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J. Biol. Chem., Vol. 277, Issue 18, 15432-15438, May 3, 2002

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The Effects of Modifying the Surface Charge on the Catalytic Activity of a Thermolysin-like Protease^*

Arno de Kreij^§, Bertus van den Burg^¶, Gerard Venema, Gert Vriend^**, Vincent G. H. Eijsink, and Jens E. Nielsen^§§¶¶

From the  Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands, the ^** Centre for Molecular and Biomolecular Informatics, Katholieke Universiteit Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, the  Department of Chemistry and Biotechnology, Agricultural University of Norway, P. O. Box 5040, N-1432 Ås, Norway, and the ^§§ European Molecular Biology Laboratory, BIOcomputing, Meyerhofstrasse 1, Heidelberg D-69117, Germany

Received for publication, January 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The impact of long range electrostatic interactions on catalysis in the thermolysin-like protease from Bacillus stearothermophilus was studied by analyzing the effects of inserting or removing charges on the protein surface. Various mutations were introduced at six different positions, and double-mutant cycle analysis was used to study the extent to which mutational effects were interdependent. The effects of single point mutations on the k_cat/K_m were non-additive, even in cases where the point mutations were located 10 Å or more from the active site Zn²⁺ and separated from each other by up to 25 Å. This shows that catalysis is affected by large electrostatic networks that involve major parts of the enzyme. The interdependence of mutations at positions as much as 25 Å apart in space also indicates that other effects, such as active site dynamics, play an important role in determining active site electrostatics. Several mutations yielded a significant increase in the activity, the most active (quadruple) mutant being almost four times as active as the wild type. In some cases the shape of the pH-activity profile was changed significantly. Remarkably, large changes in the pH-optimum were not observed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The acceleration of reaction rates by enzymes is one of the essential prerequisites for life as we know it, and the multitude and diversity of enzymes shows that it should be possible to design an enzyme that will catalyze almost any reaction under almost any set of conditions. To achieve a high rate of acceleration, enzymes rely on charged groups in their active site that stabilize the transition state or function as acid or base catalysts in the reaction. The kinetic parameters of enzymes therefore display a significant pH dependence, which is determined by the pK_a values of the active site groups.

Catalysis depends on intricate electrostatic interactions, which may be noticeable over distances that are large compared with short range of interactions such as hydrogen bonds and hydrophobic contacts. Thus, larger parts of an enzyme may be involved in optimizing its catalytic center than previously thought. The long range character of electrostatic effects is illustrated by a, very limited, number of examples in the literature, showing that changes in surface charge at locations as far as 15 Å from a catalytic center may affect enzyme activity (1, 2). Unfortunately, electrostatic interactions are hard to handle theoretically not only because of their long range character but also because of intrinsic theoretical difficulties. For example, most electrostatic models still use a single rigid protein structure and, at most, two dielectric constants to account for all the dynamics of the protein. This clearly is an oversimplification of reality (3).

We have studied the contribution of long range electrostatic interactions to catalysis by analyzing the effects of a series of charge mutations scattered over a larger part of the surface of a thermolysin-like protease from Bacillus stearothermophilus (TLP-ste).¹ Thermolysin-like proteases (TLPs) are members of the peptidase family M4 (4) of which thermolysin (EC 3.4.24.27) is the prototype. One of their characteristics is a zinc ion bound in the catalytic center. The amino acid sequences of several TLPs have been determined (see Ref. 4, or the Merops data base at www.merops.co.uk/merops/famcards/m4.htm), and the three-dimensional structures of TLPs isolated from several bacteria (Bacillus thermoproteolyticus (5), Bacillus cereus (6), Pseudomonas aeruginosa (7), and Staphylococcus aureus (8)) have been solved. TLPs consist of an -helical C-terminal domain and a -rich N-terminal domain. These two domains are connected by a central -helix, which is located at the bottom of the active site cleft and which contains several of the catalytically important residues. From x-ray structures of thermolysin·inhibitor complexes (5, 9-13), the active site residues have been identified and a mechanism has been proposed (13-15). Recently, an alternative mechanism has been proposed that has gained some support (16, 17). In both proposed mechanisms residues Glu-143, His-231, Tyr-157, and a Zn²⁺ bound water play important roles during catalysis.

Mutations were introduced at six surface positions in TLP-ste, located at 10-15 Å from the catalytic center. The single and multiple mutants that were obtained displayed varying effects on catalytic efficiency, including considerable increases in activity. Double-mutant cycle analysis (18) was used to study the additivity of mutational effects, revealing remarkable interdependence of the mutated residues. The results provide insight in the complexity of predicting and interpreting electrostatic effects in catalysis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Modeling and Mutant Design-- A three-dimensional model of TLP-ste was built with WHAT-IF (19) using the crystal structure of thermolysin as template, as described elsewhere (20). The high sequence identity between thermolysin and TLP-ste (86%) indicates that the TLP-ste model is sufficiently reliable for prediction and analysis of the effects of most amino acid substitutions (20, 21). Indeed, the TLP-ste model has been used successfully for the design of various stabilizing mutations (22-24). Throughout this report, residues are numbered according to the corresponding residues in thermolysin.

Mutant selection was based on the following three criteria: 1) The mutated residue should be polar and located on the surface of the protein and it should have minimal influence on protein structure and substrate binding. Thus, residues were selected that were close to but not in the active site, and they were replaced by residues for which predicted major rotamers gave no bumps. A residue was considered to be at the surface if all of its polar side chain atoms were fully solvent accessible in at least one of the predicted major rotamers. Residues involved in salt bridges were not selected. 2) The mutation should affect one of the major active site residues (Glu-143, His-231) much more than the other. Because all mutated residues are located at the surface, this selection criterion was in practice a distance criterion: Mutated residues should clearly be closer to the one active site residue than to the other. 3) The mutated residue should only affect active site residues; i.e. the major rotamers in the backbone-specific rotamer search should not only fit well but should also point in the direction of the residue that was supposed to be influenced, and there should be nothing "in-between" the new charge and the point where it should exert its influence. Because these three criteria tend to contradict each other, compromises had to be made. We always kept rule 1 and were less stringent on 2 and even less on 3. The search for mutable positions was done manually but was exhaustive, i.e. everything within 10-15 Å of the active site was studied rigorously. Potentially interesting mutations were subjectively sorted; nine of them were constructed and are presented here.

Molecular Biology-- The nprT gene encoding the TLP of B. stearothermophilus CU21 (25) (TLP-ste) was cloned, subcloned, and expressed as described previously (26). Site-directed mutagenesis was performed on subcloned fragments of the TLP-ste gene using the QuikChange site-directed mutagenesis kit from Stratagene, La Jolla, CA. The nucleotide sequences of mutated fragments of the nprT gene were verified by DNA sequencing and the mutated fragments were subsequently cloned into variants of the Bacillus expression vector pGE501 (26) containing the TLP-ste gene with a deletion of the previously subcloned fragment.

Production and Characterization of Mutant Enzymes-- Production and purification of the enzymes were performed as described elsewhere (27). Prior to determining the kinetic parameters, protease preparations were desalted using pre-packed PD-10 gel filtration columns supplied by Amersham Biosciences, Inc., Uppsala, Sweden.

Determination of Kinetic Constants-- The k_cat/K_m values of the enzymes for the furylacryloylated tripeptide 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine obtained from Bachem AG, Bubendorf, Switzerland were determined at 37 °C, in a thermostatted PerkinElmer Life Sciences Lambda 11 spectrophotometer. The reaction mixture (1 ml) contained 50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris-HCl), 50 mM 4-morpholineethanesulfonic acid (MES), pH 4.4-8.4. with an interval of 0.4 units, 5 mM CaCl₂, 1% Me₂SO, 1% isopropanol, 0.01% Triton X-100, and 100 µM of substrate. The reaction was followed by measuring the decrease in absorption at 345 nm (₃₄₅ = 317 M¹·cm¹) (28). The stock solution of the furylacryloylated tripeptide was prepared by dissolving the peptide in Me₂SO. Apparent second order rate constants (k_cat/K_m) were determined by varying the enzyme concentrations over a 50-fold range under pseudo-first-order conditions and measuring the initial activity, essentially according to the method described by Feder (28). All k_cat/K_m values are the result of a linear regression analysis of at least 14 independent measurements. The error margins, defined by the highest and lowest value of the 95% confidence interval of the linear regression analysis, were at most 15% of the values given.

Double-mutant Cycle Analysis-- Determining the contribution of a single amino acid to the activity or stability of a protein by mutating that residue alone is often misleading. This is because the neighboring residues often change their position slightly to compensate for the mutation. A very illustrative recent example of this effect is provided by Albeck and Schreiber (29, 30) for the TEM·BLIP complex. To overcome this problem the method of double-mutant cycle analysis may be exploited (18, 31, 32).

When dealing with catalysis, double-mutant cycle analysis can be performed using the energy difference between free enzyme plus substrate and the enzyme·substrate complex in the transition state (18, 31, 33). This energy difference, referred to as G, reflects binding energy in the transition state and can be derived from measured k_cat/K_m values using,

(Eq. 1)

Now, consider two residues A and B in the enzyme, which do not interact and which are mutated to A' and B', respectively. The effects of mutating these residues will be independent and therefore additive. Expressed in terms of Fig. 1,

(Eq. 2)

G₁ stands for the change in G upon introducing the A  A' mutation in a wild type background, whereas G₁' stands for the change in G upon introducing the A  A' mutation after residue B has been mutated to B'. G₂ stands for the change in G upon introducing the B  B' mutation in a wild type background, whereas G₂' stands for the change in G upon introducing the B  B' mutation after residue A has been mutated to A'. These values are calculated as follows (example for G₁, using the annotation of Fig. 1),

(Eq. 3)

(Eq. 4)

If the residues symbolized by A and B in Fig. 1 somehow influence each other, the effect of mutating one may become dependent on whether or not the other is mutated too. Expressed in the terms of Fig. 1,

(Eq. 5)

In which the term |G₁  G₁| is defined as the coupling energy. This term is zero if the effects of the mutations are independent.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Double-mutant cycles. Residue A is mutated to A' and residue B to B'. G is the energy difference between free enzyme plus substrate and the enzyme-substrate complex in the transition state. G1 is the change in G upon mutating residue A, G2 is the change in G upon mutating residue B. See text for details.

The experimental error in the calculated G values is derived by inserting the highest and lowest values of the 95% confidence interval of the k_cat/K_m values in the equation for G. The experimental error of the coupling factor is the sum of the errors in the G values. The coupling factor is significantly non-zero when the error is smaller than the value of the coupling factor.

Electrostatic Calculations-- The change in the electrostatic potential at the N1 of His-231, the O1 of Gly-143, and at the oxygen of the water molecule bound to the catalytic zinc ion was calculated using WHAT-IF (19) interfaced to DelPhi II (34). A dielectric constant of 4 was applied in the interior of the protein, and a dielectric constant of 80 was assigned to the solvent phase (35). The ionic strength in the calculations was set to match the experimental conditions at pH 7.0 (210 mM). The pK_a can be calculated from the electrostatic potential difference using the following formulas,

(Eq. 6)

In which Q is the charge,  the electrostatic potential energy in volts, and G the free-energy. Rearrangement of these formula's leads to an equation for the p K_a,

(Eq. 7)

Effects on Specific Activity and Thermostability-- The specific activities of the TLP-ste variants toward casein were determined according to a method adapted from Fujii et al. (25): ~0.5 µg of protease was incubated in 1 ml of 50 mM Tris-HCl, 50 mM MES, pH 6.8, 5 mM CaCl₂, 0.01% Triton X-100 containing 0.8% (w/v) casein at 37 °C for 1 h. The reaction was quenched by the addition of 1 ml of a solution containing 100 mM trichloroacetic acid, pH 3.5. One unit of activity is defined as the amount of enzyme activity needed to liberate a quantity of acid soluble peptide corresponding to an increase in A_275 nm of 0.001/min. The temperature optimum of the TLP-ste variants was determined by determining the specific activity toward casein at various temperatures.

For the determination of the thermal stability 0.1 µM purified protease solutions (in 20 mM sodium acetate, pH 5.3, 5 mM CaCl₂, 0.01% Triton X-100, 0.5% 2-propanol, and 62.5 mM NaCl) were incubated at various temperatures for 30 min, after which the residual proteolytic activity was determined with casein as a substrate (25). Thermal stability was quantified by T₅₀, being the temperature giving 50% residual activity after a 30-min period of incubation (20, 36).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Mutant Design and Production of Mutant Proteins-- All mutants were constructed as described under "Materials and Methods." Fermentation and purification yields were normal for all single and multiple mutants. Table I summarizes the characteristics of the single mutants, compared with the wild type. Fig. 2 gives an overview of the stereochemical relationship of the mutated surface residues to each other and to the catalytically active residues and the Zn²⁺. The closest contact distance between the mutated residues and the active site Zn²⁺ is generally between 10 and 15 Å. The closest contact distance between the most remote mutated residues is ~25 Å, between residue 225 and residues 116 and 119. 

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Stereo picture showing the active site and mutated residues. The active site Zn2+ (small cross at center), and active site residues Glu-143 and His-231 are indicated.

Characterization of Mutant Proteases-- Kinetic parameters for the reaction of TLP-ste variants with the furylacryloylated tripeptide substrate 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine at different pH values are shown in Fig. 3. The pH-activity profiles generally show minimal changes in the pH optimum, the only exception being the relatively inactive mutant N227D, for which the profile shows a small acidic shift. The profiles do show some conspicuous changes in shape, in particular around the "second" pH optimum near pH 6.0. 

View larger version (17K): [in this window] [in a new window]   Fig. 3.   pH-dependent activity profiles of TLP-ste and the single surface charge mutants. Left panel: , TLP-ste; , N227D; , Q119R; , D150Q; , D150N; and , D213E. Right panel: , TLP-ste; , N116D; , D150E; , Q225R; and ×, Q225E. Experimental errors are less than 15% of the values given.

Charge Effects-- To analyze the possible electrostatic effects on the active site, we analyzed mutational effects on the (calculated) pK_a values of relevant groups in the catalytic center (Table I). Generally, the calculated effects on pK_a values were small and there were no clear overall correlations between the effects on activity and the effects on pK_a values in the active site. However, the various mutations at position 150 showed a relatively clear correlation; comparison of D150D (wild type) with D150N and comparison of D150E with D150Q (Table I) shows that removal of charge at position 150 leads to a relatively large decrease in active site pK_a values, which is accompanied by a decrease in activity. Multiple mutations were also made at position 225, but here no clear correlation was observed: Although replacement of the uncharged Gln-225 by either a negative (Glu) or a positive (Arg) amino acid had opposite effects on the calculated pK_a values, the effects on the pH-activity profile and on the activity at the pH optimum were marginal and almost identical.

Possible Non-charge Effects-- Residues 116 and 119 are located in the same surface loop and share a hydrogen bond in the wild type enzyme. Interestingly, changing the interaction between residues 116 and 119 by replacement of either one of those residues leads to an increase in activity, regardless of the net change in charge. This suggests that the increase in activity is due to effects other than electrostatics. One might speculate that the interaction between residues 116 and 119 affects the structure and/or mobility of the 116-119 surface loop and that these effects on the loop contribute to the observed mutational effects.

The mutations at position 150 also indicate that other effects may play a role, in addition to the expected (and calculated) effects on the pK_a values of active site groups (Table I). This is clearly seen in a comparison of the activities of D150N and D150Q at pH 6.8. Both mutations remove the charge at this position and are expected to yield nearly equal pK_a values. However, D150N displays a 60% drop in activity, whereas D150Q shows a 25% increase in activity. Repositioning the charge at position 150 as in D150E resulted in a 90% increase in activity at pH 6.8.

Taken together, these observations show that other factors, such as changes in loop flexibility due to disrupted or changed hydrogen bonds and relatively small changes in the spatial localization of relevant charges, contribute to the observed mutational effects.

Combining Mutations-- To obtain more active enzymes and to study the additivity of mutational effects, double, triple, and quadruple mutants were constructed. Table II summarizes the characteristics of the purified multiple mutants and Fig. 4 shows pH-activity profiles. The most active mutants displayed a 3.7-fold increase in activity at pH 6.8. Like for the single mutants, changes in pH optimum were marginal, but some multiple mutants displayed distinct changes in the shape of the pH-activity profile. The "second" pH optimum at pH 6.0 that is observed in the wild type enzyme, has completely disappeared in some of the most active mutants, in particular in the triple mutant N116D/Q119R/Q225R and the quadruple mutant N116D/Q119R/D150Q/Q225R. Because the double optimum must be a result of the ionization constants of the catalytic residues, the disappearance of this double optimum indicates a change in active site electrostatics. However, there was no obvious correlation between the mutational effects on catalysis and either pK_a or charge, as was observed for the single mutants. Interestingly, the results indicate considerable non-additivity of mutational effects. For example, addition of the Q225R mutation to D150E increases activity considerably, whereas the Q225R mutation had only marginal effects when introduced into the wild type enzyme.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   pH-dependent activity profiles of TLP-ste and the multiple surface charge mutants. Left panel: , TLP-ste; , N116D/D150Q; , N116D/Q225R; and , D150Q/Q225R. Right panel: , D150E/Q225R; , N116D/Q119R/Q225R; , N116D/Q119R/D150E/Q225R; and , N116D/Q119R/D150Q/Q225R. Experimental errors were less than 15% of the values given.

Calculation of Coupling Energies-- To determine the interdependence of the residues mutated in this study, double-mutant cycle analyses were performed as described under "Materials and Methods." Fig. 5 shows the double-mutant cycles, which can be constructed from the available data. The G values and the coupling factors were calculated from the k_cat/K_m values presented in Tables I and II.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Double-mutant cycle analysis. The G values were calculated from the kcat/Km values shown in Tables I and II. The coupling factor |G1  G1'| (= |G2  G2'|) is indicated in the center of each cycle. A non-zero coupling factor indicates that the effects of mutations are dependent on each other. See "Materials and Methods" for further details.

The coupling factor in Fig. 5A indicates that the effects of mutations at position 116 and 150 are dependent on each other. The effects of mutating positions 116 and 225 are also dependent on each other, as indicated by their coupling factor in Fig. 5B. Fig. 5 (C and D) shows the dependence of mutations at position 150 and 225 (note that only the cycle of Fig. 5C shows a significant coupling energy). From these cycles it can be concluded that the effects of mutations at positions 116, 150, and 225 are dependent on each other.

Because the roles of residues 116, 150, and 225 in catalysis are dependent on each other, demonstrating that the role of residue 119 depends on any of these residues, would show that the effects of all four residues are dependent on each other. Fig. 5E indicates that the effect of mutating residue 119 is indeed dependent on the residues present at positions 116 and 225. Therefore, the effects of mutating 116, 119, 150, and 225 are all dependent on each other. This is a remarkable observation considering that the closest contact distance between some of the mutated amino acids is almost 25 Å (between residues 116, 119, and 225; Fig. 2) and considering that the closest contact distance between each of the four residues and the active site residues is in the range of 10-15 Å.

Fig. 5 (F and G) shows a double-mutant cycle to determine whether the effect of mutating residue 150 depends on the presence of the combined mutations N116D/Q119R/Q225R. Remarkably, although the effect of D150Q depends on the individual mutations N116D (Fig. 5A) and Q225R (Fig. 5C), Fig. 5G shows no significant dependence of the D150Q mutation on the presence of the 116/119/225 combination. Even more remarkable is that the cycle involving another mutation at position 150 (D150E) and the 116/119/225 combination shows the largest coupling factor observed in this study (Fig. 5F). These results indicate that even though the effects of mutating certain positions are dependent on each other, some mutations might appear to be independent of each other, depending on which combination of amino acids is present.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Modification of the surface charge should, according to electrostatic theory, lead to a change in the active site electrostatics (1). Therefore, the catalytic performance of an enzyme may be modified without structurally disturbing the active site. Here, we have engineered a considerable increase in activity by modifying surface charges in TLP-ste. The most active mutants were approximately four times more active than the wild type. It is noteworthy that this increase was achieved by mutations that are all far from the active site. The distances between the catalytically important Zn²⁺ and the mutated residues varies from 10 to 15 Å.

Mutational effects on the activity toward tripeptide substrate were more pronounced than mutational effects on specific activity toward casein (Tables I and II). The latter effects were marginal in most cases, but two of the mutants that were most active toward the tripeptide also displayed increased specific activity toward casein (Table II). The pH optimum of these mutants was the same regardless of the substrate used,² indicating that there are no fundamental differences in the ways in which the different substrates are hydrolyzed. In most published studies on engineering protease activity, only peptide substrates were used for mutant characterization. In the few cases in which proteinaceous substrates were used (e.g. Refs. 37, 38; and by others in Refs. 39, 40) one generally observed that the results differed considerably from those obtained with peptide substrates. Usually, mutational effects were much less pronounced for proteinaceous substrates, as is also the case in the present study. One possible explanation may be that the contribution of binding to catalysis is much larger for complex substrates, thus veiling effects of e.g. subtle changes in active site electrostatics. Analysis of mutational effects on activity toward complex proteinaceous substrates is intrinsically complex, due to the large number of titratable (ionized) groups in the substrate and the fact that the substrate has many different productive binding modes. Clearly, analysis of mutational effects on active site electrostatics requires the use of short substrates that have only one productive binding mode and whose binding to the enzyme has a marginal (and constant) effect on the dielectric constant and charge in the active site.

Several elements in the results indicate that the effects of surface charge mutations on catalysis result from factors that are independent of the changes in charge per se. In general, with few exceptions, the observed changes in the pH-activity profile and activity do not correlate with expected changes in the pK_a values. This suggests that values other than those of charge-induced pK_a effects, that is, effects that are not accounted for in the software used for calculating pK_a values, are important.

One indication for the occurrence of other than charge effects comes from the studies on additivity of mutational effects. A priori, one may expect the effects of surface charge mutations to be additive as long as the residues do not have direct interactions (2, 41). Accordingly, we observed increased effects upon combination of several mutations. However, the mutational effects were far from additive, as shown by the double-mutant cycle analysis. This observation is not surprising for residues 116 and 119, which share a hydrogen bond in the wild type enzyme. However, it is highly surprising to find that all residues mutated in this study affect each other, that is, they seem to interact over distances as long as 25 Å. The fact that the interdependent residues are so far apart excludes the possibility of direct contacts and also makes it unlikely that charge effects alone account for the mutational effects.

Another indication of the occurrence of non-charge effects is the discrepancy between the observed effects on activity, and the observed effects on the pH optimum. If the change in activity is a result of a change in active site electrostatics, then a change in the pH optimum would also be expected, at least in some cases. Although changes in the shape of the pH profile were observed, the pH optimum itself was not changed in the most active enzyme variants.

The notion that complex long range interactions may affect activity without having direct effects on the pK_a values of reactive groups is supported by earlier work in which replacing uncharged residues with other uncharged residues resulted in changes in activity and in the pH-activity profile that were similar to, or larger than, the changes observed after introducing or removing charges (42). It has been suggested that changes in active site dynamics are in part responsible for such counter-intuitive results. Alternatively, the electrostatic network on the surface (and throughout) an enzyme may be of an unappreciated complexity that cannot yet be properly described, making the effects of a surface charge mutation unpredictable.

One might argue that changes in active site dynamics would cause changes in thermal stability and the temperature optimum for activity. However, none of the mutants described in this study displayed large changes in thermal stability or temperature optimum (results not shown). The lack of effects on thermal stability is not surprising, because it is known that the thermal stability of thermolysin-like proteases is mainly determined by local unfolding in a surface-located region in the N-terminal domain (Refs. 36, 43, 44; see also 45, 46). None of the mutations described here are located in this surface region. The lack of stability effects does not necessarily mean that the mutations did not affect flexibility. We have previously made a series of Gly  Ala mutations in the enzyme that were aimed at reducing flexibility (37). Some of these mutations (e.g. G135A and G136A) had profound effects on activity, but the mutations (eight in total) did not significantly affect stability, apart from one that was located in the stability-determining region in the N-terminal domain (37). These mutations did not significantly affect the temperature optimum of the enzyme either.³ Interestingly, there is recent evidence in the literature suggesting that an increase in catalytically relevant flexibility does not necessarily result in decreased stability (47).

The fact that residues 25 Å apart are coupled has serious consequences for modeling and calculation of the effects of charge mutations, because it implies that the effect of any mutation on the surface of an enzyme is dependent on the rest of the surface residues. The coupling also implies that larger parts of an enzyme are involved in optimizing its catalytic center. If this is true, then nature probably optimized considerably more than just the active site of enzymes during evolution. The large size of enzymes could be explained by the need to balance all the interactions on the surface of an enzyme and their influence on the catalytic center.

Here we show that considerable increases in catalytic activity can be obtained by modification of surface charge. The most active mutant obtained was almost four times as active as wild type TLP-ste. However, the results show that this achievement cannot easily be rationalized, e.g. by correlating it to changes in the pK_a values of active site residues. Factors that are not included in current electrostatic models, e.g. conformational dynamics and unknown complexities in electrostatic networks, probably play major roles. Reliable models and predictions as to how to modify the activity and pH-activity profile of an enzyme requires better understanding of these factors.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Danisco Innovation, Langebrogade 1, Copenhagen DK-1001, Denmark.

^¶ Present address: IMEnz Bioengineering BV, Kerklaan 30, 9751 NN Haren, The Netherlands.

^¶¶ Present address: Dept. of Biochemistry, University of California-San Diego, La Jolla, CA 92093-0365.

To whom correspondence should be addressed. Tel.: 31-50-363-2132; Fax: 31-50-363-2348; E-mail: g.venema@biol.rug.nl.

Published, JBC Papers in Press, February 21, 2002, DOI 10.1074/jbc.M200807200

² A. de Kreij, unpublished observations.

³ B. van den Burg, O. R. Veltman, and V. G. H. Eijsink, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: TLP-ste, thermolysin-like protease from B. stearothermophilus; TLP, thermolysin-like protease; MES, 4-morpholineethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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