| Texto completo | |
| Autor(es): |
Otsuka, Felipe A. M.
;
Chagas, Rafael S.
;
Almeida, Vitor M.
;
Marana, Sandro R.
Número total de Autores: 4
|
| Tipo de documento: | Artigo Científico |
| Fonte: | Protein Science; v. 29, n. 9, p. 11-pg., 2020-07-13. |
| Resumo | |
In this work, we investigated how activity and oligomeric state are related in a purified GH1 beta-glucosidase fromSpodoptera frugiperda(Sf beta gly). Gel filtration chromatography coupled to a multiple angle light scattering detector allowed separation of the homodimer and monomer states and determination of the dimer dissociation constant (K-D), which was in the micromolar range. Enzyme kinetic parameters showed that the dimer is on average 2.5-fold more active. Later, we evaluated the kinetics of homodimerization, scanning the changes in the Sf beta gly intrinsic fluorescence over time when the dimer dissociates into the monomer after a large dilution. We described how the rate constant of monomerization (k(off)) is affected by temperature, revealing the enthalpic and entropic contributions to the process. We also evaluated how the rate constant (k(obs)) by which equilibrium is reached after dimer dilution behaves when varying the initial Sf beta gly concentration. These data indicated that Sf beta gly dimerizes through the conformational selection mechanism, in which the monomer undergoes a conformational exchange and then binds to a similar monomer, forming a more active homodimer. Finally, we noted that conformational selection reports and experiments usually rely on a ligand whose concentration is in excess, but for homodimerization, this approach does not hold. Hence, since our approach overcomes this limitation, this study not only is a new contribution to the comprehension of GH1 beta-glucosidases, but it can also help to elucidate protein interaction pathways. (AU) | |
| Processo FAPESP: | 18/25952-8 - Catálise e Termoestabilidade em Enzimas: Efeitos da Oligomerização, Redes Estruturais e Dinâmica |
| Beneficiário: | Sandro Roberto Marana |
| Modalidade de apoio: | Auxílio à Pesquisa - Regular |
| Processo FAPESP: | 18/18537-4 - Relação da estrutura quaternária e atividade enzimática da beta-glicosidase de Spodoptera frugiperda (Sfbgly) |
| Beneficiário: | Felipe Akihiro Melo Otsuka |
| Modalidade de apoio: | Bolsas no Brasil - Doutorado Direto |
| Processo FAPESP: | 16/22365-9 - Redes estruturais e dinâmica em enzimas |
| Beneficiário: | Sandro Roberto Marana |
| Modalidade de apoio: | Auxílio à Pesquisa - Regular |