Kinetic and anion inhibition studies of a β-carbonic anhydrase (FbiCA 1) from the C4 plant Flaveria bidentis

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1626–1630

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Kinetic and anion inhibition studies of a b-carbonic anhydrase (FbiCA 1) from the C4 plant Flaveria bidentis Simona Maria Monti a,⇑, Giuseppina De Simone a, Nina A. Dathan a, Martha Ludwig b, Daniela Vullo c, Andrea Scozzafava c, Clemente Capasso d, Claudiu T. Supuran c,e,⇑ a

Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134 Napoli, Italy School of Chemistry and Biochemistry [M310], The University of Western Australia, Crawley, Western Australia 6009, Australia Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy d Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131 Napoli, Italy e Università degli Studi di Firenze, NEUROFARBA Department, Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy b c

a r t i c l e

i n f o

Article history: Received 22 December 2012 Revised 17 January 2013 Accepted 20 January 2013 Available online 30 January 2013 Keywords: Carbonic anhydrase Beta-class enzyme Anion Enzyme inhibitor Flaveria bidentis Photosynthesis

a b s t r a c t Several b-carbonic anhydrases (CAs, EC 4.2.1.1) are present in all land plants examined thus far. Here we report the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C4 plant Flaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO2 hydrase catalytic activity (kcat of 1.2  105 and kcat/Km of 7.5  106 M1  s1) and was moderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (KIs in the range of 4–60 lM). Such inhibitors may be used as tools to better understand the role of various b-CA isoforms in photosynthesis. Ó 2013 Elsevier Ltd. All rights reserved.

The majority of land plants fix atmospheric CO2 into complex carbohydrates using the C3 photosynthetic pathway. The primary carboxylating enzyme of this pathway is ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO), which uses atmospheric CO2 directly. However, O2 competes with CO2 for the active site of RUBISCO, and the oxygenase activity of the enzyme catalyzes the first step in the photorespiratory pathway, which can lead to 25% of the fixed carbon being lost from C3 plants.1 A number of plant lineages have evolved mechanisms that result in increased concentrations of CO2 around RUBISCO, decreasing its oxygenase activity and significantly reducing photorespiration. One of these biochemical CO2 concentrating mechanisms (CCM) is the C4 photosynthetic pathway, which has evolved more than 60 independent times within the flowering plants from C3 ancestors.1,2 A combination of changes in leaf anatomy and biochemistry are responsible for the increased efficiency of C4 plants, for example, the primary carboxylating enzyme is phosphoenolpyruvate carboxylase (PEPC),

⇑ Corresponding authors. Tel.: +39 0812534583; fax: +39 0812534574 (S.M.M.); tel.: +39 0554573005; fax: +39 0554573385 (C.T.S.). E-mail addresses: [email protected] (S.M. Monti), claudiu.supuran@unifi.it (C.T. Supuran). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.01.087

which requires inorganic carbon in the form of bicarbonate as a substrate, and does not recognize O2.1,2 The genus Flaveria contains closely related species showing different types of photosynthesis, including C3, C3–C4, C4-like and C4 photosynthesis.2 Flaveria spp. thus are an excellent model for examining the molecular evolution of the C4 photosynthetic pathway.2 Work with the enzyme carbonic anhydrase (CA, EC 4.2.1.1),3– 8 which catalyzes the reversible hydration of CO2 to bicarbonate, the two forms of inorganic carbon used by C3 and C4 Flaveria congeners, respectively, has added significant insight into the understanding of such processes.2 Three different b-CA isoforms have been identified in these plants: In Flaveria pringlei, a C3 species, CA 1 and CA 3 showed high expression in leaves (and their products localized to the chloroplast).2 In contrast, while CA 1 was also found to be a chloroplastic enzyme in the C4 species, F. bidentis, CA 3 transcripts were the most highly abundant CA mRNA in leaves and the enzyme localized to the cytosol of leaf mesophyll cells. CA 2 transcript levels were low in all organs examined in both F. pringlei and F. bidentis, and the gene was found to encode a cytosolic enzyme.2 In addition to catalyzing the first step in the C4 pathway, the role of CA in providing HCO 3 for photosynthesis has also been well documented in marine algae and cyanobacteria.9,10 However, an

S. M. Monti et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1626–1630 Table 1 Kinetic parameters for the CO2 hydration reaction catalysed by the human cytosolic isozymes hCA I and II (a-class CAs) at 20 °C and pH 7.5 in 10 mM HEPES buffer and 20 mM Na2SO4, and the b-CAs Can2, CalCA (from C. neoformans and C. albicans, respectively), SceCA (from S. cerevisiae), and the Flaveria bidentis CA (FbiCA 1) measured at 20 °C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO417 Isozyme

Activity level

kcat (s1)

kcat/Km (M1  s1)

KI (acetazolamide) (nM)

hCA I hCA II Can2 CalCA SceCA FbiCA 1

Moderate Very high Moderate High High Low

2.0  105 1.4  106 3.9  105 8.0  105 9.4  105 1.2  105

5.0  107 1.5  108 4.3  107 9.7  107 9.8  107 7.5  106

250 12 10.5 132 82 27

Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido1,3,4-thiadiazole-2-sulfonamide) are also provided.

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even more general CCM system seems to be present in most plants. This so-called basal CCM (bCCM) is proposed to be composed of a mitochondrial CA belonging to the b-CA class, and a c-type CA domain of the mitochondrial NADH dehydrogenase complex.5 Together, these two enzymes reduce leakage of CO2 from plant cells and allow efficient recycling of mitochondrial CO2 for carbon fixation in chloroplasts.11 Ultimately, CA functions in three primary modes in photosynthetic systems: (a) to convert HCO 3 to CO2, for fixation by Rubisco; (b) to convert CO2 to HCO 3 for fixation by PEPC; and (c) to provide rapid equilibration between CO2 and HCO 3 so that facilitated diffusion of CO2 is enhanced. CAs belonging to the a-,12 b-13 and c-CA14 families have been described from many photosynthetic organisms, including the cyanobacteria Synechoccus spp.,12a and Microcoleus chthonoplastes,13c the unicellular algae Chlamydomonas reinhardtii11 and Coccomyxa,12b

Figure 1. Alignment of the predicted amino acid sequences of selected b-CAs from plant, yeast, fungal and bacterial organisms. Pisum sativum numbering system was used. Zinc ligands are indicated in red; amino acids involved in the enzyme catalytic cycle are indicated in blue; amino acids forming the continuous hydrophobic surface in the binding pocket are indicated in green. The asterisk (⁄) indicates identity at a position; the symbol (:) designates conserved substitutions, while (.) indicates semi-conserved substitutions. Multiple alignment was performed with the program Clustal W, version 2.1. Legend: FbiCA_plant, Flaveria bidentis, isoform I (Accession number: AAA86939.2); VraCA_plant, Vigna radiata (Accession number: AAD27876); PsaCA_plant, Pisum sativum (Accession number: AAA33652); Can2_fungus; Cryptococcus neoformans (Accession number: GI: 219109194); SceCA_yeast, Saccharomyces cerevisiae (Accession number: GAA26059); CalCA_fungus, Candida albicans (Accession number: XP_721672.1); HpyCA_bacterium, Helicobacter pylori (Accession number: BAF34127.1).

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Figure 2. Phylogenetic tree of the b-CAs from selected prokaryotic and eukaryotic species. The tree was constructed using the program PhyML 3.0. Branch support values are reported at branch points. FbiCA_plant, VraCA_plant, PsaCA_plant, Can2_fungus, SceCA_yeast, CalCA_fungus and HpyCA_bacterium are indicated in the legend of Fig. 1; PgiCA_bacterium, Porphyromonas gingivalis ATCC 33277 (Accession number: YP_001929649.1); MinCA_bacterium, Myroides injenensis M09-0166 (Accession number: ZP_10784819); AbaCA_bacterium, Acinetobacter baumannii AB307-0294 (Accession number: YP_002326524); DmeCA_insect, Drosophila melanogaster (Accession number: NP_649849); Cab_bacterium, Methanobacterium thermoautotrophicum (Accession number: GI: 13786688); EcoCa_bacterium, Escherichia coli (Accession number: ACI70660); LpnCA_bacterium, Legionella pneumophila 2300/99 (Accession number: YP_003619232); DbrCA_yeast, Dekkera bruxellensis AWRI1499 (Accession number: EIF49256); OpaCA_fungus, Ogataea parapolymorpha DL-1 (Accession number: EFW97556); AfuCA_yeast, Aspergillus fumigatus Af293 (Accession number: XP_751704); SpoCA_yeast, Schizosaccharomyces pombe (Accession number: CAA21790); CspCA_alga, Coccomyxa sp. (Accession number: AAC33484.1); CreCA_alga, Chlamydomonas reinhardtii (Accession number: XP_001699151.1); BsuCA_bacterium, Brucella suis 1330 (Accession number: NP_699962.1); BthCA_bacterium, Burkholderia thailandensis Bt4 (Accession number: ZP_02386321); TasCA_fungus, Trichosporon asahii CBS 8904 (Accession number: EKD04029); SmaCA_fungus, Sordaria macrospora (Accession number: CAT00781); AthCA_plant, Arabidopsis thaliana (Accession number: AAA50156); ZmaCA_plant, Zea mays (Accession number: NP_001147846.1); OsaCA_plant, Oryza sativa (Accession number: AAA86943).

and the land plants Arabidopsis thaliana,12b Lotus japonicus,12c Zea mays,13d and Pisum sativum.13e In most photosynthetic species, representatives belonging to several CA families have been found11–14 and typically multiple isoforms of the enzymes are present. It should be noted that few X-ray crystal structures are available for such enzymes. The a-CA from Chlamydomonas reinhardtii,12a as well as the b-CAs from Coccomyxa,13a and Pisum sativum13e are among the few representatives for which detailed structural information is available. Interestingly, for the Coccomyxa b-CA, adducts of the enzyme with several inhibitors, including iodide, thiocyanate and acetazolamide,13a showed an interesting inhibition pattern for this b-class enzyme, thought earlier to be true only for a-CAs. For example, acetazolamide and thiocyanate coordinate the active site Zn(II) ion along with two Cys and one His residues from the protein, in a tetrahedral geometry.13a This is reminiscent of the inhibition mechanism of a-CAs with anion or sulfonamide inhibitors,3,4 although thiocyanate binds in a trigonal bipyramidal geometry with the Zn(II) ion to human (h) hCA II. In this adduct, the Zn(II) is coordinated by the three His residues from the protein, one water molecule and the anion inhibitor. In the Coccomyxa b-CA, the most interesting inhibitor binding mode was observed for iodide, which was not coordinated with the Zn(II) ion, but anchored to the zincbound water/hydroxide ion, an inhibition mechanism observed

earlier (in the a-CAs) for phenols, polyamines and one ester.15,16 It is thus of great interest to investigate inhibition of such enzymes with various classes of inhibitors, especially in that very few such studies have been done with plant enzymes. These studies may reveal novel inhibition patterns and also help in understanding the involvement of these enzymes in photosynthesis and other biological mechanisms (e.g., calcification, in corals or other marine organisms).1b Although, as mentioned above, three b-CAs have been reported in Flaveria spp., no detailed kinetic studies have been reported, and neither their catalytic activity nor their inhibition profile has been investigated. Here we report the characterization of one isoform of F. bidentis b-CA, FbiCA 1, with respect to its inhibition profile with anions and other small molecules known to interfere with the activity of CAs. To our knowledge, this is the first detailed inhibition study of a plant b-CA.17,18 Table 1 shows the kinetic parameters for the CO2 hydration reaction catalyzed by FbiCA 1 as well as several well-investigated CAs,17 including the two a-CAs of human (h) origin, hCA I and II,3,4 and b-CAs from the fungal pathogens Cryptococcus neoformans (Can2)7b and Candida albicans (CalCA),19 and the yeast from Saccharomyces cerevisiae (SceCA).20 FbiCA 1 has significant catalytic activity as a CO2 hydrase, with a kcat of 1.2  105 and a kcat/Km of

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7.5  106 M1  s1. It is also highly sensitive to the clinically used sulfonamide acetzolamide, with an inhibition constant of 27 nM. Among the investigated CAs shown in Table 1, FbiCA 1 is the most inefficient catalyst, with other CA kcat/Km values in the range of 107–108 M1  s1. Nevertheless the value measured for FbiCA 1, even if outside this range, demonstrates that the enzyme is a good catalyst for the conversion of CO2 to bicarbonate and protons. To rationalize the kinetic data in Table 1, an alignment of the amino acid sequence of FbiCA 1 with that of selected b-CAs from other plants (VraCA, from Vigna radiata;14a PsaCA, from Pisum sativum);13e fungi (Can2, from Cryptococcus neoformans;7b CalCA, from Candida albicans);19 yeasts (SceCA, from Saccharomyces cerevisiae);20 as well as bacteria (HpyCA, from Helicobacter pylori)21 is shown in Figure 1. FbiCA 1 has all the amino acid residues involved in catalysis, as is the case for the other members of the family (Fig 1): (i) the three Zn(II) ligands, Cys160, His220 and Cys223 (Pisum sativum CA numbering system);13e (ii) the Asp162–Arg164 catalytic dyad, which forms a hydrogen bond network with the water coordinated to the Zn(II) ion, enhancing its nucleophilicity;7b,13e and (iii) a cluster of hydrophobic amino acid residues considered to be involved in the binding of the substrate (and inhibitors), including those in position 151, 179 and 184.7b,13e Indeed, Q151, F179 and V184 are conserved in FbiCA 1 and in almost all the eukaryotic enzymes used in the alignment, irrespective of their source (plant, yeast, fungus). Figure 2 presents a phylogenetic analysis of selected b-CAs for which predicted amino acid sequence is available in data bases. We included sequences from a number of plants, algae, yeasts, fungi, and bacteria, even if the corresponding enzymes were not yet characterized completely, to get an overview on the phylogenetic relationship of this family of CAs. FbiCA 1 clusters with corresponding enzymes from the other land plants, and most tightly with the CAs from other dicotyledonous species (Pisum sativum, Vigna radiata, Arabidopsis thaliana). The enzymes from the fungi Trichosporon asahii (TasCA) and Sordaria macrospora (SmaCA) are sister to the plant CAs. Some algal, bacterial and fungal enzymes cluster in a sister clade while the yeast CAs and most of the bacterial enzymes are more distantly related to FbiCA1. It is interesting to note that the only arthropod bCA characterized so far, the enzyme from Drosophila melanogaster DmeCA,21 clustered with the bacterial and archaeal enzymes. This may not be so much surprising considering the fact that DmeCA is a mitochondrial enzyme,22 and mitochondria originated from bacteria which have been ‘captured’ by other cells during evolution of the Eukaryotes. Table 2 shows the inhibition data for FbiCA 1 with respect to a rather wide range of simple and complex inorganic anions as well as several small molecules observed in previous studies to act as CA inhibitors (CAIs).5,6,20,21 The analogous data for the inhibition of hCA II and the bacterial enzyme SspCA (from the extremophile Sulfurihydrogenibium yellostonense) 5c—belonging to the a-CA class—as well as HpyCA and SceCA (b-CAs) are provided for comparison. The inhibition profile of FbiCA 1 is distinct from that of a- or other b-CAs as follows: (i) The anions with the lowest propensity to coordinate metal ions, perchlorate and tetrafluroborate, were not inhibitors of FbiCA 1 up to concentrations of 200 mM. The same is true for the human isoform hCA II, as well as the bacterial/yeast enzymes tested (Table 2). (ii) Several other anions, such as hydrogensulfite, selenate, perrhutenate and iminidisulfonate were also weak inhibitors of the plant enzyme FbiCA 1, with inhibition constants in the range of 24.5–55.3 mM. It should be noted that all of these compounds are much better inhibitors of the a- or other b-CAs investigated earlier.6a

Table 2 Inhibition constants of anionic inhibitors against a-CA isozymes derived from human (hCA II), and bacterial (SspCA) sources as well as the b-CA from a bacterium (H. pylori) HpyCA, one yeast Saccharomyces cerevisiae (SceCA) and the plant Flaveria bidentis isoform 1 (FbiCA 1), at 20 °C by a stopped flow CO2 hydrase assay17 Inhibitora

KIb (mM) hCA II



F Cl Br I CNO SCN CN N 3 HCO 3

c

SspCA

d

HpyCAe

SceCAf

FbiCA 1g

>300 200 63 26 0.03 1.6 0.02 1.51 85 73

41.7 8.3 49 0.86 0.8 0.71 0.79 0.49 33.2 39.3

0.67 0.56 0.38 0.63 0.37 0.68 0.54 0.8 0.5 0.42

2.85 0.85 0.011 0.01 31.7 55.6 16.8 27.9 0.78 0.76

0.71 0.74 0.67 0.71 0.93 0.83 0.62 0.46 0.66 0.84

SnO2 3

35 63 0.04 89 0.83

0.86 0.48 0.58 21.1 0.52

0.78 0.67 0.58 0.63 0.48

13.9 0.46 0.33 0.33 nt

0.78 0.57 0.86 55.3 0.53

SeO2 4

112

0.57

0.65

nt

24.5

TeO2 4

0.92

0.53

0.45

nt

0.9

P2 O4 7

48.5

0.69

0.75

nt

0.83

V2 O4 7

0.57

0.66

0.18

nt

0.66

B4 O2 7 ReO 4 RuO 4

0.95

0.67

0.68

nt

0.86

0.75 0.69 0.084

0.8 0.69 84.6

0.82 1.1 0.93

nt nt nt

0.52 26.1 0.87

0.086 0.0088

0.07 0.06

0.97 0.21

nt nt

0.88 0.06

3.1 >200

0.004 0.82

0.0074 0.57

nt 0.58

0.008 0.62

>200 >200 0.46 0.76

>200 >200 0.73 0.75

6.5 >200 0.75 0.7

>200 >200 nt nt

>200 >200 0.69 50.9

1.13 0.39 23.1 49.2

0.009 0.042 0.041 0.005

0.072 0.094 0.073 0.092

0.0087 0.84 38.2 0.4

0.004 0.005 0.008 0.006

CO2 3 NO 3 NO 2 HS HSO 3

S2 O2 8 SeCN CS2 3 Et2 NCS 2 SO2 4  ClO4  BF4 FSO 3 NHðSO3 Þ2 2 H2NSO2NH2 H2NSO3H Ph-B(OH)2 Ph-AsO3H2 a

As sodium salt; nt = not tested. Errors were in the range of 3–5% of the reported values, from three different assays. c From Ref. 6a. d From Ref. 5c. e From Ref. 21. f From Ref. 20. g This work. b

(iii) Most of the investigated anions, including the halides, pseudohalides (cyanide, cyanate, thiocyanate, azide), bicarbonate, carbonate, nitrate, nitrite, hydrogensulfide, stannate, tellurate, diphosphate, divanadate, tetraborate, perrhenate, peroxydisulfate, selenocyanide, sulfate, and fluorosulfate, inhibited FbiCA 1 in the sub-millimolar range, with KIs of 0.46–0.93 mM. This is a rather unexpected, flat inhibition profile, considering the data for the a- and b-CAs shown in Table 2. For example, hydrogensulfide was 21.5 times a better hCA II than FbiCA 1 inhibitor. The small variation in inhibition of FbiCA 1 seen between the halides and pseudohalides is interesting and contrasts with that observed for other enzymes, especially those belonging to the a-class, for example, between fluoride and iodide, or between the light halogenides (F, Cl) and pseudohalogenides, such as cyanide, cyanate or azide (Table 2). Thus, anions known to easily complex metal ions in solution (or within the active sites of metalloenzymes), such as cyanide, azide, thiocyanate, or anions with less strong such properties

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(e.g., sulfate, fluorosulfate) showed similar inhibitory effects on FbiCA 1. The significance of this activity is not clear at this time, largely because of the lack of an X-ray crystal structure of the plant enzyme. (iv) A group of anions/molecules, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, showed low micromolar affinity for FbiCA 1, with KIs in the range of 4.0–60 lM. The best inhibitors of FbiCA 1 were sulfamide, sulfamate and phenylarsonic acid, with KIs of 4–6 lM (Table 2). Some of these compounds, for example, phenylboronic acid, are much weaker inhibitors of the yeast and human enzymes SceCA/ hCA II, but they effectively inhibited other such enzymes (SspCA, HpyCA). In conclusion, we report the first detailed biochemical characterization of a b-CA isoform, FbiCA 1, from the plant F. bidentis, which has been cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO2 hydrase catalytic activity (kcat of 1.2  105 and kcat/Km of 7.5  106 M1  s1) and was moderately inhibited by most simple/complex inorganic anions. More potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (KIs in the range of 4– 60 lM). Such inhibitors may be used as tools to better understand the role of various b-CA isoforms in photosynthesis.

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17.

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22.

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L.; Katinakis, P.; Labrou, N. E.; Flemetakis, E. Biochim. Biophys. Acta 2011, 1814, 496. (a) Huang, S.; Hainzl, T.; Grundström, C.; Forsman, C.; Samuelsson, G.; SauerEriksson, A. E. PLoS ONE 2011, 6, e28458; (b) Long, B. M.; Rae, B. D.; Badger, M. R.; Price, G. D. Photosynth. Res. 2011, 109, 33; (c) Kupriyanova, E. V.; Sinetova, M. A.; Markelova, A. G.; Allakhverdiev, S. I.; Los, D. A.; Pronina, N. A. J. Photochem. Photobiol., B. 2011, 103, 78; (d) Tems, U.; Burnell, J. N. Plant Physiol. Biochem. 2010, 48, 945; (e) Kimber, M. S.; Pai, E. F. EMBO J. 2000, 19, 1407. (a) Klodmann, J.; Braun, H. P. Phytochemistry 2011, 72, 1071; (b) Gawryluk, R. M.; Gray, M. W. BMC Evol. Biol. 2010, 10, 176. (a) Nair, S. K.; Ludwig, P. A.; Christianson, D. W. J. Am. Chem. Soc. 1994, 116, 3659; (b) Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.; Supuran, C. T. J. Med. Chem. 2010, 53, 5511. (a) Davis, R. A.; Hofmann, A.; Osman, A.; Hall, R. A.; Mühlschlegel, F. A.; Vullo, D.; Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2011, 54, 1682; (b) Martin, D. P.; Cohen, S. M. Chem. Commun. 2012, 48, 5259. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An applied photophysics stoppedflow instrument has been used for assaying the CA catalysed CO2 hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5, for a-CAs) or 20 mM TRIS (pH 8.3 for the b-CAs) as buffers, and 20 mM NaClO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (10 mM) were prepared in distilled–deionized water and dilutions up to 0.01 lM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E–I complex or for the eventual active site mediated hydrolysis of the inhibitor. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, as reported earlier,15b,16a and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier.15b,16a. A truncated version of FbCAI, without the chloroplast transit peptide, to render it more stable, was cloned in pETM11 (EMBL), with an N-terminal 6His tag. Recombinant protein was expressed in BL21(DE3) 16 h at 20 °C, 225 rpm. Purification was performed on 1 ml HisTrapFF (GE Healthcare) in 20 mM Tris, 8.0, 500 mM NaCl, 1 mM DTT, TEV-digested 20 h in dialysis at 4 °C and the resulting cleaved protein passed over MonoQ in a linear salt gradient, at pH 8.0. Purity and homogeneity of FbCA 1 was assessed by SDS/PAGE and LC-ESI-MS. (a) Mogensen, E. G.; Janbon, G.; Chaloupka, J.; Steegborn, C.; Fu, M. S.; Moyrand, F.; Klengel, T.; Pearson, D. S.; Geeves, M. A.; Buck, J.; Levin, L. R.; Mühlschlegel, F. A. Eukaryot. Cell 2006, 5, 103; (b) Bahn, Y. S.; Mühlschlegel, F. A. Curr. Opin. Microbiol. 2006, 9, 572; (c) Innocenti, A.; Mühlschlegel, F. A.; Hall, R. A.; Steegborn, C.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18, 5066. (a) Isik, S.; Kockar, F.; Arslan, O.; Ozensoy Guler, O.; Innocenti, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2008, 18, 6327; (b) Isik, S.; Kockar, F.; Aydin, M.; Arslan, O.; Ozensoy Guler, O.; Innocenti, A.; Supuran, C. T. Bioorg. Med. Chem. 2009, 17, 1158. (a) Nishimori, I.; Minakuchi, T.; Kohsaki, T.; Onishi, S.; Takeuchi, H.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2007, 17, 3585; (b) Nishimori, I.; Onishi, S.; Takeuchi, H.; Supuran, C. T. Curr. Pharm. Des. 2008, 14, 622; (c) Maresca, A.; Vullo, D.; Scozzafava, A.; Supuran, C.T. J. Enzyme Inhib. Med. Chem. 28, in press. Syrjänen, L.; Tolvanen, M.; Hilvo, M.; Olatubosun, A.; Innocenti, A.; Scozzafava, A.; Leppiniemi, J.; Niederhauser, B.; Hytönen, V. P.; Gorr, T. A.; Parkkila, S.; Supuran, C. T. BMC Biochem. 2010, 11, 28.