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Temperature effects on stereochemistry of enzymatic reactions
Temperature effects on stereochemistry of enzymatic reactions Robert S. Phillips Departments o f Chemistry and Biochemistry, University o f Geol\gia, Athens, GA
Introduction The stereochemical properties of enzymatic reactions are of considerable value in the preparation of optically active substances. The control of enzyme stereoselectivity remains an elusive goal of research in biocatalysis. Optimization of stereochemical yields in chemical as well as enzymatic reactions requires detailed knowledge of the effects of reaction conditions on the stereoselectivity. Remarkably few studies have focused on the effects of changing reaction conditions such as temperature and organic cosolvents on the stereochemistry of enzymatic reactions. Recently, Fitzpatrick and Klibanov ~ have found that enzymatic transesterification reactions catalyzed by subtilisin in organic media can have dramatically altered stereoselectivity in solvents of different polarity. Bjorkling et al. 2 found that addition of 25-50% DMSO improves the stereoselectivity of esterase-catalyzed hydrolysis of methyl alkyl dimethylmalonates. In contrast, Jones and Mehes 3 reported that chymotrypsin exhibits diminished enantiospecificity for hydrolysis of phenylalanine esters in the presence of organic solvents. It is widely believed that enzymes (and other catalysts) must exhibit their highest stereoselectivity at low temperatures. This belief has been supported by some experimental observations. For example, Lam et al. 4 found that esterase-catalyzed hydrolysis of 3-methyiglutarate diesters gives optimum optical yields in 20% methanol at - 1 0 ° C . 4 Willaert and coworkers 5 found that the diastereospecificity of reduction of 3-cyano4,4-dimethylcyclohexanone by H L A D H was greater at 5°C than 45°C. Keinan et al. 6 reported that the reduction of 2-pentanone to (S)-2-pentanol by the alcohol dehydrogenase from Thermoanaerobium brockii gave the highest stereoselectivity at 5°C, decreasing at higher temperatures. H o w e v e r , these effects of temperature on stereochemistry have been determined empiri-
Address reprint requests to Dr. Phillips at the Departments of Chemistry and Biochemistry, University of Georgia, Athens, GA 30602 Received 3 January 1992
©
1992Butterworth-Heinemann
cally, and no efforts have been made to establish a theoretical foundation. In order to obtain a rational understanding of the effects of temperature on the stereochemical behavior of enzymatic reactions, we have been studying the effects of temperature on the stereoselectivity of the reactions of alcohol dehydrogenases from Thermoanaerobacter ethanolicus, and of porcine liver esterase.
Theory of Temperature Effects on Stereochemistry The stereochemistry of a reaction can be defined by the enantiomeric ratio, E (Equation 1). 7 Under kinetic E = R/S = (kc,/Km)R/(kc,jKm) s
(I)
control, the ratio of observed products is equivalent to the ratio of the rate constants (kcat/Km) of formation (or reaction) of the enantiomers. The differential free energy of activation, AAG, is then given by Equation 2. The temperature dependence of AAG can then be given by the AAG = - R T l n E = AAG~ -- AAG s
(2)
AAG = AAH - TAAS
(3)
thermodynamic relationship in Equation 3. If there is no enantioselectivity in a reaction, E = I, and thus AAG = 0 from Equation 2. Equation 3 then can be rearranged to Equation 4, where T r is the " r a c e m i c t e m p e r a t u r e " for a T r = AAH/AAS
(4)
reaction at which there will be no stereochemical discrimination. At temperatures less than T r, the stereochemical course of the reaction is dominated by the activation enthalpy difference, and the stereochemical purity of products will be expected to decrease with increasing temperature; however, at temperatures greater than T r, the reaction is controlled by the activation entropy difference, and the stereochemical purity of the reaction products will increase with increasing temperature. H o w e v e r , the major product obtained at T > Tr will be the antipode to that obtained at T < T r,
Enzyme Microb. Technol., 1992, vol. 14, May
417
Literature Survery and a temperature-dependent reversal of stereochemistry is predicted.
Table 1 Enzyme
Reaction of Alcohol Dehydrogenases Of the alcohol dehydrogenases, horse liver alcohol dehydrogenase (HLADH) has been most widely studied with some spectacular results. For example, HLADHcatalyzed oxidation of meso-diols occurs with pro-S selectivity,8 and reduction of highly symmetrical cisand trans-decalindiones gives complete pro-R selectivity to produce chiral ketoalcohols. 9 HLADH has also been used to resolve racemic 3-methylcyclohexanone to optically active (R)- and (S)-3-methylcyciohexanone. J0However, HLADH has low activity and stereoselectivity for reduction of aliphatic ketones, limited thermal stability, and sensitivity to organic cosolvents. These limitations have stimulated interest in the properties of alcohol dehydrogenases from thermophilic microorganisms. An alcohol dehydrogenase from Thermoanaerobium brockii (TBADH) has been studied by Keinan and coworkers. 6 This enzyme has high activity with secondary alcohols and reduces some ketones to give (S)-alcohols with very high enantiomeric purity. However, short chain aliphatic ketones such as 2-butanone are reduced by TBADH to give (R)-alcohols with low stereoselectivity. Another anaerobic thermophilic bacterium, Thermoanaerobacter ethanolicus, has been investigated by Ljungdahl and coworkers. ~ This organism ferments a wide range of hexoses and pentoses as well as starch and xylan at 50-60°C. T. ethanolicus was found to contain two distinct alcohol dehydrogenases; both of these are NADP+-dependent, contain Zn '-+, and are thermostable to at least 70°C. ~ One of these enzymes is a primary alcohol dehydrogenase (PADH) that oxidizes straight chain and branched-chain primary alcohols and 1,2-diols and reduces aldehydes, but is weakly active with secondary alcohols and ketones. The other enzyme is a secondary alcohol dehydrogenase (SADH) similar to TBADH that is highly active with secondary alcohols and ketones while primary alcohols and aldehydes are poor substrates. Reduction of 2-butanone by SADH at 37°C gives (R)-2-butanol with modest stereoselectivity (28% e.e.) while 2-pentanone is reduced to (S)-2-pentanol (44% e.e.). ~2,13This unexpected reversal of stereoselectivity was also found by Keinan et al. 6 in the reduction of these ketones by TBADH. We studied the oxidation reactions of secondary alcohols with SADH in order to obtain the activation parameters for the reactions. At temperatures below 26°C, (S)-2-butanol is the preferred substrate but above 26°C (R)-2-butanol reacts more rapidly. The reaction of 2-pentanol shows a more gradual decrease in enantiospecificity with temperature, with the (S)isomer the preferred substrate at temperatures below 70°C. These data represent the first experimental demonstration of temperature-dependent reversal of enantiospecificity in an enzymatic reaction. 12 The activation parameters for the reactions of 2-butanol and 2-pentanol with SADH are given in Table 1.13 The differential enthalpies of activation for both
418
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14, M a y
SADH SADH PADH
Activation parameters f o r PADH and SADH reactions Substrate
AAH a
AAS b
mrc
2-Butanol 2-Pentanol 2-Methylbutanal
+ 8.37 -- 5.88 - 7.50
+ 27.9 + 17.3 - 26.5
26 ° 70 ° 10 °
Values are in kcal/mol, and represent the difference of R-S. b Values are in cal/deg-mol, and represent the difference o f R-S. c Values are in degrees C. a
2-butanol and 2-pentanol demonstrate that the (S)-alcohols have the best binding interactions with the active site of SADH. In contrast, the differential activation entropies show that reaction of the (R)-alcohols is entropically favored. It is this dichotomy between enthalpy and entropy that results in the observed temperature dependent reversal of enantioselectivity. A plot of the natural logarithm of the k c J K m values for the reaction of the (R)- and (S)-alcohols at 50°C versus the number of methylene groups in the carbon chain results in a reasonable linear relationship for the (S)-alcohols suggesting that the mechanism is constant from (S)-2-butanol to (S)-2-hexanol. The slope of the line is -AAG./RT, where AAG, is the incremental change in free energy of activation per methylene group. From these data we calculate that the addition of each methylene results in an increased free energy of activation of I. 11 kcal/mol at 50°C. In contrast, the data for the (R)-alcohols do not give a good linear fit, and the line through these points is convex. This implies that there is a change in rate-determining step in the reaction of the (R)-alcohols, with the break occurring between (R)-2-pentanol and (R)-2-hexanol. These results suggest a model in which there is a large alkyl-binding site and a small alkyl-binding site in the enzyme active site. The binding of (S)-alcohols, which puts the larger alkyl substituent in the larger binding pocket, is most favorable as shown by the activation enthalpy values in Table 1. The major binding forces are expected to be due to hydrophobic effects and van der Waals contacts. The small cavity can apparently easily accomodate methyl and ethyl groups, but n-propyl appears to be the limit, as shown by the curvature of the plot for the reactions of the (R)-alcohols, while the large site can readily hold groups as large as n-butyl. The binding of an (R)-alcohol would place the methyl group in the large site and the larger alkyl substituent in the small alkyl binding site. However, the data demonstrate that the activation entropy is more favorable for the reaction of the (R)-alcohols. This suggests that there is more rigidity in the transition states of the enzyme-substrate-NADP ternary complexes in the reaction of (or leading to) the (S)-alcohols. PADH from T. ethanolicus reduces aldehydes rapidly but has very low activity with ketones.l~ We have studied the reduction of racemic 2-methylbutyraldehyde by PADH at several temperatures in order to evaluate the effects of temperature on the reaction ste-
Temperature effects of stereochemistry: Phillips reospecificity. In the temperature range studied, (S)-2methylbutanal is the preferred substrate; however, the stereospecificity increases dramatically when the reaction temperature is raised from 15° (14% e.e.) to 35°C (51% e.e.) (Andrade, F.A.C. and Phillips, R.S., unpublished observations). Thus, the reduction of 2-methylbutanal by PADH at these temperatures is clearly an entropically controlled reaction. The activation parameters for this reduction are given in Table 1 ; the activation enthalpy and entropy values are quite similar to those of the reactions of SADH. These results can also be explained by a model similar to that for SADH. As with SADH, the stereochemicai discrimination is between a methyl and an ethyl group binding to two alkyl binding sites, also driven by hydrophobic effects and van der Waals forces.
Porcine liver esterase One of the most widely studied hydrolyases is the esterase isolated from porcine liver. This enzyme is capable of kinetic resolution of a wide range of esters and also can catalyze stereoselective hydrolysis of meso- and prochiral diesters.14 The stereoselective hydrolysis of methyl alkyl dimethylmalonates is particularly interesting, and results in optically active malonic half-esters with moderate to high enantiomeric purity (50-90% e.e.). 2'15'16Remarkably, there is a reversal of stereoselectivity with alkyl chain length, methyl butyl dimethylmalonate giving the (S)-monomethylester, but methyl heptyl dimethylmalonate gives the (R)-monomethylester. LsThis reversal of stereochemistry is similar to that seen in the reduction of ketones by SADH and TBADH. However, the effects of temperature on this reaction have not been previously determined. We were interested to see if this reversal of stereochemistry is due to entropic control, as with SADH and PADH discussed above. Thus, we have carried out the reaction of porcine liver esterase with alkyl methyl dimethylmalonates at various temperatures. Between 15°C and 35°C, we find either no change or modest increases in enantiomeric purities of the halfester products. ~7 These results contrast with those of L a m e t al.,4 who found that optimal stereoselectivity in the hydrolysis of 3-alkyl dimethylglutarates was obtained at -10°C. The differences in stereoselectivity observed in the malonate system are too small for an accurate estimation of the activation parameters. Nevertheless, the increase in stereoselectivity observed at higher temperatures suggests that the activation entropy also plays a significant role in controlling the stereochemical outcome of this reaction. This conclusion is supported by the effect of 25% DMSO, which also moderately increases the stereoselectivity of the hydrolysis,2 and simultaneously eliminates the effect of temperature, r This suggests that the temperature and solvent effects are due to the same mechanism. The DMSO effect is likely due to alteration of the medium polarity, which changes the hydrophobic contribution to substrate binding and reaction. These results are of practical importance for the optimization of esterase reactions. Our data suggest that esterase-
catalyzed reactions of prochiral malonate esters should be performed at 35°C in the presence of 25% DMSO for best results.
Conclusions and Future Directions Our results represent the frst demonstration of temperature-dependent reversal of enantiospecificity in an enzymatic reaction. However, we believe this phenomenon may be observed in a much wider range of enzymatic and chemical reactions. Pracejas and Tille 18 observed temperature-dependent reversal ofdiastereoselectivity in the reaction of ketenes with optically active amines. Similar temperature-dependent reversals of elution order of enantiomers have been observed recently in gas chromatographic separations on chiral phases. ~9'2° From a practical viewpoint, our results demonstrate that temperature can be a critical variable in the stereochemical optimization of biocatalytic reactions. Under enthalpic control, the highest stereochemical purities are obtained at low reaction temperatures. In contrast, reactions performed under entropic control will give highest stereochemical purities at the highest reaction temperature compatible with the enzyme, substrate, and cofactor system.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fitzpatrick, P. A. and Klibanov, A. M. J. Am. Chem. Soc. 199[, 113, 3166-3171. Bjorkling, F., Boutelje, J., Gatenbeck, S., Hult, K., Norin, T. and Szmulik, P. Bioorg. Chem. 1986, 14, 176-181. Jones, J. B. and Mehes, M. M. Can. J. Chem., 1979, 57, 2245-2248. Lain, L. K. P., Hui, R. A. H. F. and Jones, J. B. J. Org. Chem. 1988, 51, 2047-2050. Willaert, J. J., Lemiere, G. L., Joris, L. A., Lepoivre, J. A. and Alderweireldt, F. C. Bioorg. Chem. 1988, 16, 223-231. Keinan, E., Hafeli, F. V., Seth, K. K. and Lamed, R. J. Am. Chem. Soe. 1986, 108, 162-169. Sih, C. J. and Chen, C.-S. Angew. Chem. Int. Ed. Eng. 1984, 23, 570-578. Irwin, A. J. and Jones, J. B. J. Am. Chem. Soc. 1977, 99, 556-561. Dodds, D. R. and Jones, J. B. J. Chem. Soc., Chem. Commun. 1982, 1080-1081. Van Osselaer, T. A., Lemiere, G. L., Lepoivre, J. A., Alderweireldt, F. C. Bull. Soc. Chim. Belg. 1980, 89, 133-149. Bryant, F. O., Wiegel, J. and Ljungdahl, L. G. Appl. Environ. Microbiol. 1988, 45, 460-465. Pham, V. T., Phillips, R. S. and Ljungdahl, L. G. J. Am. Chem. Soc. 1989, 111, 1935-1936. Pham, V. T. and Phillips, R. S. J. Am. Chem. Soc. 1990, 112, 3629-3632. Zhu, L.-M. and Tedford, M. C. Tetrahedron 1990, 46, 6587-6611. Bjorkling, F., Boutelje, J., Gatenbeck, S., Hult, K. and Norin, T. Tetrahedron Letts. 1985, 26, 4957-4958. Bjorkling, F., Boutelje, J., Gatenbeck, S., Hult, K., Norin, T. and Szmulik, P. Tetrahedron 1985, 41, 1347-1352. Andrade, F. A. C., Andrade, M. A. C. and Phillips, R. S. Bioorg. Med. Chem. Letts., 1991, 1, 373-376. Pracejus, H. and Tille, A. Chem. Ber. 1963, 96, 854-865. Watabe, K., Charles, R. and Gil-Av, E. Angew. Chem., Int. Ed. Engl. 1989, 28, 192-194. Schurig, V., Ossig, J. and Link, R. Angew. Chem., Intl. Ed. Engl. 1989, 28, 194-196.
Enzyme Microb. Technol., 1992, vol. 14, May
419
Introduction The stereochemical properties of enzymatic reactions are of considerable value in the preparation of optically active substances. The control of enzyme stereoselectivity remains an elusive goal of research in biocatalysis. Optimization of stereochemical yields in chemical as well as enzymatic reactions requires detailed knowledge of the effects of reaction conditions on the stereoselectivity. Remarkably few studies have focused on the effects of changing reaction conditions such as temperature and organic cosolvents on the stereochemistry of enzymatic reactions. Recently, Fitzpatrick and Klibanov ~ have found that enzymatic transesterification reactions catalyzed by subtilisin in organic media can have dramatically altered stereoselectivity in solvents of different polarity. Bjorkling et al. 2 found that addition of 25-50% DMSO improves the stereoselectivity of esterase-catalyzed hydrolysis of methyl alkyl dimethylmalonates. In contrast, Jones and Mehes 3 reported that chymotrypsin exhibits diminished enantiospecificity for hydrolysis of phenylalanine esters in the presence of organic solvents. It is widely believed that enzymes (and other catalysts) must exhibit their highest stereoselectivity at low temperatures. This belief has been supported by some experimental observations. For example, Lam et al. 4 found that esterase-catalyzed hydrolysis of 3-methyiglutarate diesters gives optimum optical yields in 20% methanol at - 1 0 ° C . 4 Willaert and coworkers 5 found that the diastereospecificity of reduction of 3-cyano4,4-dimethylcyclohexanone by H L A D H was greater at 5°C than 45°C. Keinan et al. 6 reported that the reduction of 2-pentanone to (S)-2-pentanol by the alcohol dehydrogenase from Thermoanaerobium brockii gave the highest stereoselectivity at 5°C, decreasing at higher temperatures. H o w e v e r , these effects of temperature on stereochemistry have been determined empiri-
Address reprint requests to Dr. Phillips at the Departments of Chemistry and Biochemistry, University of Georgia, Athens, GA 30602 Received 3 January 1992
©
1992Butterworth-Heinemann
cally, and no efforts have been made to establish a theoretical foundation. In order to obtain a rational understanding of the effects of temperature on the stereochemical behavior of enzymatic reactions, we have been studying the effects of temperature on the stereoselectivity of the reactions of alcohol dehydrogenases from Thermoanaerobacter ethanolicus, and of porcine liver esterase.
Theory of Temperature Effects on Stereochemistry The stereochemistry of a reaction can be defined by the enantiomeric ratio, E (Equation 1). 7 Under kinetic E = R/S = (kc,/Km)R/(kc,jKm) s
(I)
control, the ratio of observed products is equivalent to the ratio of the rate constants (kcat/Km) of formation (or reaction) of the enantiomers. The differential free energy of activation, AAG, is then given by Equation 2. The temperature dependence of AAG can then be given by the AAG = - R T l n E = AAG~ -- AAG s
(2)
AAG = AAH - TAAS
(3)
thermodynamic relationship in Equation 3. If there is no enantioselectivity in a reaction, E = I, and thus AAG = 0 from Equation 2. Equation 3 then can be rearranged to Equation 4, where T r is the " r a c e m i c t e m p e r a t u r e " for a T r = AAH/AAS
(4)
reaction at which there will be no stereochemical discrimination. At temperatures less than T r, the stereochemical course of the reaction is dominated by the activation enthalpy difference, and the stereochemical purity of products will be expected to decrease with increasing temperature; however, at temperatures greater than T r, the reaction is controlled by the activation entropy difference, and the stereochemical purity of the reaction products will increase with increasing temperature. H o w e v e r , the major product obtained at T > Tr will be the antipode to that obtained at T < T r,
Enzyme Microb. Technol., 1992, vol. 14, May
417
Literature Survery and a temperature-dependent reversal of stereochemistry is predicted.
Table 1 Enzyme
Reaction of Alcohol Dehydrogenases Of the alcohol dehydrogenases, horse liver alcohol dehydrogenase (HLADH) has been most widely studied with some spectacular results. For example, HLADHcatalyzed oxidation of meso-diols occurs with pro-S selectivity,8 and reduction of highly symmetrical cisand trans-decalindiones gives complete pro-R selectivity to produce chiral ketoalcohols. 9 HLADH has also been used to resolve racemic 3-methylcyclohexanone to optically active (R)- and (S)-3-methylcyciohexanone. J0However, HLADH has low activity and stereoselectivity for reduction of aliphatic ketones, limited thermal stability, and sensitivity to organic cosolvents. These limitations have stimulated interest in the properties of alcohol dehydrogenases from thermophilic microorganisms. An alcohol dehydrogenase from Thermoanaerobium brockii (TBADH) has been studied by Keinan and coworkers. 6 This enzyme has high activity with secondary alcohols and reduces some ketones to give (S)-alcohols with very high enantiomeric purity. However, short chain aliphatic ketones such as 2-butanone are reduced by TBADH to give (R)-alcohols with low stereoselectivity. Another anaerobic thermophilic bacterium, Thermoanaerobacter ethanolicus, has been investigated by Ljungdahl and coworkers. ~ This organism ferments a wide range of hexoses and pentoses as well as starch and xylan at 50-60°C. T. ethanolicus was found to contain two distinct alcohol dehydrogenases; both of these are NADP+-dependent, contain Zn '-+, and are thermostable to at least 70°C. ~ One of these enzymes is a primary alcohol dehydrogenase (PADH) that oxidizes straight chain and branched-chain primary alcohols and 1,2-diols and reduces aldehydes, but is weakly active with secondary alcohols and ketones. The other enzyme is a secondary alcohol dehydrogenase (SADH) similar to TBADH that is highly active with secondary alcohols and ketones while primary alcohols and aldehydes are poor substrates. Reduction of 2-butanone by SADH at 37°C gives (R)-2-butanol with modest stereoselectivity (28% e.e.) while 2-pentanone is reduced to (S)-2-pentanol (44% e.e.). ~2,13This unexpected reversal of stereoselectivity was also found by Keinan et al. 6 in the reduction of these ketones by TBADH. We studied the oxidation reactions of secondary alcohols with SADH in order to obtain the activation parameters for the reactions. At temperatures below 26°C, (S)-2-butanol is the preferred substrate but above 26°C (R)-2-butanol reacts more rapidly. The reaction of 2-pentanol shows a more gradual decrease in enantiospecificity with temperature, with the (S)isomer the preferred substrate at temperatures below 70°C. These data represent the first experimental demonstration of temperature-dependent reversal of enantiospecificity in an enzymatic reaction. 12 The activation parameters for the reactions of 2-butanol and 2-pentanol with SADH are given in Table 1.13 The differential enthalpies of activation for both
418
Enzyme
Microb.
Technol.,
1992, vol.
14, M a y
SADH SADH PADH
Activation parameters f o r PADH and SADH reactions Substrate
AAH a
AAS b
mrc
2-Butanol 2-Pentanol 2-Methylbutanal
+ 8.37 -- 5.88 - 7.50
+ 27.9 + 17.3 - 26.5
26 ° 70 ° 10 °
Values are in kcal/mol, and represent the difference of R-S. b Values are in cal/deg-mol, and represent the difference o f R-S. c Values are in degrees C. a
2-butanol and 2-pentanol demonstrate that the (S)-alcohols have the best binding interactions with the active site of SADH. In contrast, the differential activation entropies show that reaction of the (R)-alcohols is entropically favored. It is this dichotomy between enthalpy and entropy that results in the observed temperature dependent reversal of enantioselectivity. A plot of the natural logarithm of the k c J K m values for the reaction of the (R)- and (S)-alcohols at 50°C versus the number of methylene groups in the carbon chain results in a reasonable linear relationship for the (S)-alcohols suggesting that the mechanism is constant from (S)-2-butanol to (S)-2-hexanol. The slope of the line is -AAG./RT, where AAG, is the incremental change in free energy of activation per methylene group. From these data we calculate that the addition of each methylene results in an increased free energy of activation of I. 11 kcal/mol at 50°C. In contrast, the data for the (R)-alcohols do not give a good linear fit, and the line through these points is convex. This implies that there is a change in rate-determining step in the reaction of the (R)-alcohols, with the break occurring between (R)-2-pentanol and (R)-2-hexanol. These results suggest a model in which there is a large alkyl-binding site and a small alkyl-binding site in the enzyme active site. The binding of (S)-alcohols, which puts the larger alkyl substituent in the larger binding pocket, is most favorable as shown by the activation enthalpy values in Table 1. The major binding forces are expected to be due to hydrophobic effects and van der Waals contacts. The small cavity can apparently easily accomodate methyl and ethyl groups, but n-propyl appears to be the limit, as shown by the curvature of the plot for the reactions of the (R)-alcohols, while the large site can readily hold groups as large as n-butyl. The binding of an (R)-alcohol would place the methyl group in the large site and the larger alkyl substituent in the small alkyl binding site. However, the data demonstrate that the activation entropy is more favorable for the reaction of the (R)-alcohols. This suggests that there is more rigidity in the transition states of the enzyme-substrate-NADP ternary complexes in the reaction of (or leading to) the (S)-alcohols. PADH from T. ethanolicus reduces aldehydes rapidly but has very low activity with ketones.l~ We have studied the reduction of racemic 2-methylbutyraldehyde by PADH at several temperatures in order to evaluate the effects of temperature on the reaction ste-
Temperature effects of stereochemistry: Phillips reospecificity. In the temperature range studied, (S)-2methylbutanal is the preferred substrate; however, the stereospecificity increases dramatically when the reaction temperature is raised from 15° (14% e.e.) to 35°C (51% e.e.) (Andrade, F.A.C. and Phillips, R.S., unpublished observations). Thus, the reduction of 2-methylbutanal by PADH at these temperatures is clearly an entropically controlled reaction. The activation parameters for this reduction are given in Table 1 ; the activation enthalpy and entropy values are quite similar to those of the reactions of SADH. These results can also be explained by a model similar to that for SADH. As with SADH, the stereochemicai discrimination is between a methyl and an ethyl group binding to two alkyl binding sites, also driven by hydrophobic effects and van der Waals forces.
Porcine liver esterase One of the most widely studied hydrolyases is the esterase isolated from porcine liver. This enzyme is capable of kinetic resolution of a wide range of esters and also can catalyze stereoselective hydrolysis of meso- and prochiral diesters.14 The stereoselective hydrolysis of methyl alkyl dimethylmalonates is particularly interesting, and results in optically active malonic half-esters with moderate to high enantiomeric purity (50-90% e.e.). 2'15'16Remarkably, there is a reversal of stereoselectivity with alkyl chain length, methyl butyl dimethylmalonate giving the (S)-monomethylester, but methyl heptyl dimethylmalonate gives the (R)-monomethylester. LsThis reversal of stereochemistry is similar to that seen in the reduction of ketones by SADH and TBADH. However, the effects of temperature on this reaction have not been previously determined. We were interested to see if this reversal of stereochemistry is due to entropic control, as with SADH and PADH discussed above. Thus, we have carried out the reaction of porcine liver esterase with alkyl methyl dimethylmalonates at various temperatures. Between 15°C and 35°C, we find either no change or modest increases in enantiomeric purities of the halfester products. ~7 These results contrast with those of L a m e t al.,4 who found that optimal stereoselectivity in the hydrolysis of 3-alkyl dimethylglutarates was obtained at -10°C. The differences in stereoselectivity observed in the malonate system are too small for an accurate estimation of the activation parameters. Nevertheless, the increase in stereoselectivity observed at higher temperatures suggests that the activation entropy also plays a significant role in controlling the stereochemical outcome of this reaction. This conclusion is supported by the effect of 25% DMSO, which also moderately increases the stereoselectivity of the hydrolysis,2 and simultaneously eliminates the effect of temperature, r This suggests that the temperature and solvent effects are due to the same mechanism. The DMSO effect is likely due to alteration of the medium polarity, which changes the hydrophobic contribution to substrate binding and reaction. These results are of practical importance for the optimization of esterase reactions. Our data suggest that esterase-
catalyzed reactions of prochiral malonate esters should be performed at 35°C in the presence of 25% DMSO for best results.
Conclusions and Future Directions Our results represent the frst demonstration of temperature-dependent reversal of enantiospecificity in an enzymatic reaction. However, we believe this phenomenon may be observed in a much wider range of enzymatic and chemical reactions. Pracejas and Tille 18 observed temperature-dependent reversal ofdiastereoselectivity in the reaction of ketenes with optically active amines. Similar temperature-dependent reversals of elution order of enantiomers have been observed recently in gas chromatographic separations on chiral phases. ~9'2° From a practical viewpoint, our results demonstrate that temperature can be a critical variable in the stereochemical optimization of biocatalytic reactions. Under enthalpic control, the highest stereochemical purities are obtained at low reaction temperatures. In contrast, reactions performed under entropic control will give highest stereochemical purities at the highest reaction temperature compatible with the enzyme, substrate, and cofactor system.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
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