Interfacial enzyme reactions are ubiquitous both in vivo and in technical applications, but analysis of their kinetics remains controversial. In particular, it is unclear whether conventional Michaelis–Menten theory, which requires a large excess of substrate, can be applied. Here, an extensive experimental study of the enzymatic hydrolysis of insoluble cellulose indeed showed that the conventional approach had a limited applicability. Instead we argue that, unlike bulk reactions, interfacial enzyme catalysis may reach a steady-state condition in the opposite experimental limit, where the concentration of enzyme far exceeded the molar concentration of accessible surface sites. Under this condition, an “inverse Michaelis–Menten equation”, where the roles of enzyme and substrate had been swapped, proved to be readily applicable. We suggest that this inverted approach provides a general tool for kinetic analyses of interfacial enzyme reactions and that its analogy to established theory provides a bridge to the accumulated understanding of steady-state enzyme kinetics. Finally, we show that the ratio of parameters from conventional and inverted Michaelis–Menten analysis reveals the density of enzyme attack sites on the substrate surface as probed by one specific enzyme. This density, which is an analogue to a molar substrate concentration for interfacial reactions, was shown to vary strongly even among related enzymes. This difference reflects how the enzyme discriminates between local differences in surface structure on the substrate.

The Sabatier principle states that optimal catalysis occurs when interactions between catalyst and substrate are of intermediary strength. Although qualitative in nature, this concept has proven extremely useful within (nonbiochemical) heterogeneous catalysis. In the current work, we show that the principle may be applied to an interfacial enzyme reaction. Specifically, we studied the breakdown of cellulose by different cellulases (wild types and variants) and found that the results could be rationalized in so-called volcano plots that are emblematic of the principle. This implies that the rate of the complex enzymatic reaction can be described by a single parameter (binding strength), and we show how this may help elucidating e.g. rate-controlling steps and relationships of substrate load and enzymatic efficacy. On a more general level, we propose that the Sabatier principle may be widely applicable to interfacial enzyme processes and hence open an avenue to the application within biocatalysis of some of the principles and practices originally developed for heterogeneous catalysis.

Background:To elucidate the rate-determining steps of cellobiohydrolase Cel7A from T. reesei, variants with lower substrate affinity were designed. Results: Mutant (W38A) had reduced substrate affinity but a 2-fold increase in the maximum quasi-steady-state rate. Conclusion: Dissociation of stalled TrCel7A is the rate-limiting step in the initial phase of hydrolysis. Significance: This work offers a new perspective for the design of faster cellulases.

Interfacial enzyme reactions are common in Nature and in industrial settings, including the enzymatic deconstruction of poly(ethylene terephthalate) (PET) waste. Kinetic descriptions of PET hydrolases are necessary for both comparative analyses, discussions of structure–function relations and rational optimization of technical processes. We investigated whether the Sabatier principle could be used for this purpose. Specifically, we compared the kinetics of two well-known PET hydrolases, leaf-branch compost cutinase (LCC) and a cutinase from the bacterium Thermobifida fusca (TfC), when adding different concentrations of the surfactant cetyltrimethylammonium bromide (CTAB). We found that CTAB consistently lowered the strength of enzyme–PET interactions, while its effect on enzymatic turnover was strongly biphasic. Thus, at gradually increasing CTAB concentrations, turnover was initially promoted and subsequently suppressed. This correlation with maximal turnover at an intermediate binding strength was in accordance with the Sabatier principle. One consequence of these results was that both enzymes had too strong intrinsic interaction with PET for optimal turnover, especially TfC, which showed a 20-fold improvement of k cat at the maximum. LCC on the other hand had an intrinsic substrate affinity closer to the Sabatier optimum, and the turnover rate was 5-fold improved at weakened substrate binding. Our results showed that the Sabatier principle may indeed rationalize enzymatic PET degradation and support process optimization. Finally, we suggest that future discovery efforts should consider enzymes with weakened substrate binding because strong adsorption seems to limit their catalytic performance.

Background: Substrate interactions in the long tunnel of processive cellulases govern both their catalytic activity and stepwise movement along a cellulose strand. Results: The energetics of enzyme-substrate interactions at different depths of the tunnel are reported. Conclusion:The affinity for the substrate varies strongly through the tunnel. Significance: Quantitative information on interactions is required to understand the complex processive mechanism.