The Human UDP-Glucuronosyltransferases: Studies on Substrate Binding and Catalytic Mechanism

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Proton translocation coupled to electron transfer reactions in terminal oxidases

Terminal oxidases are the final proteins of the respiratory chain in eukaryotes and some bacteria. They catalyze most of the biological oxygen consumption on Earth done by aerobic organisms. During the catalytic reaction terminal oxidases reduce dioxygen to water and use the energy released in this process to maintain the electrochemical proton gradient by functioning as a redox-driven proton pump. This membrane gradient of protons is extremely important for cells as it is used for many cellular processes, such as transportation of substrates and ATP synthesis. Even though the structures of several terminal oxidases are known, they are not sufficient in themselves to explain the molecular mechanism of proton pumping. In this work we have applied a complex approach using a variety of different techniques to address the properties and the mechanism of proton translocation by the terminal oxidases. The combination of direct measurements of pH changes during catalytic turnover, time-resolved potentiometric electrometry and optical spectroscopy, made it possible to obtain valuable information about various aspects of oxidase functioning. We compared oxygen binding properties of terminal oxidases from the distinct heme-copper (CcO) and cytochrome bd families and found that cytochrome bd has a high affinity for oxygen, which is 3 orders of magnitude higher than that of CcO. Interestingly, the difference between CcO and cytochrome bd is not only in higher affinity of the latter to oxygen, but also in the way that each of these enzymes traps oxygen during catalysis. CcO traps oxygen kinetically - the molecule of bound dioxygen is rapidly reduced before it can dissociate. Alternatively, cytochrome bd employs an alternative mechanism of oxygen trapping - part of the redox energy is invested into tight oxygen binding, and the price paid for this is the lack of proton pumping. A single cycle of oxygen reduction to water is characterized by translocation of four protons across the membrane. Our results make it possible to assign the pumping steps to discrete transitions of the catalytic cycle and indicate that during in vivo turnover of the oxidase these four protons are transferred, one at a time, during the P→F, F→OH, Oh→Eh, and Eh→R transitions. At the same time, each individual proton translocation step in the catalytic cycle is not just a single reaction catalyzed by CcO, but rather a complicated sequence of interdependent electron and proton transfers. We assume that each single proton translocation cycle of CcO is assured by internal proton transfer from the conserved Glu-278 to an as yet unidentified pump site above the hemes. Delivery of a proton to the pump site serves as a driving reaction that forces the proton translocation cycle to continue.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) : Identification of novel TNFRSF1A mutations and intracellular signalling defects

The systemic autoinflammatory disorders are a group of rare diseases characterized by periodically recurring episodes of acute inflammation and a rise in serum acute phase proteins, but with no signs of autoimmunity. At present eight hereditary syndromes are categorized as autoinflammatory, although the definition has also occasionally been extended to other inflammatory disorders, such as Crohn s disease. One of the autoinflammatory disorders is the autosomally dominantly inherited tumour necrosis factor receptor-associated periodic syndrome (TRAPS), which is caused by mutations in the gene encoding the tumour necrosis factor type 1 receptor (TNFRSF1A). In patients of Nordic descent, cases of TRAPS and of three other hereditary fevers, hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), chronic infantile neurologic, cutaneous and articular syndrome (CINCA) and familial cold autoinflammatory syndrome (FCAS), have been reported, TRAPS being the most common of the four. Clinical characteristics of TRAPS are recurrent attacks of high spiking fever, associated with inflammation of serosal membranes and joints, myalgia, migratory rash and conjunctivitis or periorbital cellulitis. Systemic AA amyloidosis may occur as a sequel of the systemic inflammation. The aim of this study was to investigate the genetic background of hereditary periodically occurring fever syndromes in Finnish patients, to explore the reliability of determining serum concentrations of soluble TNFRSF1A and metalloproteinase-induced TNFRSF1A shedding as helpful tools in differential diagnostics, as well as to study intracellular NF-κB signalling in an attempt to widen the knowledge of the pathomechanisms underlying TRAPS. Genomic sequencing revealed two novel TNFRSF1A mutations, F112I and C73R, in two Finnish families. F112I was the first TNFRSF1A mutation to be reported in the third extracellular cysteine-rich domain of the gene and C73R was the third novel mutation to be reported in a Finnish family, with only one other TNFRSF1A mutation having been reported in the Nordic countries. We also presented a differential diagnostic problem in a TRAPS patient, emphasizing for the clinician the importance of differential diagnostic vigiliance in dealing with rare hereditary disorders. The underlying genetic disease of the patient both served as a misleading factor, which possibly postponed arrival at the correct diagnosis, but may also have predisposed to the pathologic condition, which led to a critical state of the patient. Using a method of flow cytometric analysis modified for the use on fresh whole blood, we studied intracellular signalling pathways in three Finnish TRAPS families with the F112I, C73R and the previously reported C88Y mutations. Evaluation of TNF-induced phosphorylation of NF-κB and p38, revealed low phosphorylation profiles in nine out of ten TRAPS patients in comparison to healthy control subjects. This study shows that TRAPS is a diagnostic possibility in patients of Nordic descent, with symptoms of periodically recurring fever and inflammation of the serosa and joints. In particular in the case of a family history of febrile episodes, the possibility of TRAPS should be considered, if an etiology of autoimmune or infectious nature is excluded. The discovery of three different mutations in a population as small as the Finnish, reinforces the notion that the extracellular domain of TNFRSF1A is prone to be mutated at the entire stretch of its cysteine-rich domains and not only at a limited number of sites, suggesting the absence of a founder effect in TRAPS. This study also demonstrates the challenges of clinical work in differentiating the symptoms of rare genetic disorders from those of other pathologic conditions and presents the possibility of an autoinflammatory disorder as being the underlying cause of severe clinical complications. Furthermore, functional studies of fresh blood leukocytes show that TRAPS is often associated with a low NF-κB and p38 phosphorylation profile, although low phosphorylation levels are not a requirement for the development of TRAPS. The aberrant signalling would suggest that the hyperinflammatory phenotype of TRAPS is the result of compensatory NF-κB-mediated regulatory mechanisms triggered by a deficiency of the innate immune response.

Lewis Acid-Lewis Base Mediated Metal-Free Hydrogen Activation and Catalytic Hydrogenation

Organocatalysis, the use of organic molecules as catalysts, is attracting increasing attention as one of the most modern and rapidly growing areas of organic chemistry, with countless research groups in both academia and the pharmaceutical industry around the world working on this subject. The literature review of this thesis mainly focuses on metal-free systems for hydrogen activation and organocatalytic reduction. Since these research topics are relatively new, the literature review also highlights the basic principles of the use of Lewis acid-Lewis base pairs, which do not react irreversibly with each other, as a trap for small molecules. The experimental section progresses from the first observation of the facile heterolytical cleavage of hydrogen gas by amines and B(C6F5)3 to highly active non-metal catalysts for both enantioselective and racemic hydrogenation of unsaturated nitrogen-containing compounds. Moreover, detailed studies of structure-reactivity relationships of these systems by X-ray, neutron diffraction, NMR methods and quantum chemical calculations were performed to gain further insight into the mechanism of hydrogen activation and hydrogenation by boron-nitrogen compounds.