Tris inhibits a GH1 β-glucosidase by a linear mixed inhibition mechanism

Autor(es)

Chagas, Rafael S. ; Marana, Sandro R.

• Tipo de documento: Artigo Científico
• Fonte: PLoS One; v. 20, n. 3, p. 11-pg., 2025-03-25.

Resumo

Here we demonstrate that Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol), largely used as a buffering agent, is a linear mixed inhibitor (Ki = 12 +/- 2 mM and alpha = 3 +/- 1) of the GH1 beta-glucosidase from the insect Spodoptera frugiperda (Sf beta gly). Such an inhibition mechanism implies the formation of a non-productive ESI complex involving Sf beta gly, substrate, and Tris. In addition, Tris binding reduces by 3 fold the enzyme affinity for the substrate. Hence, at concentrations higher than the Ki, Tris can completely abolish Sf beta gly activity, whereas even at lower concentrations the presence of Tris causes underestimation of beta-glucosidase kinetic parameters (Km and kcat). In agreement with the inhibition mechanism, computational docking showed that Tris could bind to a pocket placed at the lateral of the active site opening in the Sf beta gly-substrate complex, hence leading to the formation of an ESI complex. In agreement with the crystallographic data available, computational docking also showed that Tris may find binding spots in the interior of the active site of the Sf beta gly and several GH1 beta-glucosidases. Moreover, the variety of their active site shapes results in a multiplicity of binding profiles, foreseeing different inhibition mechanisms. Thus, Tris inhibition may affect other GH1 beta-glucosidases. This remark should be taken into account in their study, highlighting the importance of the appropriate buffer for accurate enzyme characterization. (AU)

• Processo FAPESP: 21/10577-0 - Centro de Pesquisa em Biologia de Bactérias e Bacteriófagos (CEPID B3)
• Beneficiário: Shaker Chuck Farah
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 21/03967-6 - Caracterização da interface dimérica de uma beta-glicosidase
• Beneficiário: Sandro Roberto Marana
• Modalidade de apoio: Auxílio à Pesquisa - Regular