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Doctoral Thesis
DOI
https://doi.org/10.11606/T.46.1999.tde-08082008-130612
Document
Author
Full name
Sandro Roberto Marana
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 1999
Supervisor
Committee
Terra, Clélia Ferreira (President)
Araújo, Pedro Soares de
Gambarini, Ângelo Geraldo
Marinotti, Osvaldo
Nader, Helena Bonciani
Title in Portuguese
Purificação e caracterização das β-glicosidases digestivas de Spodoptera frugiperda (Lepidoptera)
Keywords in Portuguese
Beta-glicosidases
Beta-glucosidase
Enzimas
Spodoptera frugiperda
Abstract in Portuguese
Foram purificadas através de uma combinação de cromatografias as duas β- glicosidases digestivas (Mr 47.000 e 50.000 - denominadas β47 e β50, respectivamente) encontradas na larva de S. frugiperda. Experimentos de competição entre substratos e modificação química mostraram que a β47 possui dois sítios ativos. Um desses sítios denominado aril&$946;glicosidase apresenta um subsítio -1 que liga galactose mais eficientemente do enquanto ,que o subsítio +1 prefere pequenos grupos hidrofóbicos cíclicos. O segundo sítio, denominado celobiase, possui um subsítio -1 que prefere glicose. Já a região de ligação do aglicone apresenta 4 subsítios, que ligam glicose com afinidade decrescente à medida que afastam-se do ponto de clivagem do substrato. O cDNA que codifica a β50 foi clonado e sequenciado. Alinhamentos de sequência de aminoácidos, experimentos de competição entre substratos e inibição mostraram que esta enzima possui apenas um sítio ativo. O subsítio -1, cuja especificidade é controlada por uma rede de pontes de hidrogênio, foi estudado comparando-se os parâmetros cinéticos (Kcat e KcaUKm) para a hidrólise de NPβglicosídeos. A região de posicionamento do aglicone, uma fenda hidrofóbica composta de 3 subsítios, foi caracterizada utilizando-se alquil β-glucosídeos e oligocelodextrinas como inibidores. O alinhamento da sequência de aminoácidos da β50 com outras glicosil hidrolases sugeriu quais aminoácidos participariam da ligação do substrato e que o GlU187 (doador de prótons - pKa = 7,5) e o GIU399 (nucleófilo - pKa = 4,5) estão diretamente envolvidos na catálise. Além disso, a Arg97 e a Tyr331 participam indiretamente modulando o pKa do GIU399. r .
Title in English
Purification and characterization of digestive beta-glycosidases from Spodoptera frugiperda (Lepidoptera)
Keywords in English
Beta-glucosidases
Beta-glycosidases
Enzymes
Spodoptera frugiperda
Abstract in English
Two digestive β-glycosidases (MW 47,000 and 50,000, named βgly47 and βgly50, respectively) whose are found in the S. frugiperda larvae were purified by a combination of chromatographic steps. Substrate competition experiments and chemical modification data showed that βgly47 has two active sites. One of them was called aryl β-glycosidase and presents a -1 subsite that prefers galactose while the +1 subsite binds small cyclic hydrophobic groups. The other active site was called cellobiase and presents 4 subsites that bind glucose residues weaker as they get far from the cleavage point. The cDNA that codes the βgly50 was cloned and sequenced. Amino acid sequence alignment, substrate competition experiments and inhibitions proved that this enzyme has just one active site. The -1 subsite specificity is controlled by a hydrogen bond network as it was showed comparing the kinetic parameters (Kcat and KcatlKm) for some NPβglycosides hydrolysis. The aglycone binding region, a hydrophobic cleft, was studied with alkyl β-glucosides and oligocellodextrins as competitive inhibitors. Amino acid sequence alignment between the βgly50 and other glycosil hydrolases showed the amino acids responsible for the substrate binding and that the GIU (proton donor - pKa = 7.5) and GIU399 (nucleophile - pKa = 4.5) are directly involved in the catalysis. Beside this, Arg97 and Tyr331 participate indirectly in the catalysis, modulating the nucleophile pKa
 
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Publishing Date
2008-08-18
 
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