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Rational design of enantioselective enzymes requires considerations of entropy

Department of Biotechnology, Royal Institute of Technology, SE-100 44 Stockholm, Sweden

Reprint requests to: Karl Hult, Department of Biotechnology, Royal Institute of Technology, SE-100 44 Stockholm, Sweden; e-mail: kalle{at}biochem.kth.se; fax: +46-8-224601.

(RECEIVED April 12, 2001; FINAL REVISION May 22, 2001; ACCEPTED May 30, 2001)

¹ Present address: AstraZeneca R&D, Södertälje, Medicinal Chemistry, Novum Unit, SE-141 57 Huddinge, Sweden.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.13501.

	Abstract

TOP Abstract Introduction Theory Results Discussion Conclusion Materials and methods References

 
Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (_R-SH) and entropy (_R-SS) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. _R-SS was in all cases equally significant as _R-SH to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in _R-SH and _R-SS. Although the changes in _R-SH and _R-SS were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger _R-SH than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in _R-SS.

Keywords: Enthalpy; entropy; enantiomeric ratio; lipase; Candida antarctica; site-directed mutagenesis

	Introduction

TOP Abstract Introduction Theory Results Discussion Conclusion Materials and methods References

 
With the advances in protein engineering the vision of tailor-made proteins is becoming a reality. Modified proteins with improved properties have an enormous commercial potential (Rubingh 1997; Nixon and Firestine 2000). For instance, enzymes modified for new or improved properties would greatly improve their usability as catalysts in organic chemistry. The use of enzymes as biocatalysts has already become a popular alternative to classic chemistry because of their advantageous high selectivity and activity under mild reaction conditions (Liese and Filho 1999; Schulze and Wubbolts 1999; Carrea and Riva 2000; Faber 2000). However, finding the optimal biocatalyst and reaction conditions for a specific reaction still requires a great deal of experimental screening. Enabling rational design of enzymes would relieve this need for screening large numbers of biocatalysts to suit a target reaction and allow the introduction of new enzymatic activities. Present-day knowledge of protein structure–function relationships is, however, insufficient to predict the effects of point mutations on proteins.

Candida antarctica lipase B (CALB) is an enzyme that has proved to be a highly useful and versatile biocatalyst. It displays high activity in organic solvents and is very enantio- and regioselective, as well as being a robust and stable enzyme (Anderson et al. 1998). A model of CALB's mechanism for resolving the enantiomers of sec-alcohols based on a combination of experimental and molecular modeling research has been proposed (Orrenius et al. 1998a). This model involves the binding of the two enantiomers in two different modes in transition state to allow for catalysis (Fig. 1). The slow-reacting enantiomer, usually S, is stericly more hindered and hence, has a much lower reaction rate, yielding the possibility to kinetically resolve the two enantiomers. However, it has been shown that differences in both the enthalpic and the entropic components of the free energy of activation are important to enantioselectivity (Pham et al. 1989; Philips 1992, 1996; Galunsky et al. 1997; Sakai et al. 1997; Jönsson et al. 1999; Overbeeke et al. 1999; Ottosson and Hult 2001). Differential activation entropy is not accounted for in this two-mode model. Attempts have been made to modify this already popular biocatalyst to improve thermostability, oxidation stability as well as enantioselectivity through site-directed mutagenesis, and has been somewhat successful (Patkar et al. 1997,1998; Rotticci et al. 2001). This paper presents a thermodynamic analysis, which determines the entropic as well as the enthalpic components of enantioselectivity toward 3-methyl-2-butanol of the T103G, W104H, S47A, T42V, T42VS47A variants of CALB (Scheme 1 and Fig. 1), and compares the components to those of the wild type. The aim is to determine the importance of entropy and enthalpy in enzyme enantioselectivity with the ultimate goal of understanding how these are affected by variations in the enzyme and establishing their implications for rational design of proteins.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Simplified scheme of the two productive binding modes in the model proposed by Orrenius et al. (1998a) that rationalizes the enantioselectivity of Candida antarctica lipase B. (A) Fast-reacting enantiomer. (B) Slow-reacting enantiomer. (M) medium-sized substituent; (L) large-sized substituent.

View larger version (5K): [in this window] [in a new window]   Scheme 1. Kinetic resolution of the enantiomers of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and its T103G, W104H, S47A, T42V, T42VS47A variants.

	Theory

TOP Abstract Introduction Theory Results Discussion Conclusion Materials and methods References

Enantioselectivity is the result of a difference in activation free energy between enantiomers, _R-SG, which is related to the enantiomeric ratio E as -RTlnE. Furthermore, _R-SG is related to the difference in activation enthalpy and entropy as _R-SG = _R-SH - T_R-SS. Therefore, the enthalpic and entropic components of the enantiomeric ratio E is determined by temperature studies and equated according to equation 1 (Philips 1992).

(1)

E will vary with temperature and a racemic temperature, T_R, at which there is no enantioselectivity, E = 1 and _R-SG = 0, can be determined as T_R = _R-SH/_R-SS (Philips 1992). In the case of the resolution of 3-methyl-2-butanol, as well as most other sec-alcohols resolved byCALB, _R-SH and _R-SS are of the same sign, resulting in a real positive T_R (Overbeeke et al. 1999; J. Ottosson and K. Hult, unpubl.). The enantiomer favored by enthalpy is disfavored by entropy and vice versa. Below T_R, E will decrease with temperature and the entropic component will counteract the enthalpic yielding a lower enantioselectivity than expected from purely enthalpic considerations. On the other hand, above T_R, the enantiopreference will change to that favored by entropy and E will increase with temperature. Generally T_R for CALB-catalyzed resolutions of sec-alcohols is well above (>100 K) the experimental temperature.

Several researchers have shown that differential activation entropy significantly affects the enzyme-catalyzed resolution of enantiomers (Pham et al. 1989; Philips 1992, 1996; Galunsky et al. 1997; Sakai et al. 1997; Jönsson et al. 1999); therefore, to create a complete model of the mechanism of CALB enantioselectivity, entropy considerations must be included (Overbeeke et al. 1999; Ottosson and Hult 2001).

	Results

TOP Abstract Introduction Theory Results Discussion Conclusion Materials and methods References

 
Table 1 summarizes the results of the thermodynamic analysis of enantioselectivity in the resolution in Scheme 1 catalyzed by wild-type CALB and five variants with one or two point mutations. One of the variants, T103G, displayed an increase in enantioselectivity as compared to the wild type. The other variants had a somewhat reduced enantioselectivity and, in the case of W104H, it was the most pronounced, the enantiomeric ratio E dropped from 970 in wild type to only 150. All systems had T_Rs significantly above the experimental temperature and hence, the entropic component counteracts the enthalpic component to E. The changes in E were due to changes in both the enthalpic (_R-SH) as well as the entropic (T_R-SS) components of the activation free energy difference between the enantiomers (_R-SG = -RTlnE = _R-SH - T_R-SS). It can be noted that although the W104H mutation caused an essential decrease in the enantiomeric ratio E, both _R-SH and _R-SS were significantly greater in absolute values than for the other variants and the wild-type enzyme. The entropic component increased in number to a greater extent than the enthalpic and E was decreased, as entropy is counterproductive to enantioselectivity below T_R. The T103G variant also displayed a significant increase in both the enthalpic and the entropic component absolute values. However, in this case the entropic component did not increase enough in number to cancel the increase of the enthalpic component, yielding a more enantioselective enzyme, with an increase in E from 970 to 2140. This shows that although enthalpy and entropy displayed somewhat compensatory changes, significant changes in E can be achieved from single point mutations.

	Discussion

TOP Abstract Introduction Theory Results Discussion Conclusion Materials and methods References

 
As previously shown, for substrate effects on CALB enantioselectivity, modifications of the protein affect both the enthalpic and the entropic components of the enantiomeric ratio E (Overbeeke et al. 1999; Ottosson and Hult 2001). This fact stresses the importance of entropy in enzyme catalysis, in general, and enantioselectivity, in particular. The observed changes in enthalpy and entropy displayed a compensatory character in that, if _R-SH was large, so was _R-SS, and vice versa. However, they were not perfectly compensatory, allowing for modifications of the enzyme to improve E. Figure 2 shows the general energy profile diagrams of enthalpy and entropy for an enzyme-catalyzed kinetic resolution. Changes in the components of enantioselectivity, _R-SH and _R-SS, caused by mutations on the enzyme must be related to one or both enantiomers shifting their individual activation energy, H and S, up or down relative to each other.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Enthalpy and entropy profile diagrams of the enantioselective reaction catalyzed by Candida antarctica lipase B. Dashed lines represent the slow-reacting enantiomer and solid lines the fast-reacting enantiomer, generally S and R, respectively. The double-headed arrows display possible changes to obtain the increase in R-SH and R-SS caused by the T103G and W104H mutations. The decrease in the thermodynamic parameters as caused by the T42V, S47A, and T42VS47A mutations are shown with single-headed arrows.

W104H variant of CALB
The W104H mutation also caused the absolute values of the enthalpic _R-SH and the entropic _R-SS components of enantioselectivity to increase. As a matter of fact _R-SS for this variant was more than eight times the value of the wild type and _R-SH was more than doubled. In this case the entropic component, which is counterproductive for enantioselectivity, more than canceled the increase in the enthalpic component, causing, in contrast to T103G, a significant decrease in E. The residue W104 is positioned deep in the active site and makes up part of the boundary of the alcohol-binding site and its stereospecificity pocket (Fig. 1). The mutation of a tryptophan to a histidine liberates a volume of the active site in close contact with the chiral alcohol. Whether this causes structural changes or leaves an empty space within the enzyme remains unknown until the structure is solved. The fact that _R-SH significantly increased for W104H would result in an erroneous prediction of a very high enantioselectivity if only enthalpic energy considerations were made, as is done in most contemporary molecular modeling of enzyme catalysis where the entropic components generally are neglected (Hæffner et al. 1998; Ke et al. 1998; Orrenius et al. 1998a,b). Attempts to calculate activation entropy in enzyme catalysis with computer modeling have recently appeared in literature (Strajbl et al. 2000; Villa et al. 2000).

T42V, S47A, and T42VS47A
Amino acid residues 42 and 47 as well as 104 constitute part of the boundaries of the active alcohol-binding site. In a successful attempt to rationally design an enzyme enantioselective toward halohydrins these residues were changed by site-directed mutagenesis to T42V, S47A, and T42VS47A (Rotticci et al. 2001). These residues were chosen as they constitute part of the stereoselectivity pocket in the alcohol-binding site (Fig. 1). By exchanging the hydroxyl groups for a methyl group, T42V, or hydrogen, S47A, the enantioselectivity of sec-alcohols containing halogens on the medium substituent was hypothesized to increase due to the removal of the repulsion between the halogen and the hydroxyls. Table 2 shows how the enantiomeric ratio for 1-chloro-2-octanol and 1-bromo-2-octanol increased for the enzyme variants containing the S47A mutation. E has also decreased for the resolution of 3-methyl-2-butanol catalyzed by the same variants. This is in agreement with the model, as the variants were designed to improve E for compounds with halogenated medium substituent and as a side effect, lower E for those with alkyl substituent. However, on closer inspection of the thermodynamic components of the resolution of 3-methyl-2-butanol (Table 1) one notes that the largest changes in _R-SH and _R-SS are found for the T42V variant, the mutation that from inspection of E only seems to yield the smallest or no effect. Although the design of the S47A and T42VS47A variants was based on a model including only enthalpic and no entropic considerations, these variants were successful in improving the enantioselectivity toward the halohydrins, as seen in Table 2. However, the results from the resolution of 3-methyl-2-butanol show that the largest changes in activation enthalpy and entropy were found for the T42V mutation. This underlines the importance of not drawing premature conclusions from an enzyme model that excludes considerations of entropy.

	Conclusion

TOP Abstract Introduction Theory Results Discussion Conclusion Materials and methods References

	Materials and methods

TOP Abstract Introduction Theory Results Discussion Conclusion Materials and methods References

 
Site-directed mutagenesis
The T42V, S47A, and T42VS47A variants were obtained by using the overlap-PCR technique (Ho et al. 1989). The following primers were used to insert the desired mutations: fCALBT42V, 5'-GGAACCGGCGTCACAGGTCC; rCALBT42V, 5'-GGACCT GTGACGCCGGTTCC; fCALBS47A, 5'-TCCACAGGCTTTC GACTCGAA; and rCALBS47A, 5'-GTTCGAGTCGAAAGCCT GTGG. The CALB gene was inserted in the plasmid YpDC541 using XhoI and NotI restriction sites, placing the lipase gene directly after the -factor secretion signal sequence, as previously described by Rotticci-Mulder et al. (2001). Concerning the W104H variant, the Transformer site-directed mutagenesis kit was used as described by the manufacturer (Clontech Laboratories Inc.). The plasmid YpCALB (Rotticci-Mulder et al. 2001) was mutagenized using the mutagenic primer 5'-GCTTCCCGTGCT TACCCACTCCCAGGGTGG and the selection primer 5'-GCT GTTCCAGGGATCGCAGTGGTGAG. The selection primer allowed a silent mutation in the plasmid that removed the unique restriction-site Sma I. All different CALB variants were sequenced on both strands using Sanger sequencing reactions with dye terminators (Sanger et al. 1977) and were analyzed on an ABI Prism 377 DNA sequencer (Perkin-Elmer, Wellesley, MA). Plasmids containing the desired mutated lipase gene were transformed by electroporation into the methylotrophic yeast Pichia pastoris. One lipase-expressing transformant of each mutant was selected for further cultivation.

Protein production and purification
Wild-type lipase and its variants were produced in a 3-L fermentor according to instructions given by the manufacturer (Invitrogen, Carlsbath, CA). The cultivation medium had a volume of 1 L at the beginning of the fermentation. Using the fermentor the expression levels, measured after the protein purification, increased 20 times as compared to production in shake flasks. The T42VS47A variant was produced in shake flasks. The lipase variants were purified by hydrophobic interaction chromatography and gel filtration (Rotticci-Mulder et al. 2001). Fermentor cultivation not only increased the amount of desired protein, but also drastically decreased the amount of alcohol oxidase that was released into the medium, making the gel filtration step unnecessary.

Kinetic resolution of 3-methyl-2-butanol in hexane
The enzymes, immobilized on Accurel MP (Membrana GMBH, Obernburg, Germany), were equilibrated in the reaction vessels to a water activity of 0.11 (LiCl, sat. aq.) for more than 10 h. Dried (molecular sieves) 3-methyl-2-butanol (0.43 M) and hexane were added to the reaction vessel and equilibrated in temperature. A low enzyme load and high substrate concentrations were chosen to avoid the mass transport limitations on E, as pointed out by Rotticci et al. (2000). The reaction was started by the addition of vinyl octanoate (0.43 M). Samples were removed with a syringe at regular intervals for conversions of the substrates 50%.

Determination of enantioselectivity
The samples were analyzed for the enantiomeric excess of the remaining substrate 3-methyl-2-butanol, ee_s, and the produced octanoate ester, ee_p, with chiral capillary GC. The column used was Chirasil-Dex CB column from Chrompack, the Netherlands. The enantiomeric ratio E was calculated as an average of 5–12 samples at conversions 0–50% according to Rakels et al. (1993) from the enantiomeric excess of both substrate ee_s and product ee_p.

Molecular modeling
Molecular dynamic calculations were performed as previously published by Raza et al. (2001).

	Acknowledgments

 
This work was supported by the European Union (grant number BIO4-CT95–0231) and the Swedish Research Council for Engineering Sciences (TFR). Novo Nordisk A/S, Denmark, kindly provided the enzyme, its gene, and the T103G CALB variant. The authors also wish to thank Mehmedalija Jahic for assistance with the fermentations.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

	References

TOP Abstract Introduction Theory Results Discussion Conclusion Materials and methods References

 
Anderson, E.M., Larsson, K.M., and Kirk, O. 1998. One biocatalyst—many applications: The use of Candida antarctica. Biocatal. Biotransform. 16: 181–204.

Carrea, G. and Riva, S. 2000. Properties and synthetic applications of enzymes in organic solvents. Angew. Chem. Int. Ed. 39: 2226–2254.[CrossRef]

Faber, K. 2000. Biotransformations in organic chemistry, 4th ed. Springer, Heidelberg, Germany.

Galunsky, B., Ignatova, S., and Kasche, V. 1997. Temperature effects on S₁- and S'₁-enantioselectivity of -chymotrypsin. Biochim. Biophys. Acta 1: 130–138.

Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R. 1989. Site-directed mutagenesis using the polymerase chain reaction. Gene 77: 51–59.[CrossRef][Medline]

Hæffner, F., Norin, T., and Hult, K. 1998. Molecular modeling of the enantioselectivity in lipase-catalyzed transesterification reactions. Biophys. J. 74: 1251–1262.[Abstract/Free Full Text]

Jönsson, Å., Wehtje, E., Adlercreutz, P., and Mattiasson, B. 1999. Thermodynamic and kinetic aspects on water vs. organic solvent as reaction media in the enzyme-catalysed reduction of ketones. Biochim. Biophys. Acta 1430: 313–322.[CrossRef][Medline]

Ke, T., Tidor, B., and Klibanov, A.M. 1998. Molecular-modeling calculations of enzymatic enantioselectivity taking hydration into account. Biotechnol. Bioeng. 57: 741–745.[CrossRef][Medline]

Liese, A. and Filho, M.V. 1999. Production of fine chemicals using biocatalysis. Curr. Opin. Biotechnol. 10: 595–603.[CrossRef][Medline]

Nixon, A.E. and Firestine, S.M. 2000. Rational and "irrational" design of proteins and their use in biotechnology. IUBMB Life 49: 181–187.[CrossRef][Medline]

Ollis, D.L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S.M., Harel, M., Remington, S.J., Silman, I., Schrag, J., Sussman, J.L., Verschueren, K.H. G., and Goldman, A. 1992. The /ß-hydrolase fold. Protein Eng. 5: 197–211.[Abstract/Free Full Text]

Orrenius, C., Hæffner, F., Rotticci, D., Öhrner, N., Norin, T., and Hult, K. 1998a. Chiral recognition of alcohol enantiomers in acyl transfer reactions catalysed by Candida antarctica lipase B. Biocatal. Biotransform. 16: 1–15.

Orrenius, C., van Heusden, C., van Ruiten, J., Overbeeke, P.L.A., Kierkels, H., Duine, J.A., and Jongejan, J.A. 1998b. Simple conformation space search protocols for the evaluation of enantioselectivity of lipases. Protein Eng. 11: 1147–1153.[Abstract/Free Full Text]

Ottosson, J. and Hult, K. 2001. Influence of the acyl chain length on the enantioselectivity of Candida antarctica lipase B and its thermodynamic components in kinetic resolution of sec-alcohols. J. Mol. Catal. B: Enzymol. 11: 1025–1028.[CrossRef]

Overbeeke, P.L.A., Ottosson, J., Hult, K., Jongejan, J.A., and Duine, J.A. 1999. The temperature dependence of enzymatic kinetic resolutions reveals the relative contribution of enthalpy and entropy to enzymatic enantioselectivity. Biocatal. Biotransform. 17: 61–79.

Patkar, S.A., Svendsen, A., Kirk, O., Clausen, I.G., and Borch, K. 1997. Effect of mutation in non-consensus sequence Thr-X-Ser-X-Gly of Candida antarctica lipase B on lipase specificity, specific activity and thermostability. J. Mol. Catal. B: Enzymol. 3: 51–54.

Patkar, S., Vind, J., Kelstrup, E., Christensen, M.W., Svendsen, A., Borch, K., and Kirk, O. 1998. Effect of mutations in Candida antarctica B lipase. Chem. Phys. Lipids 93: 95–101.[CrossRef][Medline]

Pham, V.T., Phillips, R.S., and Ljungdahl, L.G. 1989. Temperature-dependent enantiospecificity of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus. J. Am. Chem. Soc. 111: 1935–1936.[CrossRef]

Philips, R.S. 1992. Temperature effects on stereochemistry of enzymatic reactions. Enzyme Microb. Technol. 14: 417–419.[CrossRef]

———. 1996. Temperature modulation of the stereochemistry of enzymatic catalysis: Prospects for exploitation. Trends Biotechnol. 14: 13–16.[CrossRef]

Rakels, J.L.L., Straathof, A.J.J., and Heijnen, J.J. 1993. A simple method to determine the enantiomeric ratio in enantioselective biocatalysis. Enzyme Microb. Technol. 15: 1051–1056.[CrossRef][Medline]

Raza, S., Fransson, L., and Hult, K. 2001. Enantioselectivity in Candida antarctica lipase B: A molecular dynamics study. Protein Sci. 10: 329–338.[Abstract/Free Full Text]

Rotticci, D., Norin, T., and Hult, K. 2000. Mass transport limitations reduce the effective stereospecificity in enzyme-catalyzed kinetic resolution. Org. Lett. 2: 1373–1376.[CrossRef][Medline]

Rotticci-Mulder, J.C., Gustavsson, M., Holmqvist, M., Hult, K., and Martinelle, M. 2001. Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose binding domain. Protein Expression Purif. 21: 386–392.[CrossRef][Medline]

Rotticci, D., Rotticci-Mulder, J.C., Denman, S., Norin, T., and Hult, K. 2001. Improved enantioselectivity of a lipase by rational protein engineering. ChemBioChem: In press.

Rubingh, D.N. 1997. Protein engineering from a bioindustrial point of view. Curr. Opin. Biotechnol. 8: 417–422.[CrossRef][Medline]

Sakai, T., Kawabata, I., Kishimoto, T., Ema, T., and Utaka, M. 1997. Enhancement of the enantioselectivity in lipase-catalyzed kinetic resolutions of 3-phenyl-2H-azirine-2-methanol by lowering the temperature to -40°C. J. Org. Chem. 62: 4906–4907.[CrossRef]

Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain-termination inhibitors. Proc. Natl. Acad. Sci. 74: 5463–5467.[Abstract/Free Full Text]

Schulze, B. and Wubbolts, M.G. 1999. Biocatalysis for industrial production of fine chemicals. Curr. Opin. Biotechnol. 10: 609–615.[CrossRef][Medline]

Strajbl, M., Sham, Y.Y., Villa, J., Chu, Z.T., and Warshel, A. 2000. Calculations of activation entropies of chemical reactions in solution. J. Phys. Chem. B 104: 4578–4584.[CrossRef]

Villa, J., Strajbl, M., Glennon, T.M., Sham, Y.Y., Chu, Z.T., and Warshel, A. 2000. How important are entropic contributions to enzyme catalysis? Proc. Natl. Acad. Sci. 97: 11899–11904.[Abstract/Free Full Text]

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