When substrate inhibits and inhibitor activates: implications of β-glucosidases

Abstract

Backgroundβ-glucosidases (BGs) catalyze the hydrolysis of β-glycosidic bonds in glucose derivatives. They constitute an important group of enzymes with biotechnological interest like supporting cellulases in degradation of lignocellulose to fermentable sugars. In the latter context, the glucose tolerant BGs are of particular interest. These BGs often show peculiar kinetics, including inhibitory effects of substrates and activating effects of inhibitors, such as glucose or xylose. The mechanisms behind the activating/inhibiting effects are poorly understood. The nonproductive binding of substrate is expected in cases where enzymes with multiple consecutive binding subsites are studied on substrates with a low degree of polymerization. The effects of inhibitors to BGs exerting nonproductive binding of substrate have not been discussed in the literature before.ResultsHere, we performed analyses of different reaction schemes using the catalysis by retaining BGs as a model. We found that simple competition of inhibitor with nonproductive binding of substrate can account for the activation of enzyme by inhibitor without involving any allosteric effects. The transglycosylation to inhibitor was also able to explain the activating effect of inhibitor. For both mechanisms, the activation was caused by the increase of k cat with increasing inhibitor concentration, while k cat/K m always decreased. Therefore, the activation by inhibitor was more pronounced at high substrate concentrations. The possible contribution of the two mechanisms in the activation by inhibitor was dependent on the rate-limiting step of glycosidic bond hydrolysis as well as on whether and which glucose-unit-binding subsites are interacting.ConclusionKnowledge on the mechanisms of the activating/inhibiting effects of inhibitors helps the rational engineering and selection of BGs for biotechnological applications. Provided that the catalysis is consistent with the reaction schemes addressed here and underlying assumptions, the mechanism of activation by inhibitor reported here is applicable for all enzymes exerting nonproductive binding of substrate.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0690-z) contains supplementary material, which is available to authorized users.

Cellulases and xylanases production by endophytic fungi by solid state fermentation using lignocellulosic substrates and enzymatic saccharification of pretreated sugarcane bagasse

“…4a we can observe that when glucose concentration is increased, β-glucosidases activities decreased, indicating that enzymes are inhibited by the product. In the enzymatic hydrolysis of lignocellulose biomass to fermentable sugars, an inevitable consequence is the accumulation of hydrolysis products at high concentrations, which may result in cellulases inhibition (Kuusk and Väljamäe, 2017), as observed in this study for β-glucosidases from the two selected fungi. According to (Guimarães et al, 2006), glucose inhibition is commonly cited in studies regarding microbial β-glucosidases.…”

Engineering the GH1 β-glucosidase from Humicola insolens: Insights on the stimulation of activity by glucose and xylose

“…The topology of the active site has also been associated with the tolerance of some enzymes to high concentration of glucose [ 28 , 29 , 35 ]. Furthermore, a recent theoretical study raised the possibility that competition of inhibitors with the non-productive binding of substrate may be a possible mechanism of glucose/xylose stimulation of β-glucosidases that have more than one glycosyl residue binding subsite in the aglycone binding site [ 36 ]. It has been recently proposed that the relative binding affinity/preference by sites at the entrance and middle of the substrate channel regulate the effects of glucose [ 37 ].…”

Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

“…The B8CYA8 crystal structure in the presence of glucose and STD-NMR studies, however, show the interaction with glucose near the bottom of the active site pocket. Glucose inhibition has been attributed to binding to other allosteric sites, and by non-productive binding to other sites in the active site pocket [11, 45] However cooperativity in B8CYA8 could not be established by measurement of hill coefficient (Supplementary file, Table S6) which were around one in the wild-type and the mutants. The binding of glucose to secondary binding site(s) is, however, yet to be experimentally proven.…”

“…Previously it was reported that the addition of small amounts of glucose could reduce the non-productive binding of substrate to +1 and +2 subsites and stimulate enzyme activity [45]. A comparison with PDB structure 3F5K and 2O9P indicates the presence of the +2 subsite in B8CYA8 which can potentially assist in the non-productive binding of the substrate.…”