MOESM1 of When substrate inhibits and inhibitor activates: implications of β-glucosidases

Additional file 1: Figure S1. Effect of inhibitor to an enzyme exerting the nonproductive binding of substrate. Figure S2. Effects of inhibitor to enzymes exerting the nonproductive binding of substrate and transglycosylation to inhibitor-formation of the second product. Figure S3. Effects of inhibitor to enzymes exerting the nonproductive binding of substrate and transglycosylation to inhibitor-formation of the transglycosylation product. Figure S4. Effects of inhibitor to enzymes exerting the transglycosylation to inhibitor that binds to the “transglycosylation-binding-site”

Michaelis-Menten kinetic parameter values on different chitin substrates.

<p>Michaelis-Menten kinetic parameter values on different chitin substrates.</p

Endo-probability and processivities measured on α-chitin.

<p>Endo-probability and processivities measured on α-chitin.</p

Progress curves of AA-CNW hydrolysis.

<p>(A) AA-CNWs (1 mg/mL) were hydrolysed with HCHT50, HCHT39, <i>Sm</i>ChiA, <i>Sm</i>ChiB or <i>Sm</i>ChiC at 37°C. The release of AA-labelled sugars and total soluble reducing ends were measured at defined time points (5, 10, 20, 40 and 60 min). Error bars show standard deviations and are from three independent experiments. (B) Progress curves at different concentrations of HCHT39.</p

Michaelis-Menten kinetics of HCHTs.

<p>α-chitin, amorphous chitin or CNWs were hydrolyzed with HCHT50 or HCHT39 at 37°C for 1 min. The solid lines represent the best fit according to the Michaelis-Menten equation. Error bars show standard deviations and are from three independent experiments.</p

NAG₂ inhibition of HCHT.

<p>(A) Activity of HCHT on MU-NAG₂ substrate as a function of substrate concentration. (B) NAG₂ inhibition of HCHT on MU-NAG₂ substrate measured at 3 different substrate concentrations—0.5 μM, 5 μM or 50 μM. (C) NAG₂ inhibition of HCHT on ¹⁴C-CNW substrate (1.0 g/L). <i>v</i>_i and <i>v</i>_{i = 0} stand for the rates measured in the presence and absence of inhibitor, respectively. Error bars show standard deviations and are from three independent experiments.</p

SDS-PAGE analysis of purified chitinases.

<p>4–6 μg purified HCHT 50 kDa and 39 kDa isoforms and <i>S</i>. <i>marcescens</i> chitinases <i>Sm</i>ChiA, <i>Sm</i>ChiB and <i>Sm</i>ChiC were loaded and the gel was stained with Coomassie Brilliant Blue G-250.</p

Processivity and probability of endo initiation of HCHTs.

<p>AA-α-chitin (1 mg/mL) or reduced α-chitin (1 mg/mL) were hydrolyzed with HCHT50, HCHT39 (10 nM) or <i>Sm</i>ChiC (1 nM) at 37°C. (A) The release of soluble reducing groups (SRGs). Shown are the combined results with AA-α-chitin and reduced α-chitin. Error bars are from six independent experiments, three made with AA-α-chitin and three with reduced α-chitin as substrate. (B) The release of AA-labelled sugars from AA-α-chitin and the formation of insoluble reducing groups (IRG) on reduced α-chitin under otherwise identical conditions. (C) Data of the hydrolysis of reduced α-chitin from panels (A) and (B) plotted in the coordinates of total reducing groups (RG_tot = IRG + SRG) <i>versus</i> IRG. The solid lines represent the best fit of linear regression (only the data points shown within the solid lines were included in linear regression analysis). The slope of the solid line from linear regression equals to apparent processivity, P^app. (D) Discrimination between different populations of HCHT bound to α-chitin. The total concentration of HCHT was 10 nM and that of α-chitin was 1 mg/mL. The concentration of total bound HCHT ([HCHT]_bound) was found as a difference between the total concentration of the enzyme and the concentration of the enzyme free in solution. The concentration of HCHT with free active site was measured by following the MU-NAG₂ hydrolyzing activity of HCHT in the presence of α-chitin. The concentration of bound HCHT with active site occupied by chitin ([HCHT] _{bound OA}) was found as a difference between the total concentration of the enzyme and that with free active site. The concentration of bound HCHT with free active site ([HCHT] _{bound FA}) was found as a difference between the [HCHT]_bound and [HCHT] _{bound OA}. Error bars show standard deviations and are from three independent experiments.</p