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Carbonic Anhydrases as Esterases and Their Biotechnological Applications
CHAPTER 21
Carbonic Anhydrases as Esterases and Their Biotechnological Applications Jean-Yves Winum*, Pedro Colinas**
*Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier, Montpellier Cedex, France **LADECOR, Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Contents 21.1 Introduction 361 21.2 CA and esterase activity 362 21.2.1 Hydrolysis of esters by CAs 362 21.2.2 Mechanistic aspects 364 21.2.3 CA esterase activity on coumarins and sulfocoumarins 364 21.3 Biotechnological applications of esterase activity of CAs 367 21.3.1 Without changes in the active site of CAs 367 21.3.2 Engineering CA active site 367 References 370
21.1 INTRODUCTION The carbonic anhydrases (CAs, EC 4.2.1.1) belong to the lyase group of enzymes and are widely distributed in all organisms starting from Archaea to higher animals including vertebrates. They are implicated in a variety of physiological and pathological processes, catalyzing the reversible carbon dioxide hydration reaction to bicarbonate and proton with maximum turnover numbers among the highest known for any enzyme. Important advancements have been achieved in the CA research field, especially in the medicinal chemistry domain where now CA isozymes are important targets for the design of inhibitors or activators with clinical applications as anticancer, antiglaucoma, anticonvulsant, antipain, antiobesity, and probably soon as anti-infective drugs (antibacterial and antifungal agents). Many important review papers have been published on this particular topic in the last years (1,2). For a long time, it was believed that CAs exhibited absolute specificity, that is, that they would only catalyze the physiological interconversion between CO2 and HCO3−. However, in the 1960s it was discovered that CAs, especially those from the a-family, can also catalyze a variety of other reactions such as the hydration of cyanate to carbamic acid or the hydration of cyanamide to urea, the aldehyde hydration to gem-diols, the
Carbonic Anhydrases as Biocatalysts. DOI: 10.1016/B978-0-444-63258-6.00021-4 Copyright © 2015 Elsevier B.V. All rights reserved
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Carbonic Anhydrases as Biocatalysts
Figure 21.1 Reactions catalyzed by a-CAs.
hydrolysis of carboxylic or sulfonic esters, and also some other hydrolytic processes (3) (Figure 21.1). No data are currently available on the physiological relevance of all these reactions; nevertheless, the particular characteristic to catalyze other hydrolytic reactions has been well investigated in the case of the esterasic properties of CAs. First demonstrated in 1962 with 1-naphthyl acetate (4), the esterase activity by CAs was then extended to different substrates such as nitrophenyl acetate and has been exploited successfully for 40 years, especially in the kinetic studies of CA inhibitors (CAIs) (5). This unique ability of CAs to catalyze hydrolytic reactions with very high specificities can be exploited industrially as well as in synthetic organic chemistry. In this chapter, we will provide an overview of the literature regarding the esterase activity of CAs and its potential applications in biotechnology.
21.2 CA AND ESTERASE ACTIVITY 21.2.1 Hydrolysis of esters by CAs The ability of CAs to catalyze the hydrolysis of esters is known for 50 years. After pioneering work on 1-naphthyl acetate (4), hydrolysis of acetate esters containing various aromatic alcohol groups, such as nitrophenyl (6–8) and other substituted phenols (9), has been investigated. Hydrolysis of other types of substrates has been demonstrated as well, for example, pyruvate esters (10), 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone (11), and recently per-O-acylated sugar-based sulfamates (12). The various ester substrates are hydrolyzed with different efficiencies by CAs, substrate recognition being based on both the acyl part and the alcohol part of the substrate. Earlier studies have shown that bovine CA I catalyzes the hydrolysis of paranitrophenyl esters with different efficiencies depending on the structure of the acyl part
Carbonic Anhydrases as Esterases and Their Biotechnological Applications
of the substrate. Ester substrates with long and bulky acyl groups are hydrolyzed less efficiently than smaller substrates (6,8). Activity of hCA II for hydrolysis of an isologous series of aliphatic para-nitrophenyl esters differing in the length of the acyl chain from one to five carbon atoms (para-nitrophenyl acetate (pNPA), para-nitrophenyl propionate (pNPP), para-nitrophenyl butyrate (pNPB), para-nitrophenyl valerate (pNPV), and para-nitrophenyl caproate (pNPC)) was described by Jonsson and collaborators. They showed that the pattern of specificity for hCA II was similar to that for the bovine enzyme, with the highest catalytic efficiency (kcat/KM) for pNPA, and steadily decreasing efficiencies for longer substrates (13) (Table 21.1). Substrate selectivity was also demonstrated with respect to the alcohol part of the substrate. Indeed, both bovine CA I and human CA II hydrolyze pNPA more efficiently than ortho-nitrophenyl acetate, while human CA I is more efficient with ortho-nitrophenyl acetate than pNPA (6,7). These substrates have similar pKa values for the corresponding nitrophenol group, and it is therefore not expected that the enzymes display activity differences based on differences in ester bond stability. In the case of human CA II, a linear relationship between the pKa of the phenolic leaving group and hCA II esterase substrate reactivity was demonstrated by Gould and Tawfik and collaborators with a series of substituted phenyl acetates (14). In their study, they showed that activated esters,where the pKa of the alcohol product is low,are the best substrates for the esterase activity of hCA II.This is exemplified by 4-nitrophenyl acetate (pKa of 4-nitrophenol = 7.14), which is the most active known hCA II substrate for ester hydrolysis, while 4-methoxyphenyl acetate (pKa of 4-methoxyphenol = 10.29) is ∼400-fold less active (Table 21.2). Table 21.1 Activity of hCA II against an isologous series of aliphatic para-nitrophenyl esters (13) Substrate
kcat/KM (M−1 s−1)
pNPA pNPP pNPB pNPV pNPC
2080 516 47 3.2 1.7
Table 21.2 Rate of hydrolysis with a series of substituted phenyl acetates with leaving groups having different pKa values (14) Substrate
Alcohol pKa
kcat/KM (M−1 s−1)
4-Methoxyphenyl acetate 4-Chlorophenyl acetate 3-Cyanophenyl acetate 2,4-Difluorophenyl acetate 4-Cyanophenyl acetate 2,3-Difluorophenyl acetate 4-Nitrophenyl acetate
10.29 9.38 8.61 8.43 7.95 7.81 7.14
5.3 53 72 133 210 1140 2050
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Carbonic Anhydrases as Biocatalysts
The observation of substrate recognition based on both the acyl part and the alcohol part of the substrate suggests the possibility of engineering hCA II variants with selectivity for a variety of different substrates (see Section 21.3).
21.2.2 Mechanistic aspects The esterase reaction apparently has many features in common with the CO2 hydration reaction.The zinc ion, which is strongly bound to the CAs, is essential for the esterase activity as well as for CO2 hydration. Moreover, both reactions are strongly inhibited by sulfonamides and, to a lesser degree, by anions. hCA II is one of the most proficient enzymes known, exhibiting a kcat value of around 106 turnovers per second and a kcat/KM value close to the diffusion limit (108 s−1 M−1) for CO2 hydration reaction. hCA II also exhibits a weak and promiscuous esterase activity toward activated esters such as pNPA (kcat/KM ∼ 103 s−1 M−1). Resolution of X-ray crystal structure of several a-CAs allowed understanding this particular feature of these enzymes. Indeed, the three-dimensional structures of the different isoforms revealed that CA catalysis takes place in a large, cone-shaped cavity where the zinc ion is found at the bottom, coordinated by three histidine residues. In the neighborhood of the metal ion is found a hydrophobic pocket composed of the residues Val121, Val143, Leu198, and Trp209 where the CO2 is located and that can accommodate other ligands much larger than CO2 that can undergo esterase activity (3). This promiscuous esterase activity probably stems from the mechanistic similarity between hydration of the carbonyl of CO2 and that of an ester (14). In Figure 21.2 is reported a schematic representation of the proposed catalytic mechanisms for the CO2 hydration reaction and ester hydrolysis, both catalyzed by CAs. The active form of the enzyme is the basic one with hydroxide bound to Zn(II) ion (A). This strong nucleophile attacks the CO2 molecule (B) or the ester molecule (B9) bound in the hydrophobic pocket in its neighborhood leading to the formation of bicarbonate (C) or acid (C9) coordinated to Zn(II). The bicarbonate or acid molecule is then displaced by a water molecule and liberated into solution, leading to the acid form of the enzyme with water coordinated to Zn(II) (D) that is catalytically inactive. The generation of the basic form (A) is then accomplished via a proton transfer reaction from the active site to the environment that may be assisted by active site residues or by buffers present in the medium.
21.2.3 CA esterase activity on coumarins and sulfocoumarins Promiscuous esterase activity of CAs was also demonstrated on cyclic ester substrates. This was first showed by Supuran and coworkers who reported in 2009 the esterase activity of hCA II on a natural coumarin, 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2Hchromen-2-one. This compound showed significant inhibitory activity against hCA II acting as “prodrug” inhibitor, being hydrolyzed by the enzyme to the corresponding cis-2-hydroxy-cinnamic acid derivative 2, which is the de facto CAI as demonstrated by X-ray crystallography analysis (15,16) (Figure 21.3). Thiocoumarins were then shown to behave similarly (16).
Carbonic Anhydrases as Esterases and Their Biotechnological Applications
Figure 21.2 Schematic representation of the proposed catalytic mechanisms for the CA-catalyzed CO2 hydration (left side) and CA-catalyzed ester hydrolysis (right side).
Figure 21.3 Hydrolysis of the natural coumarin 1 or simple coumarin 3 by hCA II.
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Several interesting facts emerged from studies on coumarins and thiocoumarins as CAIs: (i) this new class of CAIs binds in hydrolyzed form at the entrance of the CA active site (which differs significantly between the various isoforms) and does not interact with the metal ion, thus constituting a new category of mechanism-based inhibitors; (ii) the formed substituted cinnamic acids were observed bound within the CA active site as either the cis- or trans-2-hydroxycinnamic acids (although these derivatives are stable in solution only as trans isomers); (iii) a very high level of isoform selectivity has been observed for many coumarins/thiocoumarins assayed so far. This finding led to important consequences for the design of inhibitors with clinical applications opening novel strategies for the design of antitumor therapies. A large number of coumarin derivatives possessing CA isoform-selective behavior were identified, and among these highly selective hCA IX/XII inhibitors were discovered. One such compound, the highly soluble glycosyl-substituted coumarin 6, being a selective nanomolar inhibitor of hCA IX and hCA XII, showed excellent antitumor and antimetastatic effects in an animal model of breast cancer and has been further evaluated in preclinical models of this disease (17) (Figure 21.4). Very recently Supuran and coworkers reported that the coumarin bioisoster sulfocoumarins, that is, 1,2-benzoxathiine 2,2-dioxides, possess a similar mechanism of CA inhibition as the coumarins, acting as effective inhibitors of these enzymes. The sulfocoumarins are hydrolyzed by the esterase CA activity to 2-hydroxyphenyl-vinylsulfonic acids (Figure 21.5), which thereafter bind within the enzyme active site in a region rarely occupied by classical sulfonamide inhibitors (18).
Figure 21.4 Structure of the highly selective CA IX/CA XII glycosyl coumarin inhibitor 6.
Figure 21.5 Hydrolysis of sulfocoumarin by hCA II.
Carbonic Anhydrases as Esterases and Their Biotechnological Applications
21.3 BIOTECHNOLOGICAL APPLICATIONS OF ESTERASE ACTIVITY OF CAs 21.3.1 Without changes in the active site of CAs One of the drawbacks of the use of enzymes in catalysis is their low stability to changes in the pH, temperature, etc.Thus, several methodologies have been developed to stabilize the proteins. Among them, the sol–gel method is one of the most useful in the design of sensors, catalyst supports, etc. Enzymes may be entrapped in silica glasses, retaining their chemical activities and opening possibilities for research and application in biocatalysts, biotechnology, etc. Badjic´ and Kostic´ encapsulated bovine CA II in silica monoliths by the sol–gel method (19).The authors performed the hydrolysis of pNPA in the presence of the transparent monoliths of silica doped with the enzyme. It should be noted that specific activity of the encapsulated enzyme was only 1–2% of that of the enzyme in solution. In fact, due to the slow diffusion into silica pores, most of the catalyst is due to the CA embedded near the surfaces of the monoliths. Very recently, Pack, Lee, and coworkers studied the a-CA from the halotolerant green alga, Dunaliella sp. (Dsp-CA), a duplicated enzyme composed of two tandem repeats, N-half (Dsp-CA-n) and C-half (Dsp-CA-c) repeats (20). The purified N-half domain showed very low esterase activity, but the purified C-half domain retained its activity. However, the mixture of the two half domains at the ratio 1:1 showed increased esterase activity. Although it is a preliminary study, the results suggest that the activity of a duplicated CA could be enhanced by expressing each half CA domain individually and by in vitro reconstitution. This new strategy for enhancing CA functionality may be employed for development of more efficient systems with esterase activity in a large range of salinities.
21.3.2 Engineering CA active site Several modifications have been done by engineering CA active site, to increase the activity and/or specificity of the enzyme. Lindskog and Thorslund studied the metal specificity of the bovine CA II, showing that Zn(II) and Co(II) are the only efficient reactivators of the esterase activity to the apoenzyme while Cu(II), Ni(II), Fe(II), Mn(II), and Cd(II) have no effect (6). Due to these results, no further studies have been performed on the modification of the metal ion. The replacement of amino acids at different sequence positions of the active site of hCA II has given the best results in the enhancement of the esterase activity of the enzyme. Lindskog and coworkers demonstrated that the catalytic properties of hCA II are sensitive to the nature of the active site residue Thr200 (21). In fact, the 4-nitrophenyl acetate hydrolysis of hCA II mutants, obtained through replacing nine different amino acids in position 200 by site-directed mutagenesis, showed that the variant with Asp200 has a very low esterase activity, while most of the other variants have enhanced esterase activities with activity of the mutant Thr200Arg being as much as seven-fold.
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These data indicate that probably, the substrate is positioned in the active site so that this basic residue has an orientation favoring the stabilization of a negative charge formed in a tetrahedral transition state. In accordance with this result, the negatively charged side chain in the mutant with Asp in position 200 probably destabilizes such a transition state during the hydrolysis of all investigated substrates. Later on, Lindskog and coworkers compared the rates of hydrolysis of 4-, 3-, and 2-nitrophenyl acetates catalyzed by mutant forms of hCA II at sequence positions 7, 65, and 200 (Table 21.3) (22). The results showed that the substrate specificity is affected only by some mutations at position 200. The Thr200Gly mutation results in a 380-fold enhancement of the activity with 2-nitrophenyl acetate, making this compound a better substrate than the para-substituted ester. These changes could be explained in terms of steric hindrance between the side chain of residue 200 and the nitro group of the ortho-substituted substrate. However, the mutants with basic amino acids behave quite different. As was stated above, the tetrahedral transition state develops a negative charge, which could be stabilized by basic residues.With 3-nitrophenyl acetate as substrate, some mutations (Thr200Gly, Thr200Ala, and Thr200Ser) shifted the enzyme specificity in favor of the meta-substituted ester. As for 2-nitrophenyl acetate, these changes involving mutants at position 200 could be caused by steric hindrance. It is largely known that an increased size of the acyl moiety of 4-nitrophenyl esters results in decreased hydrolysis rates. To test the hypothesis that this size dependence reflects a steric interference between the acyl chain and the wall of the hydrophobic pocket of hCA II, the authors also prepared mutants at several positions of this pocket. Change in residue 207 had no effect, probably due to the distance of it from the zinc ion. The Val121Ala and Leu198Ala mutations resulted in decreased activities with both substrates. Thus, these residues do not interact with the acyl groups of the substrates. A different situation was found with the mutants Val143Gly and Val143Ala, with which the propionyl ester was better substrate than the acetyl ester. These results could be caused by the removal of steric hindrance in Table 21.3 Activities* and specificities† of hCA II and enzyme mutants against a series of nitrophenyl acetates
hCA II Tyr7Phe Ala65Leu Thr200Ala Thr200Gly Thr200Ser Tyr7Phe/Thr200Gly Ala65Leu/Thr200Gly
pNPA
mNPA
oNPA
2.800 6.300 13.000 5.000 7.900 7.900 4.600 14.000
410 (0.15) 900 (0.14) 2.300 (0.18) 4.100 (0.82) 4.300 (0.54) 2.900 (0.37) ND ND
120 (0.042) 360 (0.057) 840 (0.066) 15.000 (3.0) 47.000 (5.9) 3.100 (0.39) 35.000 (7.6) 79.000 (5.5)
* Esterase activity kcat/KM (M−1 s−1). † Value in parentheses indicates the activity on each substrate compared with the corresponding value for the pNPA.
Carbonic Anhydrases as Esterases and Their Biotechnological Applications
the mutants and by favorable hydrophobic interactions. Closely related results have been previously found by Fierke and coworkers studying mutations of hCA II at Val143 and at Leu198 (23,24). It is interesting to note that the results discussed above are in full accordance with later results found by Tawfik’s group. In 2005, they used directed evolution to increase reactivity of hCA II toward the nonactivated 2-naphthyl acetate substrate (2NA) (14). One of the variants showed a 40-fold enhancement of the 2NA hydrolysis and had three mutations: Asp110Asn, Thr200Ala, and Ala65Val. The authors mentioned that by comparison with other mutants, only the latter two mutations are significant. The mutation of Thr200 to a smaller group enhances the availability of the active site to bulkier and more polar compounds. The triple mutant was evaluated against a series of substituted phenyl esters, and the authors found a linear relationship between the pKa of the leaving group and esterase substrate reactivity. A similar relationship was found with native hCA II (see above), suggesting that all the substrates studied have a similar binding mode. The triple mutant showed a higher esterase activity than hCA II, with up to 60-fold enhancement of the hydrolysis when 2,4-difluorophenyl was the substrate. In 2006, Jonsson and coworkers redesigned hCA II by mutations of Val121 and Val143 to allow for specific biding and efficient catalysis of several p-nitrophenyl esters with increased acyl chain lengths (Table 21.4) (13). The authors studied the hydrolysis of the esters catalyzed by three mutants:Val143Ala,Val121Ala/Val143Ala, and Val121Ala. At position 121, removal of two methyl groups resulted in a reduced activity for esters with less than five carbon atoms in the acyl chain probably due to removal of hydrophobic interactions.With longer acyl chains, opposite results were found, suggesting that the side chain in position 121 is a steric hindrance for them. The Val143Ala mutation showed similar results, but the hydrophobic interaction was found only with the pNPA. The combination of the two mutations enhanced the hydrolysis of almost all the esters studied, leading only to a less efficient hydrolysis of the acetate substrate. Also automated docking was used to probe if the observed enhancement was correlated to binding Table 21.4 Activities* of hCA II and enzyme mutants against a series of para-nitrophenyl esters
hCA II Val121Ala Val143Ala Val121Ala/143Ala Val121Ala/Val143Ala/ Thr200Ala
pNPA
pNPP
pNPB
pNPV
pNPC
pNPBenzo†
2.080 472 645 1.554 101.666
516 88 13.882 1.820 43.714
47 14.6 2.713 2.491 29.241
3.2 13.6 803 9.810 46.310
1.7 10.4 41 2.178 55.447
—‡ ND ND —‡ 625
* Esterase activity kcat/KM (M−1 s−1). † pNPBenzo: para-nitrophenyl benzoate. ‡ The activity was too low to be determined.
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in the transition state. The analysis showed that the results depended on both removal of steric hindrance and introduction of additional favorable interactions between the transition states and the enzymes. Later on, the docking experiments were performed to predict mutations that could allow the hydrolysis of p-nitrophenyl benzoate (25). In accordance with the results previously found by other groups, these studies suggested that the steric hindrance with Thr200 should be removed. Thus, Jonsson and coworkers evaluated the mutant Val121Ala/Val143Ala/Thr200Ala, which showed to be able to hydrolyze benzoate ester, while native hCA II and Val121Ala/Val143Ala mutant were inactive (Table 21.4). It is important to note that the additional Thr200Ala mutation allowed to develop the most efficient esterase compared with wild-type hCA II for all substrates tested. The mutant was tested for hydrolysis of the benzoate ester in cocaine, but no positive results were found. These findings suggest that further efforts are necessary to develop a mutant useful for industrial or medical applications.
REFERENCES 1. Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81. 2. Alterio V, Di Fiore A, D’Ambrosio K, Supuran CT, De Simone G. Multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoforms? Chem Rev 2012;112:4421–68. 3. Supuran CT, Winum J-Y. Drug design of zinc-enzyme inhibitors. Hoboken, NJ: John Wiley & Sons, Inc.; 2009. 4. Shaw CR, Syner FN, Tashian RE. New genetically determined molecular form of erythrocyte esterase in man. Science 1962;138:31–2. 5. Pocker Y, Stone JT. The catalytic versatility of erythrocyte carbonic anhydrase. III. Kinetic studies of the enzyme-catalyzed hydrolysis of p-nitrophenyl acetate. Biochemistry 1967;6:668–78. 6. Thorslund A, Lindskog S. Studies of the esterase activity and the anion inhibition of bovine zinc and cobalt carbonic anhydrases. Eur J Biochem 1967;3:117–23. 7. Verpoorte JA, Mehta S, Edsall JT. Esterase activities of human carbonic anhydrase B and C. J Biol Chem 1967;242:4221–9. 8. Pocker Y, Storm DR. The catalytic versatility of erythrocyte carbonic anhydrase. IV. Kinetic studies of enzyme-catalysed hydrolyses of p-nitrophenyl esters. Biochemistry 1968;7:1202–14. 9. Pocker Y, Beug MW. Kinetic studies of bovine carbonic anhydrase catalyzed hydrolyses of parasubstituted phenyl esters. Biochemistry 1972;11:698–707. 10. Pocker Y, Meany JE, Davis BC, Arrigoni J, Stein JE. Bifunctional substrates of erythrocyte carbonic anhydrase. Enzyme-catalyzed hydration and hydrolysis of pyruvate esters. J Am Chem Soc 1978;100:2883–5. 11. Kaiser ET, Lo KW.The carbonic anhydrase catalyzed hydrolysis of 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone. J Am Chem Soc 1969;91:4912–8. 12. Lopez M, Vu H, Wang CK, Wolf MG, Groenhof G, Innocenti A, et al. Promiscuity of carbonic anhydrase II. Unexpected ester hydrolysis of carbohydrate-based sulfamate inhibitors. J Am Chem Soc 2011;133:18452–62. 13. Höst G, Mårtensson LG, Jonsson BH. Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains. Biochim Biophys Acta 2006;1764:1601–6. 14. Gould SM,Tawfik DS. Directed evolution of the promiscuous esterase activity of carbonic anhydrase II. Biochemistry 2005;44:5444–52. 15. Maresca A, Temperini C,Vu H, Pham NB, Poulsen SA, Scozzafava A, et al. Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide inhibitors. J Am Chem Soc 2009;131: 3057–62.
Carbonic Anhydrases as Esterases and Their Biotechnological Applications
16. Maresca A,Temperini C, Pochet L, Masereel B, Scozzafava A, Supuran CT. Deciphering the mechanism of carbonic anhydrase inhibition with coumarins and thiocoumarins. J Med Chem 2010;53:335–44. 17. Touisni N, Maresca A, McDonald PC, Lou Y, Scozzafava A, Dedhar S, et al. Glycosyl coumarin carbonic anhydrase IX and XII inhibitors strongly attenuate the growth of primary breast tumors. J Med Chem 2011;54:8271–7. 18. Tars K,Vullo D, Kazaks A, Leitans J, Lends A, Grandane A, et al. Sulfocoumarins (1,2-benzoxathiine-2,2dioxides): a class of potent and isoform-selective inhibitors of tumor-associated carbonic anhydrases. J Med Chem 2013;56:293–300. 19. Badjić JD, Kostić NM. Effects of encapsulation in sol–gel silica glass on esterase activity, conformational stability, and unfolding of bovine carbonic anhydrase II. Chem Mater 1991;11:3671–9. 20. Ki MR, Kanth BK, Min KH, Lee J, Pack SP. Increased expression level and catalytic activity of internally-duplicated carbonic anhydrase from Dunaliella species by reconstitution of two separate domains. Proc Biochem 2012;47:1423–7. 21. Behravan G, Jonsson BH, Lindskog S. Fine tuning of the catalytic properties of human carbonic anhydrase II. Effects of varying active-site residue 200. Eur J Biochem 1991;195:393–6. 22. Elleby B, Sjöblom B, Lindskog S. Changing the efficiency and specificity of the esterase activity of human carbonic anhydrase II by site-specific mutagenesis. Eur J Biochem 1999;262:516–21. 23. Fierke CA. Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II. Biochemistry 1991;30:11054–63. 24. Krebs JF, Rana F, Dluhy RA, Fierke CA. Kinetic and spectroscopic studies of hydrophilic amino acid substitutions in the hydrophobic pocket of human carbonic anhydrase II. Biochemistry 1993;32: 4496–505. 25. Höst GE, Jonssonn BH. Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign. Biochim Biophys Acta 2008;1784:811–5.
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Carbonic Anhydrases as Esterases and Their Biotechnological Applications Jean-Yves Winum*, Pedro Colinas**
*Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-UM1-UM2, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier, Montpellier Cedex, France **LADECOR, Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Contents 21.1 Introduction 361 21.2 CA and esterase activity 362 21.2.1 Hydrolysis of esters by CAs 362 21.2.2 Mechanistic aspects 364 21.2.3 CA esterase activity on coumarins and sulfocoumarins 364 21.3 Biotechnological applications of esterase activity of CAs 367 21.3.1 Without changes in the active site of CAs 367 21.3.2 Engineering CA active site 367 References 370
21.1 INTRODUCTION The carbonic anhydrases (CAs, EC 4.2.1.1) belong to the lyase group of enzymes and are widely distributed in all organisms starting from Archaea to higher animals including vertebrates. They are implicated in a variety of physiological and pathological processes, catalyzing the reversible carbon dioxide hydration reaction to bicarbonate and proton with maximum turnover numbers among the highest known for any enzyme. Important advancements have been achieved in the CA research field, especially in the medicinal chemistry domain where now CA isozymes are important targets for the design of inhibitors or activators with clinical applications as anticancer, antiglaucoma, anticonvulsant, antipain, antiobesity, and probably soon as anti-infective drugs (antibacterial and antifungal agents). Many important review papers have been published on this particular topic in the last years (1,2). For a long time, it was believed that CAs exhibited absolute specificity, that is, that they would only catalyze the physiological interconversion between CO2 and HCO3−. However, in the 1960s it was discovered that CAs, especially those from the a-family, can also catalyze a variety of other reactions such as the hydration of cyanate to carbamic acid or the hydration of cyanamide to urea, the aldehyde hydration to gem-diols, the
Carbonic Anhydrases as Biocatalysts. DOI: 10.1016/B978-0-444-63258-6.00021-4 Copyright © 2015 Elsevier B.V. All rights reserved
361
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Carbonic Anhydrases as Biocatalysts
Figure 21.1 Reactions catalyzed by a-CAs.
hydrolysis of carboxylic or sulfonic esters, and also some other hydrolytic processes (3) (Figure 21.1). No data are currently available on the physiological relevance of all these reactions; nevertheless, the particular characteristic to catalyze other hydrolytic reactions has been well investigated in the case of the esterasic properties of CAs. First demonstrated in 1962 with 1-naphthyl acetate (4), the esterase activity by CAs was then extended to different substrates such as nitrophenyl acetate and has been exploited successfully for 40 years, especially in the kinetic studies of CA inhibitors (CAIs) (5). This unique ability of CAs to catalyze hydrolytic reactions with very high specificities can be exploited industrially as well as in synthetic organic chemistry. In this chapter, we will provide an overview of the literature regarding the esterase activity of CAs and its potential applications in biotechnology.
21.2 CA AND ESTERASE ACTIVITY 21.2.1 Hydrolysis of esters by CAs The ability of CAs to catalyze the hydrolysis of esters is known for 50 years. After pioneering work on 1-naphthyl acetate (4), hydrolysis of acetate esters containing various aromatic alcohol groups, such as nitrophenyl (6–8) and other substituted phenols (9), has been investigated. Hydrolysis of other types of substrates has been demonstrated as well, for example, pyruvate esters (10), 2-hydroxy-5-nitro-a-toluenesulfonic acid sultone (11), and recently per-O-acylated sugar-based sulfamates (12). The various ester substrates are hydrolyzed with different efficiencies by CAs, substrate recognition being based on both the acyl part and the alcohol part of the substrate. Earlier studies have shown that bovine CA I catalyzes the hydrolysis of paranitrophenyl esters with different efficiencies depending on the structure of the acyl part
Carbonic Anhydrases as Esterases and Their Biotechnological Applications
of the substrate. Ester substrates with long and bulky acyl groups are hydrolyzed less efficiently than smaller substrates (6,8). Activity of hCA II for hydrolysis of an isologous series of aliphatic para-nitrophenyl esters differing in the length of the acyl chain from one to five carbon atoms (para-nitrophenyl acetate (pNPA), para-nitrophenyl propionate (pNPP), para-nitrophenyl butyrate (pNPB), para-nitrophenyl valerate (pNPV), and para-nitrophenyl caproate (pNPC)) was described by Jonsson and collaborators. They showed that the pattern of specificity for hCA II was similar to that for the bovine enzyme, with the highest catalytic efficiency (kcat/KM) for pNPA, and steadily decreasing efficiencies for longer substrates (13) (Table 21.1). Substrate selectivity was also demonstrated with respect to the alcohol part of the substrate. Indeed, both bovine CA I and human CA II hydrolyze pNPA more efficiently than ortho-nitrophenyl acetate, while human CA I is more efficient with ortho-nitrophenyl acetate than pNPA (6,7). These substrates have similar pKa values for the corresponding nitrophenol group, and it is therefore not expected that the enzymes display activity differences based on differences in ester bond stability. In the case of human CA II, a linear relationship between the pKa of the phenolic leaving group and hCA II esterase substrate reactivity was demonstrated by Gould and Tawfik and collaborators with a series of substituted phenyl acetates (14). In their study, they showed that activated esters,where the pKa of the alcohol product is low,are the best substrates for the esterase activity of hCA II.This is exemplified by 4-nitrophenyl acetate (pKa of 4-nitrophenol = 7.14), which is the most active known hCA II substrate for ester hydrolysis, while 4-methoxyphenyl acetate (pKa of 4-methoxyphenol = 10.29) is ∼400-fold less active (Table 21.2). Table 21.1 Activity of hCA II against an isologous series of aliphatic para-nitrophenyl esters (13) Substrate
kcat/KM (M−1 s−1)
pNPA pNPP pNPB pNPV pNPC
2080 516 47 3.2 1.7
Table 21.2 Rate of hydrolysis with a series of substituted phenyl acetates with leaving groups having different pKa values (14) Substrate
Alcohol pKa
kcat/KM (M−1 s−1)
4-Methoxyphenyl acetate 4-Chlorophenyl acetate 3-Cyanophenyl acetate 2,4-Difluorophenyl acetate 4-Cyanophenyl acetate 2,3-Difluorophenyl acetate 4-Nitrophenyl acetate
10.29 9.38 8.61 8.43 7.95 7.81 7.14
5.3 53 72 133 210 1140 2050
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The observation of substrate recognition based on both the acyl part and the alcohol part of the substrate suggests the possibility of engineering hCA II variants with selectivity for a variety of different substrates (see Section 21.3).
21.2.2 Mechanistic aspects The esterase reaction apparently has many features in common with the CO2 hydration reaction.The zinc ion, which is strongly bound to the CAs, is essential for the esterase activity as well as for CO2 hydration. Moreover, both reactions are strongly inhibited by sulfonamides and, to a lesser degree, by anions. hCA II is one of the most proficient enzymes known, exhibiting a kcat value of around 106 turnovers per second and a kcat/KM value close to the diffusion limit (108 s−1 M−1) for CO2 hydration reaction. hCA II also exhibits a weak and promiscuous esterase activity toward activated esters such as pNPA (kcat/KM ∼ 103 s−1 M−1). Resolution of X-ray crystal structure of several a-CAs allowed understanding this particular feature of these enzymes. Indeed, the three-dimensional structures of the different isoforms revealed that CA catalysis takes place in a large, cone-shaped cavity where the zinc ion is found at the bottom, coordinated by three histidine residues. In the neighborhood of the metal ion is found a hydrophobic pocket composed of the residues Val121, Val143, Leu198, and Trp209 where the CO2 is located and that can accommodate other ligands much larger than CO2 that can undergo esterase activity (3). This promiscuous esterase activity probably stems from the mechanistic similarity between hydration of the carbonyl of CO2 and that of an ester (14). In Figure 21.2 is reported a schematic representation of the proposed catalytic mechanisms for the CO2 hydration reaction and ester hydrolysis, both catalyzed by CAs. The active form of the enzyme is the basic one with hydroxide bound to Zn(II) ion (A). This strong nucleophile attacks the CO2 molecule (B) or the ester molecule (B9) bound in the hydrophobic pocket in its neighborhood leading to the formation of bicarbonate (C) or acid (C9) coordinated to Zn(II). The bicarbonate or acid molecule is then displaced by a water molecule and liberated into solution, leading to the acid form of the enzyme with water coordinated to Zn(II) (D) that is catalytically inactive. The generation of the basic form (A) is then accomplished via a proton transfer reaction from the active site to the environment that may be assisted by active site residues or by buffers present in the medium.
21.2.3 CA esterase activity on coumarins and sulfocoumarins Promiscuous esterase activity of CAs was also demonstrated on cyclic ester substrates. This was first showed by Supuran and coworkers who reported in 2009 the esterase activity of hCA II on a natural coumarin, 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2Hchromen-2-one. This compound showed significant inhibitory activity against hCA II acting as “prodrug” inhibitor, being hydrolyzed by the enzyme to the corresponding cis-2-hydroxy-cinnamic acid derivative 2, which is the de facto CAI as demonstrated by X-ray crystallography analysis (15,16) (Figure 21.3). Thiocoumarins were then shown to behave similarly (16).
Carbonic Anhydrases as Esterases and Their Biotechnological Applications
Figure 21.2 Schematic representation of the proposed catalytic mechanisms for the CA-catalyzed CO2 hydration (left side) and CA-catalyzed ester hydrolysis (right side).
Figure 21.3 Hydrolysis of the natural coumarin 1 or simple coumarin 3 by hCA II.
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Several interesting facts emerged from studies on coumarins and thiocoumarins as CAIs: (i) this new class of CAIs binds in hydrolyzed form at the entrance of the CA active site (which differs significantly between the various isoforms) and does not interact with the metal ion, thus constituting a new category of mechanism-based inhibitors; (ii) the formed substituted cinnamic acids were observed bound within the CA active site as either the cis- or trans-2-hydroxycinnamic acids (although these derivatives are stable in solution only as trans isomers); (iii) a very high level of isoform selectivity has been observed for many coumarins/thiocoumarins assayed so far. This finding led to important consequences for the design of inhibitors with clinical applications opening novel strategies for the design of antitumor therapies. A large number of coumarin derivatives possessing CA isoform-selective behavior were identified, and among these highly selective hCA IX/XII inhibitors were discovered. One such compound, the highly soluble glycosyl-substituted coumarin 6, being a selective nanomolar inhibitor of hCA IX and hCA XII, showed excellent antitumor and antimetastatic effects in an animal model of breast cancer and has been further evaluated in preclinical models of this disease (17) (Figure 21.4). Very recently Supuran and coworkers reported that the coumarin bioisoster sulfocoumarins, that is, 1,2-benzoxathiine 2,2-dioxides, possess a similar mechanism of CA inhibition as the coumarins, acting as effective inhibitors of these enzymes. The sulfocoumarins are hydrolyzed by the esterase CA activity to 2-hydroxyphenyl-vinylsulfonic acids (Figure 21.5), which thereafter bind within the enzyme active site in a region rarely occupied by classical sulfonamide inhibitors (18).
Figure 21.4 Structure of the highly selective CA IX/CA XII glycosyl coumarin inhibitor 6.
Figure 21.5 Hydrolysis of sulfocoumarin by hCA II.
Carbonic Anhydrases as Esterases and Their Biotechnological Applications
21.3 BIOTECHNOLOGICAL APPLICATIONS OF ESTERASE ACTIVITY OF CAs 21.3.1 Without changes in the active site of CAs One of the drawbacks of the use of enzymes in catalysis is their low stability to changes in the pH, temperature, etc.Thus, several methodologies have been developed to stabilize the proteins. Among them, the sol–gel method is one of the most useful in the design of sensors, catalyst supports, etc. Enzymes may be entrapped in silica glasses, retaining their chemical activities and opening possibilities for research and application in biocatalysts, biotechnology, etc. Badjic´ and Kostic´ encapsulated bovine CA II in silica monoliths by the sol–gel method (19).The authors performed the hydrolysis of pNPA in the presence of the transparent monoliths of silica doped with the enzyme. It should be noted that specific activity of the encapsulated enzyme was only 1–2% of that of the enzyme in solution. In fact, due to the slow diffusion into silica pores, most of the catalyst is due to the CA embedded near the surfaces of the monoliths. Very recently, Pack, Lee, and coworkers studied the a-CA from the halotolerant green alga, Dunaliella sp. (Dsp-CA), a duplicated enzyme composed of two tandem repeats, N-half (Dsp-CA-n) and C-half (Dsp-CA-c) repeats (20). The purified N-half domain showed very low esterase activity, but the purified C-half domain retained its activity. However, the mixture of the two half domains at the ratio 1:1 showed increased esterase activity. Although it is a preliminary study, the results suggest that the activity of a duplicated CA could be enhanced by expressing each half CA domain individually and by in vitro reconstitution. This new strategy for enhancing CA functionality may be employed for development of more efficient systems with esterase activity in a large range of salinities.
21.3.2 Engineering CA active site Several modifications have been done by engineering CA active site, to increase the activity and/or specificity of the enzyme. Lindskog and Thorslund studied the metal specificity of the bovine CA II, showing that Zn(II) and Co(II) are the only efficient reactivators of the esterase activity to the apoenzyme while Cu(II), Ni(II), Fe(II), Mn(II), and Cd(II) have no effect (6). Due to these results, no further studies have been performed on the modification of the metal ion. The replacement of amino acids at different sequence positions of the active site of hCA II has given the best results in the enhancement of the esterase activity of the enzyme. Lindskog and coworkers demonstrated that the catalytic properties of hCA II are sensitive to the nature of the active site residue Thr200 (21). In fact, the 4-nitrophenyl acetate hydrolysis of hCA II mutants, obtained through replacing nine different amino acids in position 200 by site-directed mutagenesis, showed that the variant with Asp200 has a very low esterase activity, while most of the other variants have enhanced esterase activities with activity of the mutant Thr200Arg being as much as seven-fold.
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These data indicate that probably, the substrate is positioned in the active site so that this basic residue has an orientation favoring the stabilization of a negative charge formed in a tetrahedral transition state. In accordance with this result, the negatively charged side chain in the mutant with Asp in position 200 probably destabilizes such a transition state during the hydrolysis of all investigated substrates. Later on, Lindskog and coworkers compared the rates of hydrolysis of 4-, 3-, and 2-nitrophenyl acetates catalyzed by mutant forms of hCA II at sequence positions 7, 65, and 200 (Table 21.3) (22). The results showed that the substrate specificity is affected only by some mutations at position 200. The Thr200Gly mutation results in a 380-fold enhancement of the activity with 2-nitrophenyl acetate, making this compound a better substrate than the para-substituted ester. These changes could be explained in terms of steric hindrance between the side chain of residue 200 and the nitro group of the ortho-substituted substrate. However, the mutants with basic amino acids behave quite different. As was stated above, the tetrahedral transition state develops a negative charge, which could be stabilized by basic residues.With 3-nitrophenyl acetate as substrate, some mutations (Thr200Gly, Thr200Ala, and Thr200Ser) shifted the enzyme specificity in favor of the meta-substituted ester. As for 2-nitrophenyl acetate, these changes involving mutants at position 200 could be caused by steric hindrance. It is largely known that an increased size of the acyl moiety of 4-nitrophenyl esters results in decreased hydrolysis rates. To test the hypothesis that this size dependence reflects a steric interference between the acyl chain and the wall of the hydrophobic pocket of hCA II, the authors also prepared mutants at several positions of this pocket. Change in residue 207 had no effect, probably due to the distance of it from the zinc ion. The Val121Ala and Leu198Ala mutations resulted in decreased activities with both substrates. Thus, these residues do not interact with the acyl groups of the substrates. A different situation was found with the mutants Val143Gly and Val143Ala, with which the propionyl ester was better substrate than the acetyl ester. These results could be caused by the removal of steric hindrance in Table 21.3 Activities* and specificities† of hCA II and enzyme mutants against a series of nitrophenyl acetates
hCA II Tyr7Phe Ala65Leu Thr200Ala Thr200Gly Thr200Ser Tyr7Phe/Thr200Gly Ala65Leu/Thr200Gly
pNPA
mNPA
oNPA
2.800 6.300 13.000 5.000 7.900 7.900 4.600 14.000
410 (0.15) 900 (0.14) 2.300 (0.18) 4.100 (0.82) 4.300 (0.54) 2.900 (0.37) ND ND
120 (0.042) 360 (0.057) 840 (0.066) 15.000 (3.0) 47.000 (5.9) 3.100 (0.39) 35.000 (7.6) 79.000 (5.5)
* Esterase activity kcat/KM (M−1 s−1). † Value in parentheses indicates the activity on each substrate compared with the corresponding value for the pNPA.
Carbonic Anhydrases as Esterases and Their Biotechnological Applications
the mutants and by favorable hydrophobic interactions. Closely related results have been previously found by Fierke and coworkers studying mutations of hCA II at Val143 and at Leu198 (23,24). It is interesting to note that the results discussed above are in full accordance with later results found by Tawfik’s group. In 2005, they used directed evolution to increase reactivity of hCA II toward the nonactivated 2-naphthyl acetate substrate (2NA) (14). One of the variants showed a 40-fold enhancement of the 2NA hydrolysis and had three mutations: Asp110Asn, Thr200Ala, and Ala65Val. The authors mentioned that by comparison with other mutants, only the latter two mutations are significant. The mutation of Thr200 to a smaller group enhances the availability of the active site to bulkier and more polar compounds. The triple mutant was evaluated against a series of substituted phenyl esters, and the authors found a linear relationship between the pKa of the leaving group and esterase substrate reactivity. A similar relationship was found with native hCA II (see above), suggesting that all the substrates studied have a similar binding mode. The triple mutant showed a higher esterase activity than hCA II, with up to 60-fold enhancement of the hydrolysis when 2,4-difluorophenyl was the substrate. In 2006, Jonsson and coworkers redesigned hCA II by mutations of Val121 and Val143 to allow for specific biding and efficient catalysis of several p-nitrophenyl esters with increased acyl chain lengths (Table 21.4) (13). The authors studied the hydrolysis of the esters catalyzed by three mutants:Val143Ala,Val121Ala/Val143Ala, and Val121Ala. At position 121, removal of two methyl groups resulted in a reduced activity for esters with less than five carbon atoms in the acyl chain probably due to removal of hydrophobic interactions.With longer acyl chains, opposite results were found, suggesting that the side chain in position 121 is a steric hindrance for them. The Val143Ala mutation showed similar results, but the hydrophobic interaction was found only with the pNPA. The combination of the two mutations enhanced the hydrolysis of almost all the esters studied, leading only to a less efficient hydrolysis of the acetate substrate. Also automated docking was used to probe if the observed enhancement was correlated to binding Table 21.4 Activities* of hCA II and enzyme mutants against a series of para-nitrophenyl esters
hCA II Val121Ala Val143Ala Val121Ala/143Ala Val121Ala/Val143Ala/ Thr200Ala
pNPA
pNPP
pNPB
pNPV
pNPC
pNPBenzo†
2.080 472 645 1.554 101.666
516 88 13.882 1.820 43.714
47 14.6 2.713 2.491 29.241
3.2 13.6 803 9.810 46.310
1.7 10.4 41 2.178 55.447
—‡ ND ND —‡ 625
* Esterase activity kcat/KM (M−1 s−1). † pNPBenzo: para-nitrophenyl benzoate. ‡ The activity was too low to be determined.
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in the transition state. The analysis showed that the results depended on both removal of steric hindrance and introduction of additional favorable interactions between the transition states and the enzymes. Later on, the docking experiments were performed to predict mutations that could allow the hydrolysis of p-nitrophenyl benzoate (25). In accordance with the results previously found by other groups, these studies suggested that the steric hindrance with Thr200 should be removed. Thus, Jonsson and coworkers evaluated the mutant Val121Ala/Val143Ala/Thr200Ala, which showed to be able to hydrolyze benzoate ester, while native hCA II and Val121Ala/Val143Ala mutant were inactive (Table 21.4). It is important to note that the additional Thr200Ala mutation allowed to develop the most efficient esterase compared with wild-type hCA II for all substrates tested. The mutant was tested for hydrolysis of the benzoate ester in cocaine, but no positive results were found. These findings suggest that further efforts are necessary to develop a mutant useful for industrial or medical applications.
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16. Maresca A,Temperini C, Pochet L, Masereel B, Scozzafava A, Supuran CT. Deciphering the mechanism of carbonic anhydrase inhibition with coumarins and thiocoumarins. J Med Chem 2010;53:335–44. 17. Touisni N, Maresca A, McDonald PC, Lou Y, Scozzafava A, Dedhar S, et al. Glycosyl coumarin carbonic anhydrase IX and XII inhibitors strongly attenuate the growth of primary breast tumors. J Med Chem 2011;54:8271–7. 18. Tars K,Vullo D, Kazaks A, Leitans J, Lends A, Grandane A, et al. Sulfocoumarins (1,2-benzoxathiine-2,2dioxides): a class of potent and isoform-selective inhibitors of tumor-associated carbonic anhydrases. J Med Chem 2013;56:293–300. 19. Badjić JD, Kostić NM. Effects of encapsulation in sol–gel silica glass on esterase activity, conformational stability, and unfolding of bovine carbonic anhydrase II. Chem Mater 1991;11:3671–9. 20. Ki MR, Kanth BK, Min KH, Lee J, Pack SP. Increased expression level and catalytic activity of internally-duplicated carbonic anhydrase from Dunaliella species by reconstitution of two separate domains. Proc Biochem 2012;47:1423–7. 21. Behravan G, Jonsson BH, Lindskog S. Fine tuning of the catalytic properties of human carbonic anhydrase II. Effects of varying active-site residue 200. Eur J Biochem 1991;195:393–6. 22. Elleby B, Sjöblom B, Lindskog S. Changing the efficiency and specificity of the esterase activity of human carbonic anhydrase II by site-specific mutagenesis. Eur J Biochem 1999;262:516–21. 23. Fierke CA. Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II. Biochemistry 1991;30:11054–63. 24. Krebs JF, Rana F, Dluhy RA, Fierke CA. Kinetic and spectroscopic studies of hydrophilic amino acid substitutions in the hydrophobic pocket of human carbonic anhydrase II. Biochemistry 1993;32: 4496–505. 25. Höst GE, Jonssonn BH. Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign. Biochim Biophys Acta 2008;1784:811–5.
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