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

Abstract

Here we demonstrate that Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol), largely used as buffering agent, is a linear mixed inhibitor (Ki = 12 {+/-} 2 mM and = 3 {+/-} 1) of the GH1 {beta}-glucosidase from the insect Spodoptera frugiperda (Sf{beta}gly). Such inhibition mechanism implies in 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 to 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 a ESI complex. 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 mechanism. Thus, Tris inhibition is probably common among GH1 {beta}-glucosidases. This remark should be taken into account in their study, highlighting the importance of the appropriate buffer for accurate enzyme characterization.