Kinetic Modeling at Single-Molecule Resolution Elucidates the Mechanisms of Cellulase Synergy

Abstract

The mechanism of synergistic cooperation between celluloses in decomposing cellulose is revealed by resolving the molecular structures of enzymes and substrates via kinetic modeling. The emergence of endo-exo and exo-exo synergy through enzyme-enzyme and enzyme-substrate couplings was investigated with the main exoglucanases (TrCel7A and TrCel6A) and endoglucanase (TrCel7B) of the Trichoderma reesei fungus. The degree of synergy was found to depend on the interplay between two competing effects: (1) enhancement of the complexation rates of exoglucanases with the chain ends created by endoglucanases and (2) surface inhibition of processive exoglucanases by uneven layers of glucan chains on cellulose. We show that the sole effect of TrCel7B in creating more free ends for TrCel7A is insufficient to cause synergistic activity. The combined actions of TrCel7A and TrCel7B produce a rougher substrate surface that subsequently promotes blocking of processing TrCel7A enzymes. This anti-synergy can be counteracted by enhancing the rates of complexation of TrCel7A with the TrCel7B-created chain ends, allowing their synergistic cooperation. Similarly, kinetic simulations show that exo-exo synergy does not occur if TrCel6A and TrCel7A have only opposite specificities in targeting the two ends of glucan chains. Incorporating endo activity into TrCel6A with complexation rate enhancement, however, can lead to synergy with TrCel7A. Therefore, we find that endo-exo and exo-exo synergies may share the same mechanistic origin. The results of work also highlight that resolving molecular configurations in kinetic modeling allows systematic analysis for elucidating the mechanism of interfacial biocatalysis.