Figure 1.
Variation of free enzyme concentration over 4 orders of magnitude reveals multi-mode binding of TrCel7A to BC.
[Free] – [Bound] plots (A – C) and Scatchard plots (D – F) of binding of TrCel7A to BC (1 g/L). Full binding isotherm is dissected into three regions with different dominating binding modes. The low affinity binding mode dominates at free TrCel7A concentrations ([TrCel7A]Free) above 1.0 µM (A and D). The medium affinity binding mode dominates in the 0.1 µM – 1.0 µM range of [TrCel7A]Free (B and E). The high affinity binding mode dominates at [TrCel7A]Free up to 10 nM (C and F). Total bound TrCel7A (TrCel7ABound), TrCel7A bound through the active site (TrCel7ABound-OA), and bound TrCel7A with free active site (TrCel7ABound-FA). Solid lines represent best fits of Langmuiŕs one (C), two (B), and three (A) independent binding site model. Error bars are at least from three independent measurements.
Table 1.
Binding isotherm parameters for different binding modes of active site mediated and total binding of cellobiohydrolase TrCel7A to bacterial cellulose.
Figure 2.
Proposed productive and non-productive interactions between TrCel7A and BC.
(A) The high affinity binding mode corresponds to the productive binding to cellulose chain ends on the hydrophobic face of BC microcrofibril through both domains, CBM and CD. (B) The medium affinity binding mode includes non-productive binding to the hydrophobic face, where enzyme is attached through CBM only. Latter can become productive binding upon disengagement of cellulose chain into the active site by endo-mode attack. (C) The low affinity binding mode may correspond to the active site mediated binding to cellulose chain ends on the hydrophilic face. The hydrophobic face of cellulose microfibril is shown in dark gray and the cellulose chain ends available for binding through CD are depicted as protruding lines. The Amax and Kd values for corresponding binding modes are listed in Table 1.
Figure 3.
Binding of TrCel7A to BC is only partially reversible.
(A and B) Dilution experiment. BC at 1.0 g/L was incubated with 1.0 µM TrCel7A to establish equilibrium. Equilibrium was disturbed by the addition of buffer to bring up tenfold dilution and relaxation to new equilibrium was followed. (A) Binding reversibility was assessed in the basis of both, total bound TrCel7A, TrCel7ABound and active site bound TrCel7A, TrCel7ABound-OA. Dotted lines show the progression from the initial equilibrium to the disturbed equilibrium to the new equilibrium. (B) Change in the concentration of TrCel7A free from cellulose, TrCel7AFree and TrCel7A with free active site TrCel7AFA in time after disturbance of equilibrium by dilution. (C) Free enzyme depletion experiment. BC at 0.1 g/L was incubated with TrCel7A at different concentrations to establish equilibrium. Cellulose with bound enzyme was pelleted by centrifugation, 90% of the supernatant was withdrawn and pellet was resuspended in the same amount of buffer to disturb the equilibrium. The position of the new equilibrium was measured after 30 min from the disturbance. Binding reversibility was assessed in the basis of total bound TrCel7A. Dotted lines show the progression from the initial equilibrium to the disturbed equilibrium to the new equilibrium.
Figure 4.
Binding isotherm of TrCel7A depends on BC concentration.
Binding isotherms (A), and corresponding Scatchard plots (B) of binding of TrCel7A (in the level of total bound TrCel7A) to BC at different concentrations. Solid lines represent best fits of Langmuiŕs one binding site model. Error bars are at least from three independent measurements. (C) Amax/Kd values at different BC concentrations.