ITC analysis of polydisperse systems: Unravelling the impact of sample heterogeneity.

Binding interactions often involve heterogeneous samples displaying a distribution of binding sites that vary in affinity and binding enthalpy. Examples include biological samples like proteins and chemically produced samples like modified cyclodextrins. Experimental studies often ignore sample heterogeneity and treat the system as an interaction of two homogeneous species, i.e. a chemically well-defined ligand binding to one type of site. The present study explores, by simulations and experiments, the impact of heterogeneity in isothermal titration calorimetry (ITC) setups where one of the binding components is heterogeneous. It is found that the standard single-site model, based on the assumption of two homogeneous binding components, provides excellent fits to simulated ITC data when the binding free energy is normally distributed and all sites have similar binding enthalpies. In such cases, heterogeneity can easily go undetected but leads to underestimated binding constants. Heterogeneity in the binding enthalpy is a bigger problem and may result in enthalpograms of increased complexity that are likely to be misinterpreted as two-site binding or other complex binding models. Finally, it is shown that heterogeneity can account for previously observed experimental anomalies. All simulations are accessible in Google Colab for readers to experiment with the simulation parameters.

A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases.

Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei (TrCel7A), T. terrestris (TtCel7A), and Myceliophthora heterothallica (MhCel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; TrCel7A was severely impeded, TtCel7A was moderately impeded, while MhCel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate complexation. The extent of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails.

Substrate binding in the processive cellulase Cel7A: Transition state of complexation and roles of conserved tryptophan residues.

Cellobiohydrolases effectively degrade cellulose and are of biotechnological interest because they can convert lignocellulosic biomass to fermentable sugars. Here, we implemented a fluorescence-based method for real-time measurements of complexation and decomplexation of the processive cellulase Cel7A and its insoluble substrate, cellulose. The method enabled detailed kinetic and thermodynamic analyses of ligand binding in a heterogeneous system. We studied WT Cel7A and several variants in which one or two of four highly conserved Trp residues in the binding tunnel had been replaced with Ala. WT Cel7A had on/off-rate constants of 1 × 105 m-1 s-1 and 5 × 10-3 s-1, respectively, reflecting the slow dynamics of a solid, polymeric ligand. Especially the off-rate constant was many orders of magnitude lower than typical values for small, soluble ligands. Binding rate and strength both were typically lower for the Trp variants, but effects of the substitutions were moderate and sometimes negligible. Hence, we propose that lowering the activation barrier for complexation is not a major driving force for the high conservation of the Trp residues. Using so-called Φ-factor analysis, we analyzed the kinetic and thermodynamic results for the variants. The results of this analysis suggested a transition state for complexation and decomplexation in which the reducing end of the ligand is close to the tunnel entrance (near Trp-40), whereas the rest of the binding tunnel is empty. We propose that this structure defines the highest free-energy barrier of the overall catalytic cycle and hence governs the turnover rate of this industrially important enzyme.

Molecular recognition in the product site of cellobiohydrolase Cel7A regulates processive step length.

Cellobiohydrolase Cel7A is an industrial important enzyme that breaks down cellulose by a complex processive mechanism. The enzyme threads the reducing end of a cellulose strand into its tunnel-shaped catalytic domain and progresses along the strand while sequentially releasing the disaccharide cellobiose. While some molecular details of this intricate process have emerged, general structure-function relationships for Cel7A remain poorly elucidated. One interesting aspect is the occurrence of particularly strong ligand interactions in the product binding site. In this work, we analyze these interactions in Cel7A from Trichoderma reesei with special emphasis on the Arg251 and Arg394 residues. We made extensive biochemical characterization of enzymes that were mutated in these two positions and showed that the arginine residues contributed strongly to product binding. Specifically, ∼50% of the total standard free energy of product binding could be ascribed to four hydrogen bonds to Arg251 and Arg394, which had previously been identified in crystal structures. Mutation of either Arg251 or Arg394 lowered production inhibition of Cel7A, but at the same time altered the enzyme product profile and resulted in ∼50% reduction in both processivity and hydrolytic activity. The position of the two arginine residues closely matches the two-fold screw axis symmetry of the substrate, and this energetically favors the productive enzyme-substrate complex. Our results indicate that the strong and specific ligand interactions of Arg251 and Arg394 provide a simple proofreading system that controls the step length during consecutive hydrolysis and minimizes dead time associated with transient, non-productive complexes.

A steady-state approach for inhibition of heterogeneous enzyme reactions.

The kinetic theory of enzymes that modify insoluble substrates is still underdeveloped, despite the prevalence of this type of reaction both in vivo and industrial applications. Here, we present a steady-state kinetic approach to investigate inhibition occurring at the solid-liquid interface. We propose to conduct experiments under enzyme excess (E0 ≫ S0), i.e. the opposite limit compared with the conventional Michaelis-Menten framework. This inverse condition is practical for insoluble substrates and elucidates how the inhibitor reduces enzyme activity through binding to the substrate. We claim that this type of inhibition is common for interfacial enzyme reactions because substrate accessibility is low, and we show that it can be analyzed by experiments and rate equations that are analogous to the conventional approach, except that the roles of enzyme and substrate have been swapped. To illustrate the approach, we investigated the major cellulases from Trichoderma reesei (Cel6A and Cel7A) acting on insoluble cellulose. As model inhibitors, we used catalytically inactive variants of Cel6A and Cel7A. We made so-called inverse Michaelis-Menten curves at different concentrations of inhibitors and found that a new rate equation accounted well for the data. In most cases, we found a mixed type of surface-site inhibition mechanism, and this probably reflected that the inhibitor both competed with the enzyme for the productive binding-sites (competitive inhibition) and hampered the processive movement on the surface (uncompetitive inhibition). These results give new insights into the complex interplay of Cel7A and Cel6A on cellulose and the approach may be applicable to other heterogeneous enzyme reactions.

Systematic deletions in the cellobiohydrolase (CBH) Cel7A from the fungus Trichoderma reesei reveal flexible loops critical for CBH activity.

Glycoside hydrolase family 7 (GH7) cellulases are some of the most efficient degraders of cellulose, making them particularly relevant for industries seeking to produce renewable fuels from lignocellulosic biomass. The secretome of the cellulolytic model fungus Trichoderma reesei contains two GH7s, termed TrCel7A and TrCel7B. Despite having high structural and sequence similarities, the two enzymes are functionally quite different. TrCel7A is an exolytic, processive cellobiohydrolase (CBH), with high activity on crystalline cellulose, whereas TrCel7B is an endoglucanase (EG) with a preference for more amorphous cellulose. At the structural level, these functional differences are usually ascribed to the flexible loops that cover the substrate-binding areas. TrCel7A has an extensive tunnel created by eight peripheral loops, and the absence of four of these loops in TrCel7B makes its catalytic domain a more open cleft. To investigate the structure-function relationships of these loops, here we produced and kinetically characterized several variants in which four loops unique to TrCel7A were individually deleted to resemble the arrangement in the TrCel7B structure. Analysis of a range of kinetic parameters consistently indicated that the B2 loop, covering the substrate-binding subsites -3 and -4 in TrCel7A, was a key determinant for the difference in CBH- or EG-like behavior between TrCel7A and TrCel7B. Conversely, the B3 and B4 loops, located closer to the catalytic site in TrCel7A, were less important for these activities. We surmise that these results could be useful both in further mechanistic investigations and for guiding engineering efforts of this industrially important enzyme family.

pH profiles of cellulases depend on the substrate and architecture of the binding region.

Understanding the pH effect of cellulolytic enzymes is of great technological importance. In this study, we have examined the influence of pH on activity and stability for central cellulases (Cel7A, Cel7B, Cel6A from Trichoderma reesei, and Cel7A from Rasamsonia emersonii). We systematically changed pH from 2 to 7, temperature from 20°C to 70°C, and used both soluble (4-nitrophenyl ß- d-lactopyranoside [pNPL]) and insoluble (Avicel) substrates at different concentrations. Collective interpretation of these data provided new insights. An unusual tolerance to acidic conditions was observed for both investigated Cel7As, but only on real insoluble cellulose. In contrast, pH profiles on pNPL were bell-shaped with a strong loss of activity both above and below the optimal pH for all four enzymes. On a practical level, these observations call for the caution of the common practice of using soluble substrates for the general characterization of pH effects on cellulase activity. Kinetic modeling of the experimental data suggested that the nucleophile of Cel7A experiences a strong downward shift in pKa upon complexation with an insoluble substrate. This shift was less pronounced for Cel7B, Cel6A, and for Cel7A acting on the soluble substrate, and we hypothesize that these differences are related to the accessibility of water to the binding region of the Michaelis complex.

A practical approach to steady-state kinetic analysis of cellulases acting on their natural insoluble substrate.

Measurement of steady-state rates (vSS) is straightforward in standard enzymology with soluble substrate, and it has been instrumental for comparative biochemical analyses within this area. For insoluble substrate, however, experimental values of vss remain controversial, and this has strongly limited the amount and quality of comparative analyses for cellulases and other enzymes that act on the surface of an insoluble substrate. In the current work, we have measured progress curves over a wide range of conditions for two cellulases, TrCel6A and TrCel7A from Trichoderma reesei, acting on their natural, insoluble substrate, cellulose. Based on this, we consider practical compromises for the determination of experimental vSS values, and propose a basic protocol that provides representative reaction rates and is experimentally simple so that larger groups of enzymes and conditions can be readily assayed with standard laboratory equipment. We surmise that the suggested experimental approach can be useful in comparative biochemical studies of cellulases; an area that remains poorly developed.

Thermoactivation of a cellobiohydrolase.

We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a "high affinity" variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two-fold faster around 70 °C. This better thermoactivation of the high-affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability.

Anomeric Selectivity and Product Profile of a Processive Cellulase.

Cellobiohydrolases (CBHs) make up an important group of enzymes for both natural carbon cycling and industrial deconstruction of lignocellulosic biomass. The consecutive hydrolysis of one cellulose strand relies on an intricate pattern of enzyme-substrate interactions in the long, tunnel-shaped binding site of the CBH. In this work, we have investigated the initial complexation mode with cellulose of the most thoroughly studied CBH, Cel7A from Hypocrea jecorina (HjCel7A). We found that HjCel7A predominantly produces glucose when it initiates a processive run on insoluble microcrystalline cellulose, confirming the validity of an even and odd product ratio as an estimate of processivity. Moreover, the glucose released from cellulose was predominantly α-glucose. A link between the initial binding mode of the enzyme and the reducing end configuration was investigated by inhibition studies with the two anomers of cellobiose. A clear preference for ß-cellobiose in product binding site +2 was observed for HjCel7A, but not the homologous endoglucanase, HjCe7B. Possible relationships between this anomeric preference in the product site and the prevalence of odd-numbered initial-cut products are discussed, and a correlation between processivity and anomer selectivity is proposed.

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase.

Direct kinetic comparison of the two cellobiohydrolases Cel6A and Cel7A from Hypocrea jecorina.

Cellulose degrading fungi such as Hypocrea jecorina secrete several cellulases including the two cellobiohydrolases (CBHs) Cel6A and Cel7A. The two CBHs differ in catalytic mechanism, attack different ends, belong to different families, but are both processive multi-domain enzymes that are essential in the hydrolysis of cellulose. Here we present a direct kinetic comparison of these two enzymes acting on insoluble cellulose. We used both continuous- and end-point assays under either enzyme- or substrate excess, and found distinct kinetic differences between the two CBHs. Cel6A was catalytically superior with a maximal rate over four times higher than Cel7A. Conversely, the ability of Cel6A to attack diverse structures on the cellulose surface was inferior to Cel7A. This latter difference was pronounced as the density of attack sites for Cel7A was almost an order of magnitude higher compared to Cel6A. We conclude that Cel6A is a fast but selective enzyme and that Cel7A is slower, but promiscuous. One consequence of this is that Cel6A is more effective when substrate is plentiful, while Cel7A excels when substrate is limiting. These diverse kinetic properties of Cel6A and Cel7A might elucidate why both cellobiohydrolases are prominent in cellulolytic degrading fungi.

Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose.

Cel7A cellobiohydrolases perform processive hydrolysis on one strand of cellulose, which is threaded through the enzyme's substrate binding tunnel. The tunnel structure results from a groove in the catalytic domain, which is covered by a number of loops. These loops have been identified as potential targets for engineering of this industrially important enzyme family, but only few systematic studies on this have been made. Here we show that two asparagine residues (N194 and N197) positioned in the loop covering the glucopyranose subsite -4 (recently denoted B2 loop) of the thermostable Cel7A from Rasamsonia emersonii had profound effects on both substrate interactions and catalytic efficacy. At room temperature the double mutant N194A/N197A showed strongly reduced substrate affinity with a water-cellulose partitioning coefficient threefold lower than the wild type. Yet, this variant was catalytically efficient with a maximal turnover about twice as high as the wild type. Analogous but smaller changes were found for the single mutants. Analysis of these changes in affinity and kinetics as a function of temperature, led to the conclusion that replacement of N194 and particularly N197 with alanine leads to faster enzyme-substrate dissociation. Conversely, these residues appeared to have little or no effect on the rate of association. We suggest that the controlled adjustment of the enzyme-substrate dissociation prompts faster cellulolytic enzymes. Biotechnol. Bioeng. 2017;114: 53-62. © 2016 Wiley Periodicals, Inc.

Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 103 -104 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc.

Probing substrate interactions in the active tunnel of a catalytically deficient cellobiohydrolase (Cel7).

Cellobiohydrolases break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has ∼10 glucopyranose subsites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with a DP of 2-3 the binding constants were around 10(5) m(-1), and for longer ligands (DP 5-8) this value was ∼10(7) m(-1). Within each of these groups we did not find increased affinity as the ligands got longer and potentially filled more subsites. On the contrary, we found a small but consistent affinity loss as DP rose from 6 to 8, particularly at the higher investigated temperatures. Other thermodynamic functions (ΔH, ΔS, and ΔCp) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel.

Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose.

The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have implemented an electrochemical biosensor assay for product inhibition studies of cellulases acting on their natural substrate, cellulose. Using this method we measured the hydrolytic rate of Cel7A as a function of both product (inhibitor) concentration and substrate load. This data enabled analyses along the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing rate of the reverse reaction, lowering the net hydrolytic velocity as product concentrations increase. Strictly this is not a case of inhibition, as no catalytically inactive is formed. The other mechanism that matched the kinetic data was noncompetitive inhibition with an inhibition constant of 490 ± 40 µM. Noncompetitive inhibition implies that the inhibitor binds with comparable strength to either free enzyme or an enzymesubstrate complex, that is, that association between enzyme and substrate has no effect on the binding of the inhibitor. This mechanism is rarely observed, but we argue, that the special architecture of Cel7A with numerous subsites for binding of both substrate and product could give rise to a true noncompetitive inhibition mechanism. Biotechnol. Bioeng. 2016;113: 1178-1186. © 2015 Wiley Periodicals, Inc.

Kinetics of cellobiohydrolase (Cel7A) variants with lowered substrate affinity.

Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass. Identification of the rate-limiting step for cellobiohydrolases remains controversial, and recent reports have alternately suggested either association (on-rate) or dissociation (off-rate) as the overall bottleneck. Obviously, this uncertainty hampers both fundamental mechanistic understanding and rational design of enzymes with improved industrial applicability. To elucidate the role of on- and off-rates, respectively, on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp-38) in the middle of the active tunnel has been replaced with an alanine. This mutation weakens complex formation, and the population of substrate-bound W38A was only about half of the wild type. Nevertheless, the maximal, steady-state rate was twice as high for the variant enzyme. It is argued that these opposite effects on binding and activity can be reconciled if the rate-limiting step is after the catalysis (i.e. in the dissociation process).

Sabatier Principle for Rationalizing Enzymatic Hydrolysis of a Synthetic Polyester.

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.

The Sabatier principle as a tool for discovery and engineering of industrial enzymes.

The recent breakthrough in all-atom, protein structure prediction opens new avenues for a range of computational approaches in enzyme design. These new approaches could become instrumental for the development of technical biocatalysts, and hence our transition toward more sustainable industries. Here, we discuss one approach, which is well-known within inorganic catalysis, but essentially unexploited in biotechnology. Specifically, we review examples of linear free-energy relationships (LFERs) for enzyme reactions and discuss how LFERs and the associated Sabatier Principle may be implemented in algorithms that estimate kinetic parameters and enzyme performance based on model structures.

Computing Cellulase Kinetics with a Two-Domain Linear Interaction Energy Approach.

While heterogeneous enzyme reactions play an essential role in both nature and green industries, computational predictions of their catalytic properties remain scarce. Recent experimental work demonstrated the applicability of the Sabatier principle for heterogeneous biocatalysis. This provides a simple relationship between binding strength and the catalytic rate and potentially opens a new way for inexpensive computational determination of kinetic parameters. However, broader implementation of this approach will require fast and reliable prediction of binding free energies of complex two-phase systems, and computational procedures for this are still elusive. Here, we propose a new framework for the assessment of the binding strengths of multidomain proteins, in general, and interfacial enzymes, in particular, based on an extended linear interaction energy (LIE) method. This two-domain LIE (2D-LIE) approach was successfully applied to predict binding and activation free energies of a diverse set of cellulases and resulted in robust models with high accuracy. Overall, our method provides a fast computational screening tool for cellulases that have not been experimentally characterized, and we posit that it may also be applicable to other heterogeneously acting biocatalysts.