamperometric experiments. Spectroscopic and electrochemical results were compared to those obtained on carbon-supported Pt-Ru and Pt-Rh alloys, and Pt E-TEK. XAS results indicate a modification of the Pt 5d band due to geometric and electronic interactions with the Ru ant Rh substrates, but the effect of withdrawing electrons from Pt is less pronounced in relation to that for the corresponding alloys. Electrochemical stripping of adsorbed CO, which is one of the intermediates, and the currents for the oxidation of ethanol show faster kinetics on the Pt monolayer deposited on Ru nanoparticles, and an activity that exceeds that of conventional catalysts with much larger amounts of platinum. (c) 2007 Elsevier B.V. All rights reserved.

Further evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon isomerization of the ternary complex between enzyme and products

Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products. (C) 2003 Elsevier Science B.V. All rights reserved.

Electrocatalysis and electrocatalysts for low temperature fuel cells: fundamentals, state of the art, research and development

This article deals with electrocatalysis and electrocatalysts for low temperature fuel cells and also with established means and methods in electrocatalyst research, development and characterization. The intention is to inform about the fundamentals, state of the art, research and development of noble metal electrocatalysts for fuel cells operating at low temperatures.