Fig 1.
Effect of Tris on the Sfβgly activity and stability.
(A) Initial rate of 10 mM NPβglc hydrolysis catalyzed by Sfβgly in the absence (○; 13 μM.min − 1) and presence of 40 mM Tris (●; 7.9 μM.min − 1). (B) Initial rate of 10 mM NPβglc hydrolysis catalyzed by Sfβgly previously incubated with 300 mM Tris at 30 °C for 18 h (●; 15 μM.min − 1) and without Tris (○; 13 μMmin − 1). Both experiments, shown in A and B, were performed with 0.09 μM Sfβgly. Data are mean and standard deviation of three determinations of the product formed in each incubation time using three separate assays with the same enzyme sample. The substrate was prepared in 100 mM phosphate buffer pH 6.0. Activity assays were performed at 30 °C. (C) Tests aiming at the detection of the transglycosylation reaction catalyzed by Sfβgly (0.009 μM). Production of p-nitrophenolate (○) and glucose (*) from 20 mM NPβglc in the presence of 30 mM Tris. The substrate was prepared in 100 mM phosphate buffer pH 6.0. Activity assays were done at 30 °C. Data are mean and standard deviation of three determinations of the product formed in each incubation time using the same enzyme sample. (D) Standard curve of p-nitrophenolate with (○) and without (○) 120 mM Tris. Slopes are 4420 and 4721 Abs415nm. μM-1, respectively. (E) Standard curve of glucose with (○) and without (○) 120 mM Tris. Slopes are 1007 and 935 Abs415nm. μM-1, respectively. R2 are higher than 0.99.
Fig 2.
Characterization of the Tris inhibition mechanism upon Sfβgly.
(A) Lineweaver-Burk plots showing the effect of different Tris concentrations (●, 0; ■, 20 mM; ○, 40 mM; ◊, 80 mM; x, 120 mM) on the initial rate of hydrolysis of the substrate NPβglc. (B) Tris effect on the apparent Ks/k3 (calculated from the line slope). (C) Tris effect on the apparent 1/ k3 (calculated from the line intercept). NPβglc and Tris were prepared in 100 mM phosphate buffer pH 6.0. Rates were determined at 30°C. Rates are the mean of three product determinations using the same enzyme sample. Experiment performed with enzyme sample #1. Independent experiments were performed using two different enzyme samples (S1 and S2 Figs). (D) Linear mixed-type inhibition mechanism (intersecting, linear, noncompetitive). S, substrate NPβglc; I, inhibitor Tris; E, enzyme Sfβgly; P, product; Ks, dissociation constant for the ES complex; Ki, dissociation constant for the EI complex; k3, rate constant for product formation; α, factor that represents the mutual hindering effect between S and I (α > 1) [24].
Fig 3.
Characterization of the Tris inhibition mechanism upon hydrolysis of the substrate C2 catalyzed by Sfβgly.
(A) Lineweaver-Burk plots showing the effect of different Tris concentrations ●, 0; ■, 20 mM; ○, 40 mM; ◊, 80 mM; x, 120 mM) on the initial rate of C2 hydrolysis. (B) Tris effect on the apparent Ks/k3 (calculated from the line slope). (C) Tris effect on the apparent 1/ k3 (calculated from the line intercept). C2 and Tris were prepared in 100 mM phosphate buffer pH 6.0. Rates were determined at 30°C. Rates are the mean of three product determinations using the same enzyme sample. Two independent experiments were performed using two different enzyme samples (S4 Fig).
Table 1.
Parameters of the Tris inhibition mechanism upon the β-glucosidase Sfβgly.
Fig 4.
β-glucosidase Sfβgly and the Sfβgly-NPβglc complex. (A) Binding spots in the free enzyme revealed by computational docking (solutions #1 to #7 – S1 Table; S6 Fig). Residues interacting with Tris are shown in blue. (B) Binding spots in the enzyme-substrate complex revealed by computational docking (S2 Table; S7 Fig) Residues showed in the detailed structures (panels A and B) are within 3.5 Å of the Tris molecule.