AMINO ACIDS

Author: ROMERO ARAUZ ANA MARIA.
Year: 2003.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: UNIVERSIDAD DE EXTREMADURA.

Summary: The nucleótido-pirofosfatasas/fosfodiesterasas (NPP / EDP) are enzymes hydrolyzed derivatives fosfoanhídrido and fosfodiéster nucleoside 5'-monofosfatos (MPN), giving as a result. They ectoenzimas membranes which may also submit forms solubres. Its biological function is not known, although they attributed diveros roles physiological and related parts pathogenic extracellular derivatives nucleotídicos, generation of extracellular pyrophosphate, inhibition of autofosforilación receptor and insulin aspects of motility and invasiveness of tumor cells . There are no known physiological mechanisms of regulation of the enzyme activity of the NPP / EDP. This thesis has investigated the effect produced by amino acids on the inactivation of the NPP / EDP of rat liver by low concentrations of the metal chelator EDTA, which proved to be a time-dependent process, blocked by the sustrato-4-nitrofenil - dMTP. The inactivation followed a first order kinetics with respect to the concentration of NPP / EDP active. Measurements of the residual enzyme activity helped calculate the kinetic constant apparent inactivation (ki (ap)). The sensitivity EDTA has been used as a way to tap into the conformation of the enzyme. The effect of EDTA was boosted by alfa-aminoácidos free at concentrations that in the absence of chelator, did not affect the enzymatic activity. All amino acids tested acted as accelerators inactivation by ATS, but did so with different efficiency, estimated in the form of acceleration factors (ratio of the values of Ki (ap) obtained in the presence and absence of amino acid) or increases ( rates of increase in the value of ki (ap)). Experiments done with compounds whose structure differed from the alfa-aminoácidos in various aspects indicated that the presence of groups alfa-amino and alfa-carboxilo, and the distance between them, are essential elements for the observed effect. The alfa-aminoácidos can act on the NPP / EDP under physiological conditions, as they did (i) in the form of mixtures of amoniácidos prepradas in sy provide a total concentration similar to those that exist in the blood of fasting (ii) at physiological pH and (iii) on the NPP / EDP integrated raw preparations of liver membranes. The results are interpreted as a manifestation of a direct interaction aminoácidos-enzima that a change of conformation of the protein. Studies of correlation between the structure of the compounds tested, and its activity, indicating that such interaction is established by multiple structural elements, among which should include the union of amino acid to metal ions (possibly Zn2 +) linked to the enzyme. The change of conformation produced by the union to alfa-aminoácidos could have meaning in relation to actions of the NPP / EDP not dependean its catalytic activity but interactions proteína-proteína, as is the case known of the interaction of an NPP / EDP with the insulin receptor.

Author: ROMERO ARAUZ ANA M..
Year: 2003.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: UNIVERSIDAD DE EXTREMADURA.

Summary: The nucleótido-pirofosfatasas/fosfodiesterasas (NPP / EDP) are enzymes hydrolyzed derivatives fosfoanhídrido and fosfodiéster nucleoside 5'-monofosfatos (MPN), giving as a result. They ectoenzimas membranes which may also submit forms solubres. Its biological function is not known, although they attributed diveros roles physiological and related parts pathogenic extracellular derivatives nucleotídicos, generation of extracellular pyrophosphate, inhibition of autofosforilación receptor and insulin aspects of motility and invasiveness of tumor cells . There are no known physiological mechanisms of regulation of the enzyme activity of the NPP / EDP. This thesis has investigated the effect produced by amino acids on the inactivation of the NPP / EDP of rat liver by low concentrations of the metal chelator EDTA, which proved to be a time-dependent process, blocked by the sustrato-4-nitrofenil - dMTP. The inactivation followed a first order kinetics with respect to the concentration of NPP / EDP active. Measurements of the residual enzyme activity helped calculate the kinetic constant apparent inactivation (ki (ap)). The sensitivity EDTA has been used as a way to tap into the conformation of the enzyme. The effect of EDTA was boosted by alfa-aminoácidos free at concentrations that in the absence of chelator, did not affect the enzymatic activity. All amino acids tested acted as accelerators inactivation by ATS, but did so with different efficiency, estimated in the form of acceleration factors (ratio of the values of Ki (ap) obtained in the presence and absence of amino acid) or increases ( rates of increase in the value of ki (ap)). Experiments done with compounds whose structure differed from the alfa-aminoácidos in various aspects indicated that the presence of groups alfa-amino and alfa-carboxilo, and the distance between them, are essential elements for the observed effect. The alfa-aminoácidos can act on the NPP / EDP under physiological conditions, as they did (i) in the form of mixtures of amoniácidos prepradas in sy provide a total concentration similar to those that exist in the blood of fasting (ii) at physiological pH and (iii) on the NPP / EDP integrated raw preparations of liver membranes. The results are interpreted as a manifestation of a direct interaction aminoácidos-enzima that a change of conformation of the protein. Studies of correlation between the structure of the compounds tested, and its activity, indicating that such interaction is established by multiple structural elements, among which should include the union of amino acid to metal ions (possibly Zn2 +) linked to the enzyme. The change of conformation produced by the union to alfa-aminoácidos could have meaning in relation to actions of the NPP / EDP not dependean its catalytic activity but interactions proteína-proteína, as is the case known of the interaction of an NPP / EDP with the insulin receptor.

Author: LASA VENTURA MARTA.
Year: 2004.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: In this work we have studied analogues phenylalanine trans-c6Phe, cis-c5Phe and trans-c5Phe that restriction presented as a bridge alquilideno (four three carbons, respectively) between carbons a and b. We have developed synthetic routes of amino acids that allow obtain as pairs racémicos scale of several grams of a competitive basis. In the case of trans-c6Phe is considered a route based on the reaction of Diels-Alder and another in the Strecker, the latter becoming more favorable. For similar ciclopentánicos was seen as a scheme that included a synthetic reaction ciclación and another based on the reaction of Strecker, the latter being chosen again: this is a synthesis estereodivergente of cis-c5Phe and trans-c5Phe, with yields comparable and competitive . Syntheses racémicas of amino acids were completed with the resolutions of the same using high performance liquid chromatography on chiral stationary phases from polysaccharides. Applying this methodology in scale semipreparativa gave each of the enantiomers of pairs trans-c6Phe and c5Phe in pure form and determine its settings. This describes a new synthesis of the enantiomers of trans-c6Phe, and the first route to get the stereoisomers of c5Phe.Los different amino acids racémicos were incorporated into dipéptidos model formula R (S) -Pro-cnPhe-NHR '. The structural study was conducted by X-ray diffraction of peptides from each pair of diastereomers, so that we could establish configurations centers estereogénicos of them all. These peptides, in the case of c5Phe, was also synthesized from the amino acids enantiopuros which this is the method of allocating the configurations of enantiomers previously resueltos.Otra application was studied using restricted amino acids, including a derivative ( R, S) -c6Phe as ligands aminoacidato for the synthesis of organometallic complexes of ruthenium. Different complexes were synthesized, whose structures were studied using various techniques (MRI measures, X-ray circular dichroism), and also found that behave as active catalysts in processes asymmetric transfer hydrogen 2-propanol to acetophenone.

Author: TORRE FAZIO FERNANDO NICOLÁS DE LA.
Year: 2005.
University: MÁLAGA [www.uma.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS, UNIVERSIDAD DE MÁLAGA.

Summary: Nitrogen is one of the main factors limiting the growth and development of plants. The mechanisms by which this element is incorporated, assimilated and metal have been widely studied in herbaceous plants such as Arabidopsis thalian or Oryza sativa, however, the studies carried out in trees and conifers in particular has been much more limited. The previous results generated by the group led by Dr. Francis M. Cánovas Ramos have resulted in a huge step in the metabolism of nitrogen in the conifers of the genus Pinus (pine) conducting characterization of the major genes involved in the assimilation of ammonium: GS1a, GS1b, HDI, FdGOGAT, etc.. These studies have been approached from different strategies as an expression level couriers or polypeptides, analysis of location tipo-celular, obtain transgenic plants or characterization of their regions promoters. This thesis has been as comprehensive strategy to characterize the molecular, structural and kinetic key enzyme in the metabolism of nitrogen in conifers. We have generated recombinant proteins in E. coli from the coding sequences available in our laboratory for glutamine synthetase (GS), asparragina synthase (AS) and aspartate aminotransferase (AAT). The results, integrated with the information previously available, have led to propose a differential role for the isoenzymes GS1a and GS1b pine, thus suggested the essential role of GS1a in the assimilation of ammonium photosynthetic tissues and the GS1b maintaining a steady flow of glutamine, for the transport and storage, to varying concentrations of glutamate. Moreover, the characterization of the gene family of AATs in pine has allowed describe the existence of a new plant gene coding for est enzyme. This gene is filogenéticametne closest to the genes encoding the AATs in cyanobacteria and arqueobacterias that the genes encoding these proteins previously described in plants. The data comparing sequence, phylogenetic, molecular characteristics, the kinetic parameters and structural patterns of expression and the subcellular localization suggests that the physiological role of this enzyme is the conversion of glutamate aspartate in the biosynthesis of amino acids lysine , isoleucine, methionine and threonine. The phylogenetic analysis has shown that the genes coding for enzymes involved in the biosynthesis route of the amino acids methionine, lysine and threonine isoleucine originate endosimbiótico and were acquired by the plants from an ancestor of cianobacerias. The origin endosimbiótico this route may be the reason that these amino acids are essential to the nature of animals and forcing their acquisition through diet.