Biology subjects

Chagas, R. S.

Publications and source records attributed to Chagas, R. S..

3 recordsLinked to original sources

bioRxiv2025

A general model for analysis of linear and hyperbolic enzyme inhibition mechanisms

The mechanisms of reversible inhibitors with a single binding site on enzymes are usually divided into basic groups: linear and hyperbolic, also called partial. Each of them subdivided into types: competitive, non-competitive and mixed. These six mechanisms are often considered separate identities. Here, prompted by the characterization of the inhibition of the wild-type and mutant {beta}-glucosidase Sf{beta}gly by Imidazole and Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol) we developed a unifying enzyme kinetic model that integrates these six basic inhibition mechanism onto a single one. From this model we deduced a general enzyme kinetic equation that through modulation of simple parameters, i.e. the relative inhibitor affinity for two binding sites and the reactivity of the enzyme-substrate-inhibitor complex, is converted into the particular kinetic equation of each of those six inhibition mechanism. In short, we conclude that six fundamental inhibition mechanisms, linear and hyperbolic, are not isolate compartments, but actually facets of the same general model here presented.

biochemistry↗

bioRxiv2025

Impact of changes in buffer ionic concentration and mutations on a GH1 β-Glucosidase homodimer

Oligomerization is a key feature of protein function, with approximately 30% of proteins exhibiting this trait. The homodimeric form of proteins, such as the GH1 {beta}-Glucosidase from Spodoptera frugiperda (Sf{beta}gly), plays a significant role in enzyme activity. In this study, we investigate the homodimerization of Sf{beta}gly, which forms a cyclic C2 dimer with a well-defined interface. Using size exclusion chromatography and SEC-MALS, we characterized the homodimerization behavior of Sf{beta}gly at equilibrium conditions in different ionic concentrations of phosphate buffer. The dissociation constants (KD) increase with decreasing ionic concentration, suggesting that the hydrophobic effect is central to homodimer formation. Site-directed mutagenesis of key residues at the dimer interface further elucidated the contributions of specific amino acid residues to dimer stability. Mutations affecting both, apolar and hydrogen bond-forming residues, significantly increased the KD. However, mutations of hydrogen bond-forming residues caused a smaller KD change than apolar residue mutations, suggesting that while the latter is the driving factor in the dimerization, the former may play a crucial role in guiding the monomers relative orientation. These findings enhance our understanding of protein oligomerization in GH1 {beta}-Glucosidases and its implications for protein design and function.

biochemistry↗

bioRxiv2024

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

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.

biochemistry↗