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Master's Dissertation
Full name
Adriana Rios Lopes
Knowledge Area
Date of Defense
São Paulo, 1999
Terra, Walter Ribeiro (President)
Cuccovia, Iolanda Midea
Nader, Helena Bonciani
Title in Portuguese
Caracterização das tripsinas de insetos
Keywords in Portuguese
Enzima digestiva
Abstract in Portuguese
Tripsinas são enzimas comuns à maioria dos insetos e de fundamental importância para a digestão inicial de proteínas. Desta forma, tornam-se alvos importantes para orientar a construção de plantas transgênicas resistentes a insetos. As tripsinas são serina endopeptidases que clivam cadeias protéicas na porção carboxílica de resíduos de aminoácidos básicos como lisina e arginina, sendo que a hidrólise da ligação peptídica formada por um resíduo de arginina é de duas a dez vezes mais eficiente que a hidrólise da ligação peptídica formada por lisina. A purificação das tripsinas de insetos de diferentes ordens e o estudo da especificidade de seus subsítios utilizando substratos de fluorescência apagada servem de base para a seleção de um método mais eficiente de inibição da sua atividade, assim como para desvendar as tendências evolutivas da especificidade destas enzimas. O trabalho dessa dissertação levou ao desenvolvimento de processos de purificação das tripsinas de Periplaneta americana, Tenebrio molitor, Musca domestica e Diatraea saccharalis. O estudo da especificidade dos subsítios S1, S2, S3 e S1' das tripsinas demonstraram que, diferentemente das tripsinas dos outros insetos e das tripsinas de mamíferos, a tripsina de Diatraea saccharalis hidrolisa com maior eficiência substratos que apresentem lisina em P1, demonstrando uma diferença na especificidade primária desta enzima. Além disso, é possível verificar ao longo da evolução dos grupos de insetos estudados uma tendência a tornar os subsítios cada vez mais hidrofóbicos.
Title in English
Characterization of insect trypsins
Keywords in English
Digestive enzymes
Subsite specificity
Abstract in English
Trypsins are serine endopeptidases that hydrolyze peptide bonds at the carboxyl side of positively charged residues: arginine and lysine. Mammalian trypsin preferentially cleaves the peptide bond formed by arginine. Site directed mutagenesis has shown that trypsin specificity is related to residues present at the primary specificity site and to structural determinants like two surface loops. Differences in trypsins specificity may be the cause of some insects be resistant to serine endopeptidases plant inhibitors and to Bacillus thuringiensis toxins. Trypsins are usual enzymes in insects and are very important to protein digestion. There are few studies dealing with insect trypsin specificity. They generally consist in analyses of fragments formed by the action of the enzyme on peptide chains like insulin β chain. As these studies were semi-quantitative, insect trypsin specificity requires a better characterization. This dissertation describes the purification of trypsins from Periplaneta americana, Tenebrio molitor, Musca domestica and Diatraea saccharalis and the characterization of the specificity of the subsites S1, S2, S3 e S1' by the use of quenched fluorescence peptide substrates. The results showed that trypsins from the mentioned insects have different specificities, including the primary specificity. Thus, Diatraea saccharalis trypsin cleaves at Lys more efficiently than at Arg, whereas the eontrary is true for the other insects. The data also showed that trypsin subsites tend to beeome more hydrophobic as the insects are more evolved.
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