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Functional characterization of transketolase-like proteins and related model systems with respect to thiamin diphosphate mediated chemistrySchneider, Stefan 18 December 2013 (has links)
No description available.
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Rational redesign of Candida antarctica lipase BMagnusson, Anders January 2005 (has links)
This thesis describes the use of rational redesign to modify the properties of the enzyme Candida antarctica lipase B. Through carefully selected single-point mutations, we were able to introduce substrate-assisted catalysis and to alter the reaction specificity. Other single-point mutations afforded variants with greatly changed substrate selectivity and enantioselectivity. Mutation of the catalytic serine changed the hydrolase activity into an aldolase activity. The mutation decreased the activation energy for aldol addition by 4 kJ×mol-1, while the activation energy increased so much for hydrolysis that no hydrolysis activity could be detected. This mutant can catalyze aldol additions that no natural aldolases can catalyze. Mutation of the threonine in the oxyanion hole proved the great importance of its hydroxyl group in the transition-state stabilization. The lost transition-state stabilization was partly replaced through substrate-assisted catalysis with substrates carrying a hydroxyl group. The poor selectivity of the wild-type lipase for ethyl 2-hydroxypropanoate (E=1.6) was greatly improved in the mutant (E=22), since only one enantiomer could perform substrate-assisted catalysis. The redesign of the size of the stereospecificity pocket was very successful. Mutation of the tryptophan at the bottom of this pocket removed steric interactions with secondary alcohols that have to position a substituent larger than an ethyl in this pocket. This mutation increased the activity 5 500 times towards 5-nonanol and 130 000 times towards (S)-1-phenylethanol. The acceptance of such large substituents (butyl and phenyl) in the redesigned stereospecificity pocket increases the utility of lipases in biocatalysis. The improved activity with (S)-1-phenylethanol strongly contributed to the 8 300 000 times change in enantioselectivity towards 1-phenylethanol; example of such a large change was not found in the literature. The S-selectivity of the mutant is unique for lipases. Its enantioselectivity increases strongly with temperature reaching a useful S-selectivity (E=44) at 69 °C. Thermodynamics analysis of the enantioselectivity showed that the mutation in the stereospecificity pocket mainly changed the entropic term, while the enthalpic term was only slightly affected. This pinpoints the importance of entropy in enzyme catalysis and entropy should not be neglected in rational redesign.
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Electrostaticanalisys the Ras active siteKhan, Abdul Kareem 05 March 2009 (has links)
La preorganització electrostàtica del centre actiu s'ha postulat com el mecanisme genèric de l'acció dels enzims. Així, alguns residus "estratègics" es disposarien per catalitzar reaccions interaccionant en una forma més forta amb l'estat de transició, baixant d'aquesta manera el valor de l'energia dactivació g cat. S'ha proposat que aquesta preorientació electrostática s'hauria de poder mostrar analitzant l'estabilitat electrostàtica de residus individuals en el centre actiu.Ras es una proteïna essencial de senyalització i actúa com un interruptor cel.lular. Les característiques estructurals de Ras en el seu estat actiu (ON) són diferents de les que té a l'estat inactiu (OFF). En aquesta tesi es duu a terme una anàlisi exhaustiva de l'estabilitat dels residus del centre actiu deRas en l'estat actiu i inactiu. / The electrostatic preorganization of the active site has been put forward as the general framework of action of enzymes. Thus, enzymes would position "strategic" residues in such a way to be prepared to catalyze reactions byinteracting in a stronger way with the transition state, in this way decreasing the activation energy g cat for the catalytic process. It has been proposed thatsuch electrostatic preorientation should be shown by analyzing the electrostatic stability of individual residues in the active site.Ras protein is an essential signaling molecule and functions as a switch in thecell. The structural features of the Ras protein in its active state (ON state) are different than those in its inactive state (OFF state). In this thesis, an exhaustive analysis of the stability of residues in the active and inactive Ras active site is performed.
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