Substrate specificity of insect trypsins and the role of their subsites in catalysis

Substrate specificity of insect trypsins and the role of their subsites in catalysis

Autor Lopes, A. R. Google Scholar
Juliano, M. A. Google Scholar
Marana, SR Google Scholar
Juliano, L. Google Scholar
Terra, W. R. Google Scholar
Instituição Universidade de São Paulo (USP)
Universidade Federal de São Paulo (UNIFESP)
Resumo Trypsins have high sequence similarity, although the responses of insect trypsins to chemical and natural inhibitors suggest they differ in specificities. Purified digestive trypsins from insects of four different orders were assayed with internally quenched fluorescent oligopeptides with two different amino acids at P1 (Arg/Lys) and 15 amino acid replacements in positions P1', P2, P2, and P3. the binding energy (Delta G(s), calculated from K-m values) and the activation energy (Delta G(T)(++), determined from k(cat)/K-m values) were calculated. Dictyoptera, Coleoptera and Diptera trypsins hydrolyze peptides with Arg at P, at least 3 times more efficiently than peptides with Lys at P1, whereas Lepidoptera trypsins have no preference between Arg and Lys at that position. the hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. the results suggested that insect trypsin subsites become progressively more hydrophobic along evolution. Apparently, this is an adaptation to resist plant protein inhibitors, which usually have polar residues at their reactive sites. Results also suggested that, at least in lepidopteran trypsins, S3, S2, S1' and S2' significantly bind the substrate ground state, whereas in the transition state only S1' and S2' do that, supporting aspects of the presently accepted mechanism of trypsin catalysis. Homology modeling showed differences among those trypsins that may account for the varied kinetic properties (c) 2005 Elsevier B.V. All rights reserved.
Palavra-chave trypsin
substrate specificity
quenched fluorescence substrate
binding subsites
insects
activation energy
binding energy
Idioma Inglês
Data de publicação 2006-02-01
Publicado em Insect Biochemistry and Molecular Biology. Oxford: Pergamon-Elsevier B.V., v. 36, n. 2, p. 130-140, 2006.
ISSN 0965-1748 (Sherpa/Romeo, fator de impacto)
Publicador Elsevier B.V.
Extensão 130-140
Fonte http://dx.doi.org/10.1016/j.ibmb.2005.11.006
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000235269200003
Endereço permanente http://repositorio.unifesp.br/handle/11600/28723

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