Dissertations / Theses on the topic 'Substrate enzyme complex'

L’accomplissement de tout acte protéolytique implique nécessairement la formation d'un complexe entre l'enzyme et son substrat. Par différentes approches cinétiques, spectroscopiques et moléculaires nous avons cherché à caractériser les phénomènes mis en jeu au cours de l'hydrolyse, par la thermolysine, de petits peptides en milieu biomimétiques. Cette étude a été conduite à l'interface entre la biochimie, la biophysique, la chimie et la physique. Dans un premier temps, nous nous sommes intéressés au comportement catalytique de la thermolysine sur des substrats modèles et en milieu modifié. Nous avons montré d'une part, que l'ajout d'additifs polyhydroxylés influence grandement l'activité de la thermolysine et d'autre part, affine les connaissances sur la spécificité et la sélectivité de cette enzyme (en particulier, mise en évidence de l'influence du résidu P'2 dans le mécanisme). Dans un deuxième temps, nous présentons des études structurales des peptides substrats en milieu modifié. Nous avons mis en évidence l'absence d'influence du micro-environnement contenant une forte proportion de glycérol sur la conformation des molécules de substrat et le rôle possible de leur structure tridimensionnelle quant à leur hydrolyse. Ces études ont été étendues à un autre modèle peptidique, de forme cyclique ou linéaire, et corrélées aux résultats cinétiques. Dans un troisième temps, par deux approches différentes, nous avons abordé l'étude des relations structure-fonction de la thermolysine. Expérimentalement, par des études cinétiques avec l'enzyme immobilisée et des déterminations de sa structure par spectroscopie laser Raman, nous montrons que l'enzyme est très peu sensible au micro-environnement. Théoriquement en analysant, par modélisation, l'interaction de la thermolysine avec un tripeptide substrat, nous avons mis en évidence des changements de conformation du substrat et/ou des mouvements du site actif enzymatique au cours de l'acte catalytique.

Ce travail est consacre a l'etude des mecanismes responsables de la reconnaissance entre la seryl-arnt synthetase d'escherichia coli et son substrat macromoleculaire, l'arnt#s#e#r. La premiere etape de ces travaux a consiste a surexprimer l'arnt#s#e#r#2 et l'arnt#s#e#r ambre suppresseur d'e. Coli. Pour cela, les genes synthetiques des deux arnt#s#e#r ont ete construits par assemblage de sept oligonucleotides chevauchants. L'utilisation d'un plasmide dont le nombre de copies est regule par la temperature permet d'obtenir des cellules bacteriennes contenant un taux d'arnt#s#e#r vingt fois superieur a celui present dans des cellules depourvues de plasmide. Grace a cette surexpression, un protocole de purification comportant deux etapes chromatographiques a pu etre developpe. Les arnt purifies ont ensuite ete utilises pour determiner la contribution du domaine n-terminal de la seryl-arnt synthetase d'e. Coli, a l'efficacite et a la specificite de la reaction d'aminoacylation. Les resultats obtenus, montrent que le domaine n-terminal depourvu de role catalytique lors de la reaction d'activation de l'acide amine, est indispensable, d'une part, au maintien d'une efficacite de catalyse elevee lors du transfert de l'acide amine active sur l'arnt et, d'autre part, a la specificite de reconnaissance des arnt#s#e#r. Parallelement, la mesure des constantes de dissociation de l'arnt#s#e#r#2, revele que la fixation de deux molecules d'arnt sur la seryl-arnt synthetase est regit par un mecanisme de type cooperatif. La derniere partie de ce travail montre que la seryl-arnt synthetase de saccharomyces cerevisiae ne peut etre surexprimee chez e. Coli. Le contexte bacterien ne permettant pas a la seryl-arnt synthetase de s. Cerevisiae d'acquerir une structure tridimensionnelle homogene. Les etudes menees, en parallele sur la seryl-arnt synthetase de s. Cerevisiae surexprimee dans son contexte naturel, revelent une repartition de cette enzyme en deux populations distinctes, et une mobilite electrophoretique, sur sds-page, plus elevee que celle obtenue pour l'enzyme surexprimee chez e. Coli

Pour réduire notre empreinte carbone sur l’environnement, il est urgent de développer des procédés industriels utilisant une source de carbone renouvelable comme la biomasse lignocellulosique. La paroi cellulaire végétale est un enchevêtrement complexe de cellulose, d’hémicelluloses et de lignines. Elle résiste aux attaques biologiques et chimiques mais limite le développement d’une bioéconomie responsable. Dans la Nature, des enzymes multimodulaires produites par certains microorganismes peuvent la déconstruire. Toutefois, ces enzymes sont majoritairement étudiées sur des substrats artificiels ou purifiés. Dans cette thèse, nous proposons de les étudier sur des substrats broyés puis des coupes de paille de blé brutes. Les enzymes multimodulaires sont préparées à façon en utilisant la propriété d’association covalente des protéines Jo et In. Nous avons ainsi associé la xylanase NpXyn11A de N. patriciarum avec deux modules non catalytiques : le CBM3a de C. thermocellum ou le CBM2b1 C. fimi ciblant la cellulose ou les xylanes respectivement. Les propriétés biochimiques de ces protéines chimériques ont été comparées aux modules sauvages. L’activité enzymatique des protéines chimériques a ensuite été étudiée sur des substrats solubles, jusqu’à des substrats insolubles comme le son et la paille de blé, notamment par immunocytochimie. Ce travail a mis en évidence l’importance de la relation enzymes/substrats pour une caractérisation in muro d’activité enzymatique et une meilleure compréhension de la déconstruction de la biomasse végétale
In order to reduce our carbon footprint on the environment, it is more than urgent to develop new industrial process using a renewable carbon source such as lignocellulosic biomass. Plant cell walls consist of a complex network of cellulose, hemicelluloses and lignins that cross-link with each other mainly via non-covalent bonds. It is thus hardly surprising that plant biomass is rather recalcitrant to chemical or biological degradation. In the present era marked by the desire to build a green bioeconomy, this recalcitrance remains a key point. In Nature, the plant-based organic carbon contained within plant cell walls is mainly recycled by the action of cellulolytic microorganisms, producing multimodular enzymes. However, these enzymes are mainly characterized on artificial or purified substrates. In this thesis, we proposed to study multimodular enzymes on raw substrates such as wheat straw sections. The studied multimodular enzymes were associated thanks to the use of two small proteins Jo and In. Thus, we associated the xylanase NpXyn11A from N. patriciarum with two non-catalytic modules: CBM3a from C. thermocellum or CBM2b1 from C. fimi targeting cellulose or xylans respectively. Biochemical properties of these chimeric proteins and wild-type modules have been compared. The enzymatic activity of chimeric proteins has been studied on soluble substrates and compared to the activity on insoluble substrates, mainly by immunocytochemistry. This work highlighted the importance of the relationship enzymes/substrates and its key role to better understand the biomass deconstruction in muro

Ces travaux concernent l'étude cristallographique de complexes ternaires de la Glycéraldéhyde-3-phosphate déshydrogénase phosphorylante de B. Stearothermophilus. Cette enzyme catalyse la phosphorylation oxydative du Glycéraldéhyde-3-phosphate en présence d'un cofacteur, le NAD, et de phosphate inorganique. Pour fixer les groupements phosphates de ces deux substrats, l'enzyme possède deux sites de reconnaissance anionique, dénommés respectivement Pi et Ps. Cependant, la façon dont ces deux sites contribuent à la fixation des deux groupements phosphate reste encore soumise à controverse et différents modèles ont été proposés. Afin de tester la validité des ces modèles, il semblait essentiel de pouvoir disposer d'évidences structurales directes. Deux mutants ont été étudiés : le mutant C149A, inactif et le mutant C149S, très faiblement actif. Des cristaux de ces deux mutants ont été obtenus dans des conditions originales, exemptes de tout anion. Ces nouvelles conditions de cristallisation ont conduit à de nouveaux empilements cristallins. L'analyse des structures des complexes binaires met en évidence des sites Pi libres de tout anion. Par contre, les sites Ps sont occupés par un anion sulfate issu probablement de la purification. Ceci montre que le site Ps a une meilleure affinité que le site Pi vis-à-vis des anions. Ce résultat a été confirmé par l'analyse des acides aminés impliqués dans la formation des sites Pi et Ps et par le fait que le potentiel électrostatique local est plus important au niveau du site Ps qu'au niveau site Pi. Les complexes ternaires que nous avons obtenus constituent la première structure tridimensionnelle d'une GAPDH en complexe avec son substrat physiologique. L'analyse de ces complexes a permis d'apporter des informations quant aux interactions enzyme-substrat. Nous montrons sans ambigüité que dans les deux complexes obtenus, le groupement phosphate du G3P est localisé dans le site Ps. De même, les facteurs moléculaires responsables de la stéréosélectivité vis-à-vis du D-G3P ont pu être élucidés. Cependant, le débat reste ouvert concernant, d'une part, la contribution de la boucle 205-210 à la catalyse et d'autre part, la conséquence de la formation de la liaison covalente entre l'enzyme et le substrat sur le positionnement des intermédiaires réactionnels dans le site actif.

Si la GAPDH est une enzyme bien caractérisée sur le plan biochimique et structural, la contribution de ses deux sites de reconnaissance anionique lors de la catalyse reste incomprise et nécessitait la détermination des structures de l'intermédiaire thioacylenzyme et du complexe enzyme-produit. Ce manuscrit présente la mise au point des conditions de cristallisation et d'accumulation de l'intermédiaire thioacylenzyme (suivi par microspectrophotométrie) et du complexe enzyme-produit, la résolution et l'analyse des structures cristallographiques correspondantes ainsi que les implications pour le mécanisme catalytique. Les résultats obtenus mettent notamment en évidence un mouvement de "va-et-vient" du substrat au cours de la catalyse entre les deux sites de reconnaissance anionique qui est lié à l'étape d'échange du cofacteur. Bien que structuralement proche des GAPDH, l’E4PDH est une enzyme pour laquelle peu de données sont disponibles. De nombreuses questions restent donc posées concernant notamment les déterminants de l'affinité pour le cofacteur, la nature du site de reconnaissance anionique, ou encore le mécanisme d'activation de la molécule d'eau nécessaire à une étape d'hydrolyse efficace. Sont présentées dans ce manuscrit trois structures cristallographiques de l'E4PDH d'E. coli, sous forme apoenzyme, en présence de phosphate ou en complexe avec un analogue de cofacteur. L'analyse de ces structures a notamment permis de caractériser l'unique site de reconnaissance anionique de l'enzyme, de proposer des hypothèses quant à la faible affinité de l'enzyme pour le cofacteur, et d'identifier un candidat possible pour l'activation de la molécule d'eau hydrolytique
Even if GAPDH is well characterized from a biochemical and structural point of view, the contribution of its two anion recognition sites to catalysis is still matter of debate. This work presents the crystallization, the strategy for the accumulation of the thioacylenzyme intermediate and for the obtaining of the enzyme-product complex, the resolution and analysis of the corresponding crystallographic structures and the implications in terms of reaction mechanism. The results mainly shed light on the existence of a flip-flop movement of the substrate between the two anion recognition sites during catalysis which is related to the cofactor exchange step. Although structurally related to GAPDHs, the E4PDH is an enzyme fairly less characterized. Many interrogations thus remain on the determinants of cofactor affinity, on the nature of the anion recognition site or on the mechanism of activation of the water molecule that is needed for an efficient hydrolysis step. This dissertation presents the resolution of three crystallographic structures of the E4PDH from E. coli, under the apoenzyme form, in the presence of phosphate or in complex with a cofactor analog. The analysis f these structures allows the characterization of the unique anion recognition site of this enzyme, to propose structural hypotheses to explain the low affinity of this enzyme for its cofactor and also to identify possible candidates for the activation of the nucleophilic water molecule

La selectivite des oxydations catalysees par les monooxygenases a cytochrome p-450 tient compte de deux facteurs principaux: 1) le role de la chaine proteique bordant le site actif du cytochrome p450. 2) la chimioselectivite du complexe a oxygene actif implique dans la reaction. Oxydation du phenoxy-6 hexene 1 et du leucotriene b4 fait intervenir des familles de cytochromes p-450 tres differentes. L'utilisation de systemes metalloporphyriniques permet de faire, en partie, la difference entre les facteurs dus a la reactivite intriseque de l'entite a oxygene actif et ceux dus a l'environnement proteique. L'apoproteine est absente et la reconnaissance du substrat est reduite a son strict minimum puisqu'elle n'est plus excitee que par la metalloporphyrine. Mise au point de catalyseurs selectifs d'oxydation des olefine monosubstituees

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Cellulases (E.C. 3.2.1.4) are enzymes responsible for the degradation of cellulose, are molecules capable of accelerating chemical reactions and breaking the chemical bonds between glucose units. Cellulases correspond to the complex consisting of three enzymes endoglucanases, exoglucanases and beta-glucosidases, with diverse applications, being the microbial biotechnological processes responsible for a great part of the world economy, yet the costs of production are still very high. In this context, 25 strains of filamentous fungi (Ascomycetes) isolated from mangrove sediments of Rio Formoso, PE, Brazil, were investigated to investigate the production potential of the enzymes of the cellulolytic complex. The initial studies were carried out by selecting the fungi with the highest enzymatic activity, through the detection of cellulolytic activity in solid synthetic medium, with carboxymethylcellulose (CMC) as the substrate. The results indicated the presence of the cellulase enzyme through the formation of halo in 3 strains of the genus Trichoderma, 3 strains of the genus Aspergillus and 1 strain of the genus Penicillium. The most representative enzymatic indices were those of Penicillium sp. UCP 0279 with Index of 2,2, followed by Aspergillus flavus UCP 1413 with enzymatic index of 1,7. Submerged fermentations were carried out to evaluate the endoglucanase activity, exoglucanase and β-glycosidase, using agroindustrial residues, tangerine peel, pineapple peel, pineapple crown, wheat bran and corn bran as substrate. The results indicated a CMCase activity of 20.2 IU / mL for Penicillium sp. UCP 0279, with wheat bran as substrate in 72 h of fermentation and an activity of 18.3 IU / mL in 24 h with the pineapple crown. For the Aspergillus flavus UCP 1413, the yield was 14.9 IU / mL and 14.5 IU / mL with the residues of corn bran and pineapple peel respectively, and both results were obtained with 24 h of fermentation. The FPase activity for Penicillium sp. UCP 0279, using pineapple peel as substrate had 45.5 IU / mL and the tangerine peel 42.8 IU / mL, both in fermentation at 48 h. For A. flavus UCP 1413 the pineapple crown presented 25.0 IU / mL enzymatic activity in 24 h and the pineapple peel 14.4 U / mL at the same time. In the activity of the enzyme β-glycosidase, Penicillium sp. UCP 0279 showed a production of 18.2 IU / mL in 24 h, with the pineapple crown residue and the pineapple peel had 9.1 IU / mL in 48 h. The A. flavus UCP 1413 presented with 96 h of fermentation an activity of 16.9 U / mL and 14.5 U / mL, with wheat bran and corn bran, respectively.
As celulases (E.C. 3.2.1.4) são enzimas responsáveis pela degradação da celulose, são moléculas capazes de acelerar reações químicas e realizar a quebra das ligações químicas existentes entre as unidades de glicose. As celulases correspondem ao complexo constituído por três enzimas endoglucanases, exoglucanases e beta-glicosidases, com diversas aplicações, sendo os processos biotecnológicos microbianos responsáveis por uma grande parcela da economia mundial, contudo os custos de produção ainda são muito elevados. Neste contexto, foi realizada a bioprospecção de 25 linhagens de fungos filamentosos (Ascomycetes) isolados de sedimentos de mangue do município Rio Formoso, PE, Brasil, investigando o potencial de produção das enzimas do complexo celulolítico. Os estudos iniciais foram realizados selecionando os fungos com maior atividade enzimática, através da detecção da atividade celulolítica em meio sintético sólido, tendo como substrato a carboximetilcelulose (CMC). Os resultados indicaram a presença da enzima celulase através da formação do halo em 3 linhagens do gênero Trichoderma, 3 linhagens do gênero Aspergillus e 1 linhagem do gênero Penicillium. Os índices enzimáticos mais representativos foram os de Penicillium sp. UCP 0279 com Índice de 2,2, seguido de Aspergillus flavus UCP 1413 com índice enzimático de 1,7. Em seguida, foram realizadas fermentações submersas para avaliação da atividade endoglucanase, exoglucanase e β-glicosidase, utilizando resíduos agroindustriais, casca de tangerina, casca de abacaxi, coroa de abacaxi, farelo de trigo e farelo de milho como substrato. Os resultados indicaram uma atividade para CMCase de 20,2 UI/mL para Penicillium sp. UCP 0279, com farelo de trigo como substrato em 72 h de fermentação e com a coroa de abacaxi observou-se uma atividade de 18,3 UI/mL em 24 h. Para o Aspergillus flavus UCP 1413, a produção foi de 14,9 UI/mL e 14,5 UI/mL com os resíduos de farelo de milho e de casca de abacaxi respectivamente, e ambos os resultados foram obtidos com 24 h de fermentação. A atividade FPase para Penicillium sp. UCP 0279, usando casca de abacaxi como substrato apresentou 45,5 UI/mL e a casca de tangerina 42,8 UI/mL, ambos em fermentação a 48 h. Para A. flavus UCP 1413 a coroa de abacaxi apresentou 25,0 UI/mL de atividade enzimática em 24 h e a casca de abacaxi 14,4 U/mL no mesmo tempo. Na atividade da enzima β-glicosidase o Penicillium sp. UCP 0279 apresentou uma produção de 18,2 UI/mL em 24 h, com o resíduo da coroa de abacaxi e com a casca de abacaxi apresentou 9,1 UI/mL em 48 h. O A. flavus UCP 1413 apresentou com 96 h de fermentação uma atividade de 16,9 U/mL e 14,5 U/mL, com farelo de trigo e farelo de milho respectivamente.

The field of bioenergetics deals with the flow and transformation of energy within and between living organisms and their environment. The work presented in this thesis report focuses on cellular respiration and more specifically on the first enzyme of the respiratory chain, NADH:ubiquinone oxidoreductase (Complex I). This was done to clarify details about its function and its implication in disease. First, the creation of a sensor involving the biomimetically immobilized enzyme is presented and probed through a combination of surface enhanced infrared absorption spectroscopy (SEIRAS) and electrochemistry. This sensor is then tested against different substrates and inhibitors. In a second part, the interaction of Complex I with lipids, inhibitors (Zn2+ and NADH-OH) and the role of a Tyrosine residue situated in the NADH binding pocket are investigated through electrochemically induced UV-Vis and FTIR difference spectroscopies. The results gathered through these experiments are then explored under a structural perspective and a coupling mechanism between quinone reduction and proton translocation by Complex I is proposed.

The influence of substrate and enzyme concentration on the rate of saccharification of two defined, insoluble, cellulose substrates, Avicel and Solka-Floc, by the cellulase enzyme system of Trichoderma viride has been evaluated. Assays utilized enzyme concentrations ranging from 0.014 to 0.056 filter paper unit per mL and substrate concentrations up to 10% (w/v). Analysis by initial velocity methods found the maximum velocity of the enzyme to be nearly equivalent for the two substrates and the km for the two substrates to be of similar magnitude, i.e., 0.20% for Solka-Floc and 0.63% for Avicel (w/v). Studies utilizing relatively high substrate concentrations (greater than 15 times the Km) demonstrated that the enzyme exhibits very different apparent substrate inhibition properties for the two substrates. The rate of saccharification of Avicel at relatively high substrate concentrations was up to 35% lower than the maximum rate which was obtained at a lower substrate concentration. The Avicel concentration corresponding to the maximum rate of saccharification was dependent on enzyme concentration. In contrast to the results with Avicel, the enzyme did not exhibit substrate inhibition with the Solka-Floc substrate. Potential differences in the degree of substrate inhibition with different substrates, as reported in this paper, is particularly relevant to the experimental design of comparative studies.
Graduation date: 1990

N-substituted pantothenamides are analogues of pantothenate, the precursor of the essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of pantothenate kinase (PanK) in the first step of CoA biosynthesis, possessing antimicrobial activity against multiple pathogenic bacteria. This enzyme is an attractive target for drug discovery due to low sequence homology between bacterial and human PanKs. In this study, the crystal structure of the PanK from the multidrug-resistant bacterium Klebsiella pneumoniae (KpPanK) was first solved in complex with N-pentylpantothenamide (N5-Pan). The structure reveals that the N5-Pan pentyl tail is located within a highly aromatic pocket, suggesting that an aromatic substituent may enhance binding affinity to the enzyme. This finding led to the design of N-pyridin-3-ylmethylpantothenamide (Np-Pan) and its co-crystal structure with KpPanK was solved. The structure reveals that the pyridine ring adopts alternative conformations in the aromatic pocket, providing the structural basis for further improvement of pantothenamide-binding to KpPanK.

Microbial degradation of aromatic hydrocarbons is imperative for maintaining the global carbon cycle and removing potentially toxic aromatic xenobiotics. This thesis focuses on the characterization of a pyruvate-specific class II aldolase (BphI) and acetaldehyde dehydrogenase (BphJ), the final two enzymes of the bph meta-cleavage pathway in Burkholderia xenovorans LB400. This pathway is responsible for the degradation of the industrial pollutant polychlorinated biphenyls (PCB) and therefore mechanistic characterization of these enzymes can be applied to improve pollutant degradation. BphI catalyzes the aldol cleavage of 4-hydroxy-2-oxoacids to pyruvate and an aldehyde while BphJ transforms aldehydes to acyl-CoA, using NAD+ and CoASH as cofactors. Size-exclusion chromatography was used to determine that the oligomeric unit of the BphI-BphJ complex is a heterotetramer. The aldolase BphI was shown to exhibit a compulsory order mechanism and utilize 4-hydroxy-2-oxoacids with an S configuration at C4. The generation of BphI active site variants allowed for the proposal of a catalytic mechanism and a greater understanding as to how stereospecificity occurs. Using steady-state kinetic assays, Arg-16 was demonstrated to be essential for catalysis. Molecular modeling of the substrate and pH dependency (wild-type pKa of ~7, lost in H20A and H20S variants) were used to identify His-20 as the catalytic base. Tyr-290 was originally proposed to be the catalytic acid. However, this was refuted as a Tyr-290 (Y290F) variant did not affect the catalytic efficiency of the enzyme. Instead, the variant was observed to exhibit a loss of stereochemical control. From the crystal structure of an orthologous aldolase-dehydrogenase complex, solvent isotope effect studies, and a proton inventory, a water molecule was implicated as the catalytic acid. Based on their position within the crystal structure, Leu-87 and Leu-89 were implicated in substrate specificity. Replacement of Leu-89 with alanine effectively increased the length of the active site, allowing for the accommodation of longer aldehyde substrates. In contrast, Leu-87 was responsible for hydrophobic stabilization of the C4-methyl of the substrate. Double variants L87N;Y290F and L87W;Y290F were constructed to enable the binding of 4(R)-hydroxy-2-oxoacids. Polarimetric analysis confirmed that the double variants were able to synthesize 4-hydroxy-2-oxoacids of up to 8 carbons in lengths, which were of the opposite stereoisomer to those produced by the wild-type enzyme. Cys-131 was identified as the catalytic thiol that forms an acyl-enzyme intermediate in the dehydrogenase, BphJ. This enzyme was shown to exhibit similar specificity constants for acetaldehyde and propionaldehyde and utilize aliphatic aldehydes from two to five carbons in length as substrates. The enzyme was able to use either NAD+ or NADP+ as the cofactor. Finally, we demonstrated that aldehydes produced in the aldolase reaction are not released into the bulk solvent but are channeled directly to the dehydrogenase, providing the first biochemical determination of substrate channeling in any aldolase-dehydrogenase complex.
Chapter 3 - Reprinted (adapted) with permission from Baker, P., Carere, J., and Seah, S. Y. (2011) Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, BphI, Biochemistry 50: 3559-3569. Copyright (2011) American Chemical Society. Chapter 4 - Reprinted (adapted) with permission from Baker, P., and Seah, S. Y. (2012) Rational design of stereoselectivity in the class II pyruvate aldolase BphI, J Am Chem Soc 134: 507-513. Copyright (2012) American Chemical Society. Chapter 6 - Reprinted (adapted) with permission from Baker, P., Hillis, C., Carere, J., and Seah, S. Y. (2012) Protein-protein interactions and substrate channeling in orthologous and chimeric aldolase-dehydrogenase complexes, Biochemistry 51: 1942-1952. Copyright (2012) American Chemical Society.
National Science and Engineering Research Council of Canada (NSERC), Ontario Graduate Scholarship in Science and Technology

碩士
國立臺灣大學
生化科學研究所
93
Glutaminyl cyclase (QC) is a critical enzyme that is responsible for the conversion of N-terminal glutamine into pyroglutamic acid, which is an important event during protein synthesis. Many hormones, such as thyrotropin-releasing hormone and neurotensin, need QCs for modifying their N-terminal glutamine and subsequently become an active form. In the previous studies, QCs from various species, such as porcine, bovine, papaya or human have been cloned, expressed and characterized. However, the exact catalytic process still remained ambiguous due to the lack of ingormation on QC structure. The human QC has been cloned and expressed in large scale and purified successfully by Kai-Fa Huang in our group. He also determined the first QC and QC/inhibitors structures by x-ray crystallography (2005 in press). Enzyme catalytic mechanism was proposed preliminarily based on the structural alignment and the solved structures. In order to confirm the catalytic mechanism proposed by Huang, some key residues, like E201, D248, D305, Q304, H307, H319, F325 and W329, which are likely to involve in the catalytic mechanisms or play vital roles in the substrate recognition, have been chosen for site-directed mutagenesis. The catalytic ability of wild-typed human QC was assessed and characterized by high performance liquid chromatography. Coupled enzyme assay was used to evaluate the activities of the mutants. Among them, E201Q, which dramatically lost its catalytic activity, was subjected to further investigation. The E201Q/glutamine-t-butyl ester complex structure was determined. The structural information of substrate binding is supportive to the previous suggestion on enzyme catalysis. Combined with results from the biological activity analysis of human QC mutants, the proposed mechanism could be more solid and reliable.

Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.