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Analysis of substrate specificity and mechanism of GlpG, an intramembrane protease of the rhomboid family.
Peclinovská, Lucie ; Stříšovský, Kvido (advisor) ; Konvalinka, Jan (referee)
Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...
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Comparison of contactless conductivity detection and direct UV detection sensitivity for electrophoretic determination of N-acetylaminosaccharides
Malý, Michal ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
is work deals with development and optimization of a method for separation and detection of N- acetylglucosamine and its dimer and trimer using capillary zone electrophoresis with contactless conductiv- ity detection. Separation and detection of these substances is possible if sodium hydroxide is used as back- ground electrolyte. Usage of another electrolyte such as disodium phosphate leads to unacceptable baseline noise level. Optimal conditions of this method are a background electrolyte concentration of 40 mmol dm−3 and voltage of 15 kV. e method is very sensitive to background electrolyte pH, used background electrolyte also causes rapid degradation of the inner capillary wall which is why this method has proven difficult to cali- brate properly. e developed method was compared to an already existing method which uses UV detection. e existing method has lower limits of detection and quantification and is less sensitive to working condi- tions. Limits of detection and quantification of the developed method are 34 µmol dm−3 and 104 µmol dm−3 respectively. Limits of detection and quantification of the method based on UV detection are 7 µmol dm−3 and 21 µmol dm−3 respectively. Limits of quantification are that of the analyte whose detection was the least sensitive which was the dimer in both...
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Protease Inhibitors as a Research Tool: Design, Synthesis and Evaluation of HIV PR and GCPII Inhibitors
Schimer, Jiří ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Ruml, Tomáš (referee)
This dissertation thesis focuses on creating tools for the analysis and potential therapeutic intervention in the biological processes regulated by proteolysis. I focus on two important proteolytic enzymes: HIV-1 protease, which is indispensable for the polyprotein processing of the nascent virus and thus for the development of infectious viral particle, and glutamate carboxypeptidase II, a tumor marker and a neuropeptidase from the prostate and central nervous system. Rational design of inhibitors of these therapeutically relevant enzymes serves two purposes: firstly, protease inhibitors were shown to be powerful drugs (HIV protease is in fact the example of successful drug development driven by structural biology). Secondly, and in the context of this thesis perhaps more importantly, inhibitors of medicinally relevant proteases might serve as tools for the elucidation of basic biological questions concerning regulation, timing and spatiotemporal control of such key processes as virus maturation or cancer development. The experimental work described in this thesis summarizes my results in both these areas. Human Immunodeficiency Virus Protease Human immunodeficiency virus (HIV), a causative agent of AIDS, has been estimated to kill close to 40 million people during the past four decades with 1.5...
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Inhibitors of mouse serine racemase
Vorlová, Barbora ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...
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Analysis of the mechanism of action of metallacarborane inhibitors of HIV PR
Svoboda, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
English Abstract Shortly after the identification of HIV as a causative agent of AIDS, an aspartic protease was identified in the viral genetic information. The very same time protease has become one of the dominant therapeutical targets in AIDS therapy. The introduction of protease inhibitors into the antiretroviral therapy has led to a significant improvement in the quality and length of life of HIV patients. However, the virus is still able to effectively prevent the impact of an inhibitor via generating inhibitor-resistant mutated protease variants. Thus, there is a constant need for novel types of inhibitors that would be capable of effectively blocking these resistant variants and simultaneously not supporting the development of novel resistant viral strains. One way to identify such inhibitors could be searching for compounds interacting with the enzyme at different sites than the active cavity, via the mechanisms of noncompetitive or uncompetitive inhibition. The group of compounds called metallacarboranes - inorganic compounds consisting of carbon, boron, hydrogen and metall ion - were shown to exhibit such an activity against HIV-1 protease. However, for further optimization of these inhibitors, detailed biophysical investigation of the enzyme-inhibitor complex is needed. This work focuses on the...
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Mechanism of action of non-peptide inhibitors of HIV protease
Began, Jakub ; Konvalinka, Jan (advisor) ; Bořek Dohalská, Lucie (referee)
The inhibition of HIV-1 protease plays an important role in combating HIV. Nine HIV-1 protease inhibitors have been succesfully marketed for the treatment since 1995. However, their efficiencies decrease due to the resistance development. More potent compounds with novel structural motifs and mechanisms of action are therefore still needed. Several inhibitory compounds have been reported to bind to the protease at the loci different from the active site. Interestingly, darunavir, which is the last approved inhibitor with supposedly competitive mode of action, was also suggested to bind to the flap region of the protease. Two studies discussed this alternative binding mode based on the X-ray structural and kinetic analysis, respectively. Nevertheless, it is questionable, if such a mechanism is relevant also in physiological conditions or if it is only an artifact of crystallization. Another study provided a strong evidence for the alternative binding of darunavir to highly mutated HIV-1 protease. Based on thermodynamic analysis, it was shown that two molecules of darunavir bind to the protease dimer. Surprisingly, this observation was not confirmed by the X-ray structure analysis since the inhibitor was bound only within the active site. However, this protease variant was employed in further...
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Separation and determination of possible products of enzymatic cleavage of 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside using capillary electrophoresis
Velvarská, Romana ; Křížek, Tomáš (advisor) ; Kalíková, Květa (referee)
This work deals with the development and optimization of conditions of a method that can be used to compare the activity of the enzyme β-N-acetylhexosaminidase in hydrolysis of a natural substrate and a chromogenic substrate, which is often used in the study of enzyme kinetics. As a substrate, 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside was selected for cleavage. This oligosaccharide contains bond, which the enzyme cleaves in the natural substrate, and the bond that occurs in the chromogenic substrate. To determine the products arising from enzymatic hydrolysis of 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside, capillary zone electrophoresis was used. First, it was necessary to find the optimal composition of the electrolyte, its pH and concentration. The optimal background electrolyte was a solution of sodium tetraborate at a concentration of 25 mmol/l and a pH of 10.25. Subsequently, repeatability, calibration curves and linearity, limit of detection and limit of quantification were investigated. Repeatability of migration times ranged up to 0.6%, the repeatability of peak areas between 2.5 and 6.3%. Limits of detection were ranging from 0.005 to 0.120 mmol/l. Finally, the optimized method was successfully used to monitor the actual enzyme cleavage.
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Characterization of N-demethyllincomycin-methyltransferase.
Poľan, Marek ; Najmanová, Lucie (advisor) ; Petříčková, Kateřina (referee)
Lincomycin is a naturally occurring member of a lincosamide group of antibiotics. The cluster of lincomycin biosynthetic gene was already decribed and the function of many of genes has been clarified. This work, "Characterization of N-demethyllincomycin-methyltransferase", is focused on the study of the final step of lincomycin biosynthetic pathway - the methylation of nitrogen atom from the pyrollo ring of the propylproline unit of the N-demethyllicomycin (NDL). The aim of this work was the characterization of the protein LmbJ, catalysing this final biosynthetic step. All the experiments were provided for the enzyme LmbJ with N-terminal histidine tag, which had been prepared by the heterologous expression in E.coli cells. The pH and temperature optimum was determined as well as the Michaelis constants for both substrates of the reaction - N-demethyllincomycin and S-adenosyl methionine (SAM - a methyl group donor). With the exception of the pH optimum, all specified parameters have markedly differed from the data published for the enzyme isolated from the natural source. Based on the comparison of electron microscopy, blue native gel electrophoresis and gel filtration results, the hypothetical model of the LmbJ quarternary structure was created. Majority of methyltranserases, so far described occure in...
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Kinetic behavior of the NAD(P)H:Quinone oxidoreductase WrbA from Escherichia coli.
KISHKO, Iryna
This Ph.D. thesis addresses the structure-function relationship of the multimeric oxidoreductase WrbA with the principal aim being the explanation of the unusual kinetics of this enzyme in molecular terms, and thus getting an insight about its physiological role in bacteria. WrbA is a multimeric enzyme with FMN as a co-factor, catalyzing the oxidation of NADH by a two electrons transfer. Structure and function analysis of WrbA places this enzyme between bacterial flavodoxins and eukaryotic oxidoreductases in terms of its evolutionary relationship. The kinetic activity of WrbA was studied under varying conditions such as temperature, pH etc, and its kinetic mechanism was evaluated from parameters KM and Vmax and confirmed by product inhibition pattern experiments. Crystallization and proteolytic experiments also underpin the functional importance of the multimeric nature of WrbA and aid the understanding of the physiological role of this enzyme in molecular terms.
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