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Article
Sabatier Principle for Interfacial (Heterogeneous) Enzyme Catalysis
Jeppe Kari, Johan Pelck Olsen, Kenneth Jensen, Silke Flindt Badino,
Kristian Bertel Rømer Mørkeberg Krogh, Kim Borch, and Peter Westh
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b03547 • Publication Date (Web): 12 Nov 2018
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4 Sabatier Principle for Interfacial (Heterogeneous) Enzyme
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Catalysis
7 Jeppe Kari1, Johan P. Olsen2, Kenneth Jensen2, Silke F. Badino1, Kristian B.R.M Krogh2, Kim Borch2 and Peter
8 Westh1*
9 1Research Unit for Functional Biomaterials, Roskilde University, 1 Universitetsvej, DK-4000, Denmark.
10 2Novozymes A/S, Krogshøjvej 36, DK-2880, Bagsværd, Denmark.
11 *corresponding author. Email pwesth@ruc.dk
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14 ABSTRACT: The Sabatier Principle states that optimal catalysis occurs when interactions between
15 catalyst and substrate are of intermediary strength. Although qualitative in nature, this concept has
16 proven extremely useful within (non-biochemical) heterogeneous catalysis. In the current work, we
17 show that the principle may be applied to an interfacial enzyme reaction. Specifically, we studied the
18 breakdown of cellulose by different cellulases (wild types and variants) and found that the results
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could be rationalized in so-called volcano plots that are emblematic of the principle. This implies that
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21 the rate of the complex enzymatic reaction can be described by a single parameter (binding strength),
22 and we show how this may help elucidating e.g. rate-controlling steps and relationships of substrate
23 load and enzymatic efficacy. On a more general level, we propose that the Sabatier Principle may be
24 widely applicable to interfacial enzyme processes, and hence open an avenue to the application within
25 biocatalysis of some of the principles and practices originally developed for heterogeneous catalysis.
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28 KEYWORDS: cellulase; cellulose; enzyme kinetics; protein engineering; heterogeneous catalysis;
29 Sabatier principle; volcano curve; Linear free energy relationship
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32 Enzyme kinetics provides the experimental link between structure and function in biocatalysis. Most
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work in the field is at least partially rooted in the seminal model of Michaelis and Menten 1, in which
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35 the first step of the process is the association of two freely diffusible species, enzyme and substrate.
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37 This picture, however, is not representative for a large group of reactions that involve surface
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associated enzyme or insoluble substrate. It has been argued that most enzyme reactions in vivo
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40 actually occur in the heterogeneous environment of an interface 2, and interfacial reactions also
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42 dominate industrial- 3 and bioanalytical 4 applications of enzymes. In light of this, it may be useful to
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classify enzyme reactions with respect to the state of the enzyme and substrate. Indeed, this idea
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45 mirrors the immensely successful distinction within conventional (non-biochemical) catalysis,
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47 between homogeneous (bulk) and heterogeneous (interfacial) processes 5. If we transfer this concept
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to enzyme reactions, there appears to be three situations that are commonly encountered and
50 fundamentally different with respect to the state of enzyme and substrate. These classes of reactions
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52 are exemplified by the cartoons and experimental data in Fig. 1. All three panels show traditional
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54 saturation kinetics, where the reaction rate levels off towards Vmax as the substrate concentration
55 increases. The first case (Fig 1A) describes a standard (homogenous) enzyme reaction, where both
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57 enzyme and substrate are in solution, while the two other cases describe interfacial reactions. This is
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59 either surface-bound enzyme acting on a diffusible substrate as in Fig. 1B, or an insoluble substrate
60 modified by dissolved enzyme (Fig. 1C). It is tempting to suggest that the wide range of principles and

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3 practices developed within (non-biochemical) heterogeneous catalysis could be useful in attempts to
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5 rationalize the kinetics of interfacial enzyme reactions such as those in Figs. 1B and 1C. This link
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7 remains essentially unexploited perhaps because catalyst have traditionally been classified as either
8 homogenous, heterogeneous or biochemical 6-8, without much considerations of connection between
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10 the two latter. In the current work, we exemplify this connection, and demonstrate how one of the
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12 most fundamental concepts in conventional heterogeneous catalysis, the Sabatier Principle, can be
13 directly applied to an interfacial enzyme reaction of the type in Fig. 1C.
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34 FIG.1. Steady-state rates (v0) plotted against substrate concentration for three different types of enzyme
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reactions. Symbols are experimental data (see details in the Supporting Information) and solid lines are best fit
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37 to the Michaelis-Menten equation. A) Homogeneous enzyme kinetics with enzyme and substrate in aqueous
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solution. The data reflects hydrolysis of a chromogenic substrate analog by the cellulase Cel7A from Trichoderma
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40 reesei. B) Heterogeneous enzyme kinetics of a surface-immobilized enzyme (Cellobiose Dehydrogenase (CDH)
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from P. chrysosporium acting on dissolved cellobiose. C) Heterogeneous enzyme kinetics of a soluble enzyme,
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43 Cel7A, acting on an insoluble substrate (cellulose). In all cases, we see the conventional hyperbolic growth
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45 towards Vmax, where all enzyme molecules are engaged in a complex when the substrate becomes in large
46 excess. This is illustrated in the cartoons, where the enzyme is consistently green while the substrate is black.
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49 The century-old Sabatier Principle states that efficient catalysis occur when the catalyst binds its
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51 reactant with an intermediate strength 9. Weaker interaction is unfavorable because the population
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53 of intermediate (the Michaelis complex for an enzyme reaction) becomes very low. Stronger binding
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is also unfavorable for the catalytic rate because a very stable intermediate will turn over and
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56 dissociate slowly. This concept is widely applied within inorganic, heterogeneous catalysis 10-11, and
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58 although qualitative in nature, it has proven very useful as guidance both in mechanistic analyses and
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the selection or design of new catalysts. The advantage of the Sabatier Principle is that it simplifies
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3 the problem by introducing a single parameter (the stability of the intermediate) that describes
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5 catalytic efficacy. Experimentally, the principle is readily illustrated in a so-called volcano plot, which
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7 has some measure of the catalytic rate on the ordinate and the stability of the intermediate on the
8 abscissa 12. The appearance of a maximum in such plots corroborates the general principle, and its
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10 location defines the optimal binding strength for catalysis under the given conditions. The idea of an
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12 intermediary stability of the intermediate is also deeply rooted in enzymology and extensively used
13 both to explain the basics of enzyme catalysis 13-14, and in more specialized treatments of biocatalysis
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15 15-16.Nevertheless, the Sabatier Principle and volcano plots have not been widely used in this field.
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17 Recently, Lin et al. 17 reported Sabatier-like kinetic behavior of designed DNA-enzyme nanostructures,
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but it was concluded that the volcano plot relied on substrate interactions with the DNA-scaffold of
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20 the nanostructure and not actual enzyme-substrate contacts. In the current work, we show that a
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22 group of cellulases including 3 wild type enzymes and 12 enzyme variants with different affinity for
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their insoluble substrate show the characteristic Sabatier behavior. The volcano plot derived from the
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25 kinetic measurements helped us reconcile earlier controversies regarding the rate-limiting step for
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27 cellulases, and on a more general level, we suggest that the approach could provide a strong tool for
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the understanding and design of interfacial enzymes. Judging from the experiences within
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30 conventional heterogeneous catalysis, the potential of this approach could be significant.
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Results
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35 Real-time progress curves for a range of substrate loads were measured for all 15 enzymes as
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37 described in the Methods section, and the raw data can be found in the Supporting Information. As
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often seen for cellulases 18-19, we observed a transient burst in activity followed by a near-linear
40 progress curve. We defined the initial steady-state rate, v0, as the slope of the linear part of the
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42 progress curve and plotted vo as a function of the substrate load for all enzymes. Examples for the
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44 three wild types (Cel6A, Cel7A and Cel12A) are shown in Fig. 2, and the analogous plots for the variants
45 can be found in the Supporting Information. We used non-linear regression to analyze these plots with
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𝑉𝑚𝑎𝑥𝑆0
47 respect to the Michaelis Menten equation, 𝑣0 = 𝑆0 + 𝐾𝑀, and the resulting parameters are given as the
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49 turnover number, Vmax/E0, and KM, in Tab. 1.
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30 Fig.2. Michaelis Menten plots for the three wild-type enzymes, Cel6A, Cel7A and Cel12A. Symbols are
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33 the Michaelis Menten curves. The substrate was microcrystalline cellulose (Avicel).
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36 To test the applicability of the Sabatier Principle, we used the specific rate (v0/E0 in s-1) at a fixed
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38 substrate load as a measure of catalytic efficacy, and the Michalis constant, KM, as a measure of
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enzyme-substrate affinity. The choice of KM was based on the suggestion that this parameter provides
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41 a reasonable measure of the dissociation constant of the enzyme-substrate complex for this type of
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43 reaction 20. We expressed affinity as the relative standard free energy of enzyme-substrate binding,
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Go as
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K 
47 G o   RT ln  M ,i  (1)
 K 
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51 where KM,i is the Michaelis constant for the enzyme in question and KM,ref is the value for a reference
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3 Table 1. Kinetic parameters for the investigated enzymes; the three wild types, Cel6A, Cel7A and Cel12A from
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5 Trichoderma reesei, and 12 variants of Cel7A. Mutations in the Cel7A variants are given by the one-letter code.
6 Cel6A and Cel7A have separate Carbohydrate Binding Modules (CBMs) while Cel12A does not have a CBM.
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8 Variants 1-9 have the normal CBM and linker from Cel7A, but are mutated in the catalytic domain. Variants 10-
9 12 do not have a CBM and linker, and some of them are further mutated in the catalytic domain as listed.
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11 Experimental data for the Cel6A wild type 21, Cel7A wild type and variants 1, 10 and 11 are taken from an earlier
12 publication 22. The other enzymes were characterized using the same procedure.
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16 Enzyme Mutation Vmax/E0 (s-1) KM (g/l)
17 Cel7A wild type None 0.20 ±0.01 1.5 ±0.2
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19 Cel7A variant 1 W38A 0.38 ±0.01 5.9 ±0.3
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Cel7A variant 2 A100W 0.17 ±0.01 1.2 ±0.2
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22 Cel7A variant 3 Q101A 0.23 ±0.01 2.2 ±0.3
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24 Cel7A variant 4 I203T 0.25 ±0.01 2.6 ±0.3
25 Cel7A variant 5 Q101A, Y247F, I203T 0.26 ±0.01 2.2 ±0.3
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27 Cel7A variant 6 Q101A, Y247F, T246C, Y371C 0.20 ±0.01 1.9 ±0.4
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29 Cel7A variant 7 N197A, N200A 0.29 ±0.01 2.4 ±0.2
30 Cel7A variant 8 T246C, Y371C 0.21 ±0.01 1.6 ±0.4
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32 Cel7A variant 9 Q101A, Y247F, T246C, Y371C, R251A, 0.47 ±0.03 13.5 ±2.1
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34 Cel7A variant 10 Catalytic domain, W38A 0.53 ±0.03 24.3 ±2.9
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36 Cel7A variant 11 Catalytic domain, Wild type 0.34 ±0.02 12.6 ±1.8
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Cel7A variant 12 Catalytic domain Q101A, Y247F, I203T 0.49 ±0.03 16.4 ±2.3
39 Cel12A wild type None 0.54 ±0.02 28.0 ±2.2
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41 Cel6A wild type None 0.40 ±0.01 5.0 ±0.5
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44 Figure 3 shows specific rates plotted as a function of Go for all investigated enzymes at different
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46 substrate loads. Specifically, the figure shows the activity of the enzymes at 1 g/l, 10 g/l, 60 g/l and at
47 saturation where the maximal turnover, Vmax/E0 is reached (designated S0 → ∞ in the figure). This
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49 figure unveils some clear correlations between binding strength and activity, which are discussed
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Figure 3. Example of volcano plots for cellulases acting on insoluble cellulose (Avicel). Specific rates, v0/E0, at
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36 different loads of substrate were plotted as a function of the strength of enzyme-substrate interaction. The
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38 binding strength, Go, was estimated from the Michaelis constants (Tab. 1) as shown in eq. (1). Symbols
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41 location of the three wild types as illustrated above the figure. The label S0 in the box specifies saturation
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Discussion
47 The Sabatier Principle makes up an essential tool within non-biochemical, heterogeneous catalysis.
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49 Thus, the correlation between affinity and activity as expressed in a volcano plot is conceptually
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simple, easy to employ and facilitates both mechanistic analyses and identification of new catalysts 10.
52 To evaluate applicability of the Sabatier Principle to an interfacial (heterogeneous) enzyme reaction,
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54 we studied relationships between substrate affinity and catalytic efficacy for a group of 15 cellulases.
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56 We used the wild types Cel6A, Cel7A and Cel12A from the filamentous fungus Trichoderma reesei,
57 which are among the most thoroughly investigated cellulases, as well as 12 variants of Cel7A. Results
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59 in Fig. 3 showed typical volcanos when the catalytic rate was plotted against substrate affinity at
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3 intermediate substrate loads (10 and 60 g/l). This is a direct documentation of the Sabatier Principle
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5 for an enzyme reaction, and in the following, we discuss corollaries of this with respect to rate
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7 limitation of cellulolytic enzymes and interfacial enzyme catalysis in more general terms.
8 We first note that the optimal affinity for catalysis, i.e. the location of the apex in Fig. 3, Goapex ,
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10 moved towards lower values (stronger binding) as the substrate became more dilute. At 60 g/l, for
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12 example, Goapex was about +3 kJ/mol, but at 10 g/l it had fallen to around 0. An analogous shift
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induced by changes in reactant concentration has also been seen for inorganic catalysts 23-24, and
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15 probably reflects dilution-induced dissociation of the complex according to the Le Châtelier’s Principle.
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17 To compensate for a lower complex concentration under dilute conditions, the optimal affinity for
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catalysis shifts towards stronger binding. This explanation also accounts for the results at both very
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20 low- and very high substrate loads (i.e. at respectively 1 g/l and saturation with maximal turnover).
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22 Under these conditions, we did not observe maxima in Fig. 3, and we suggest that this simply reflects
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24 that Goapex fell outside the investigated range of Go. In other words, high catalytic efficacy in
25 dilute substrate suspensions will require tighter binding (lower Go) than the range covered here,
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27 and as a result, we only see the downslope on the weak-binding side of the volcano for a load of 1 g/l
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29 (red line in Fig.3). Conversely, at saturation, where all enzymes are engaged in a complex, catalysis can
30 be improved by looser binding, and we only see the upslope for the curve representing Vmax/E0 (black)
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32 in Fig. 3. One way to further assess this interpretation would be to obtain experimental data for
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34 enzymes with higher or lower affinity than those studied here. Cel7A has much stronger substrate
35 binding 25-26 than average hydrolases 27, and hence, weaker binding might be straightforward to
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37 engineer. Much stronger binding, on the other hand, could be difficult to achieve.
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39 One interpretation of the apex in the volcano plots is that it marks the boundary between dissociation-
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control and association-control of the overall enzymatic rate (see Fig 4). To illustrate this, we may
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42 consider enzymes that are located to the left of the apex (Go < Goapex). Such enzymes form stable
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44 substrate complexes that tend to accumulate, and the overall reaction rate is hence governed by their
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dissociation. For less stable complexes to the right of the apex (Go > Goapex), the overall process
47 will be accelerated by tighter binding, and one may say that the reaction is association controlled. This
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49 aspect of the volcano plot is interesting both as a general tool and in specific discussions of rate
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51 limitation for Cel7A. To illustrate the latter, we note that there has been some controversy regarding
52 the rate-limiting step for Cel7A. Some work has concluded that the rate of association (i.e. the
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54 formation of a catalytically competent complex) is slow and hence limits the overall reaction rate 28-
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56 while others have reached the opposite conclusion and suggested a dissociation-controlled
57 mechanism 30-31. The shift in the location of the maximum in Fig. 3 suggest that these controversies
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59 may rely at least in part on the influence of the substrate load. Thus, the substrate affinity of wild type
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3 Cel7A corresponds to Go = -3 kJ/mol and this is on the downslope for low substrate loads (S0=1 g/l).
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5 It follows that the reaction is association controlled under these conditions. At loads of 10 g/l or higher,
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7 on the other hand, wild type Cel7A is on the upslope suggesting dissociation-control. We emphasize
8 that this approach to rate-control is qualitative in nature and hence quite coarse compared to rigorous
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10 analyses of microkinetic reaction schemes. The big advantage, however, is that the approach is model
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12 free and hence readily applicable on the basis of experimental data without detailed microkinetic
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understanding of the reaction. This is valuable for cellulolytic reactions, which are only beginning to
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15 be understood on the level of elementary steps 32-35. We suggest that this simplifying power of volcano
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17 plots could be a very useful tool in attempts to assess rate control for a range of enzymes acting on
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insoluble substrates.
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38 Figure 4. Illustration of a Sabatier analysis in interfacial (heterogenous) enzyme catalysis. The highest activity
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40 under a given experimental condition is found when adsorption- and desorption rates is balanced. This is
41 achieved when the enzyme has an intermediate substrate affinity, such that the stability of the enzyme-
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43 substrate complex is neither too strong or too week.
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46 Many cellulases are multi-domain enzymes with one or more Carbohydrate Binding Modules (CBM).
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48 The CBM is considered non-catalytic 36, and primarily serves the purpose of promoting adsorption 37-
49 39. This role has been directly documented in adsorption studies 38, 40, and also appeared from kinetic
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51 experiments, where enzymes without CBM had a much higher KM regardless of whether a CBM had
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53 been artificially added to a CBM-less wild type or removed from a wild type with CBM 41. In addition
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to promoting affinity, CBMs have been shown to help targeting the enzyme to specific crystal planes
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56 or regions of the substrate 42-44. Although these roles of the CBM obviously appear to promote enzyme
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58 efficacy, over half of the (wild type) cellulases identified in a recent sequence analysis study did not
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3 related to the course of the volcano plot. To illustrate this, we consider the results in Tab.1, which
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5 show that removal of the CBM changed Go from -3.0 kJ/mol (for wild type Cel7A) to +2.3 kJ/mol for
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7 variant 11, which was the wild-type catalytic domain without linker and CBM. According to Fig. 3, this
8 shift of over 5 kJ/mol is associated with major change in catalytic efficacy. At a substrate load of 10
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10 g/l, for example, it would change the reaction from dissociation-control with CBM (Fig. 4, left-side of
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12 the curve) to association-control without CBM (Fig. 4, right-side of the curve). This implies that a CBM
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can be advantageous or detrimental for the activity of a given cellulase depending on the dry-matter
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15 content at which it is working and the substrate affinity of the CBM-less enzyme. As a result,
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17 evolutionary pressure could be for or against a CBM depending on the location of the apex defined by
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the specific conditions faced by the organism in question. This interpretation is in line with recent
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20 experimental studies on the effect of the CBM on activity at different substrate loads and
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22 temperatures 45-48.
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The wild types used in this study represents distinctly different types of cellulases. Not only do Cel6A,
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25 Cel7A and Cel12A belong to different Glycoside Hydrolase families with different overall folds, they
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27 also differ with respect to catalytic mechanism, mode of substrate attack, degree of processivity and
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direction of processive movement. In spite of these differences, which essentially span the whole
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30 range of hydrolytic cellulases, their activity-affinity relationships condense into a simple volcano curve,
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32 which they share with a group of Cel7A variants. This remarkable unifying power of the volcano plot
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appears to have a significant potential within research and technology of cellulases and possibly other
35 interfacial enzymes. One particularly interesting application, which is directly inspired by work on
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37 inorganic catalysts 24, is computer-assisted design of industrial enzymes. This could be used, for
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39 example, in attempts to improve catalytic activity under process conditions and it would involve two
40 steps. First, an experimental volcano plot akin to Fig. 3 should be made under relevant conditions. In
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42 the second step, variants should be tested in silico with respect to their strength of substrate binding
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44 using protein-ligand free energy calculations 49-50. The pivot of the strategy is the simple and model
45 free connection between activity and affinity, which replaces the computationally intractable problem
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47 of assessing catalytic speed with the much more amenable challenge of calculating ligand binding
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49 strength.
50 In conclusion, we have found that enzymatic efficacy within a group of cellulases could be rationalized
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52 with respect to the Sabatier Principle. Most of the cellulases had a complex structure with three
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54 domains (catalytic-, linker- and binding domain), which all interact to different extents with the
55 substrate surface 37, 39, 51. In spite of this complex mode of interaction, the enzymes shared a common
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57 volcano curve. In fact, even the four investigated enzymes without linker and binding domain (wild
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59 type of Cel12A and Variants 10-12) fell on the same curve, and this further supports the notion of a
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3 simple yet robust relationship between affinity and activity. If this relationship extends beyond the
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5 single group of enzymes (cellulases) tested here, it could provide a powerful framework for both
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7 fundamental studies of heterogeneous enzyme catalysis as exemplified in Figs. 1B and 1C, and for
8 practical enzyme design. As a first attempt to assess broader applicability of the principle, we explored
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10 the literature and found a couple of reports with comprehensive data on both affinity and activity for
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12 interfacial enzymes other than cellulases. One example addressed the breakdown of starch granules
13 by a range of amylase variants 52, while another investigated hydrolysis of precipitated Serum Albumin
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15 by variants of Cellulomonas bogoriensis chymotrypsin 53. Although these studies were not rationalized
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17 along the lines of the Sabatier Principle, they both found an apex in enzyme activity that could be
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correlated to an intermediate binding strength. These results offer some further support of a more
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20 general use of the Sabatier Principle for interfacial enzymes, but more experimental work is needed.
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22 If indeed the Sabatier Principle turns out to be widely applicable to interfacial enzyme reactions, it
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may act as a bridge between heterogeneous catalysis and biocatalysis, and hence allow enzymologists
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25 to tap into some of the sophisticated principles and practices developed for inorganic catalysis.
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Materials and methods
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30 Enzymes. Structures and properties of the investigated wild-types from Trichoderma reesei Cel6A,
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32 Cel7A and Cel12A (also known as respectively CBH2, CBH1 and EG3) have been reviewed recently 54.
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Briefly, Cel6A is an inverting cellobiohydrolase, which is composed by a catalytic domain and a
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35 Carbohydrate Binding Module (CBM) connected by a flexible, glycosylated linker. Cel6A is an exo-lytic
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37 enzyme that attacks the non-reducing end of the cellulose strand and moves processively towards the
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reducing end while cleaving off cellobiose as its main product. Cel7A is also a processive (exo-lytic)
40 cellobiohydrolase with CBM and linker, but it attacks the reducing end of the cellulose strand and uses
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42 the retaining hydrolytic mechanism. Finally, Cel12A is an endolytic enzyme with little or no
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44 processivity. It also uses the retaining mechanism, and the enzyme from Trichoderma reesei (used
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47 Mutagenesis, expression and purification. The three wild types from Trichoderma reesei described
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49 above as well as 12 variants of Cel7A (see Tab 1 for mutations) were expressed heterologously in
50 Aspergillus oryzae and purified as described elsewhere22, 55. The purified enzymes showed a single
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52 band in SDS NuPAGE 4-12% Bis-Tris gel (GE Healthcare) and their concentrations were determined by
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54 absorbance at 280 nm using extinction coefficients calculated from their amino acid composition 56.
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56 Kinetics. All measurements were made in a standard 50mM acetate buffer, 2mM CaCl2, pH 5.0 using
57
58 real time measurements with enzyme-functionalized biosensors operated in the amperometric mode.
59
60 The sensors were designed, produced and calibrated in-house as detailed elsewhere 21, 57-58.

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3 Specifically, we used sensors functionalized with cellulose dehydrogenase (CDH) from Phanerochaete
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5 chrysosporium 57 to study the retaining cellulases Cel7A and Cel12A (and all variants of Cel7A) and
6
7 sensors functionalized with pyranose dehydrogenase (PDH) from Agaricus meleagris to study the
8 kinetics of the inverting enzyme Cel6A 21. The substrate was Avicel PH101 (Sigma-Aldrich, St. Louis,
9
10 MO), which was suspended in the standard buffer without any further processing. To perform a kinetic
11
12 measurement, we transferred 3 ml suspension to a stirred, double-jacket beaker, which was
13 thermostated at 25.0 °C (c.f. Fig. 1). The reaction was started by the addition of enzyme from a syringe
14
15 pump (Fusion 100, Chemyx, Stafford, USA) to a final concentration of 0.10 µM, and the progress curve
16
17 was recorded continuously for approximately 300s. To produce Michaelis Menten plots, we made
18
progress curves at about ten different substrate loads for each of the 15 enzymes in Tab. 1. The specific
19
20 loads were chosen to be reasonably distributed above and below KM after this parameter had been
21
22 approximately determined in preliminary experiments. In most cases, we used about 10xKM for the
23
highest substrate load, but in some trials, we could not get higher than 5-8 times KM because the
24
25 highest load that could be practically handled in these experiments was about 150 g/l. Nevertheless,
26
27 KM and Vmax could be well resolved as indicated by the error margins in Tab. 1. Experimental data for
28
the Cel6A wild type, Cel7A wild type and variants 1, 10 and 11 are taken from an earlier publication 21-
29
30 22, which used the same experimental protocol.
31
32
Supporting Information
33
34
35 Progress curves and steady-state analysis of all investigated enzymes. Material and Method
36
concerning data shown in figure 1.
37
38
39 Acknowledgements
40
41
The research was supported by Innovation Fund Denmark (Grants 5150-00020 to PW) and Novo
42 Nordisk foundation (Grant number NNF15OC0016606 to PW).
43
44
45 Author contributions
46
47 JK, KB and PW conceived and coordinated the study. SB, KK and KJ provided technical assistance with
48 expression and purification of the enzymes. JK and JO designed, performed and analyzed the
49
50 experiments. JK and PW wrote the paper. All authors reviewed the results and approved the final
51
52 version of the manuscript
53
54 Competing interests
55
56 JPO, KJ, KBRMK and KB are employed at Novozymes, a major producer of industrial enzymes.
57
58
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