Carbonic anhydrases (CAs, EC 4.2.1.1) catalyse the interconversion between CO2 and bicarbonate as well as other hydrolytic reactions. Among the six genetic families known to date, the α-, β-, γ-, δ-, ζ- and η-CAs, detailed kinetic and X-ray crystallographic studies have allowed a deep understanding of the structure–function relationship in this superfamily of proteins. A metal hydroxide nucleophilic species of the enzyme, and a unique active site architecture, with half of it hydrophilic and the opposing part hydrophobic, allow these enzymes to act as some of the most effective catalysts known in Nature. The CA activation and inhibition mechanisms are also known in detail, with a large number of new inhibitor classes being described in the last years. Apart from the zinc binders, some classes of inhibitors anchor to the metal ion coordinated nucleophile, others occlude the entrance of the active site cavity and more recently, compounds binding outside the active site were described. CA inhibition has therapeutic applications for drugs acting as diuretics, antiepileptics, antiglaucoma, antiobesity and antitumour agents. Targeting such enzymes from pathogens may lead to novel anti-infectives. Successful structure-based drug design campaigns allowed the discovery of highly isoform selective CA inhibitors (CAIs), which may lead to a new generation of drugs targeting these widespread enzymes. The use of CAs in CO2 capture processes for mitigating the global temperature rise has also been investigated more recently.

INTRODUCTION

The carbonic anhydrases (CAs, EC 4.2.1.1) belong to a superfamily of metalloenzymes which use as substrates some unusually chemically simple molecules/ions, as they catalyse the interconversion between CO2 and bicarbonate [113]. Although this reaction may occur without a catalyst (reaction 1 in Figure 1), at physiological pH values it is too slow to meet metabolic needs in which CA substrates/reaction products are involved in most organisms on earth. CO2, a poorly water soluble gas, may damage cellular components (e.g. membranes) if generated in exceedingly high amounts in a cell/tissue, whereas its conversion to water soluble ions (bicarbonate and protons), may interfere with the pH balance of the cell through the generation of an acid (H+) and a buffering base (HCO3) [113]. Thus, catalysts to deal with the rapid inter-conversion between these chemical species have evolved at least six times independently, in a very interesting example of convergent/divergent evolution phenomenon, with six CA enzymatic families known to date, the α-, β-, γ-, δ-, ζ-and η-CAs [113]. The α-CAs are present in vertebrates, protozoa, algae, cytoplasm of green plants and in many Gram negative bacteria [1,3,5]; the β-CAs are found in both Gram negative and positive bacteria algae and chloroplasts of mono- as well as di-cotyledons, and also in many fungi and some Archaea [19]. The γ-CAs were found in Archaea, cyanobacteria and most types of bacteria [15], the δ- and ζ-CAs seem to be present only in marine diatoms [2,9], whereas the η-CAs in protozoa [14]. In many organisms these enzymes are involved in crucial physiological processes connected with pH and CO2 homoeostasis/sensing; biosynthetic reactions, such as gluconeogenesis, lipogenesis and ureagenesis; respiration and transport of CO2/bicarbonate; electrolyte secretion in a variety of tissues/organs; bone resorption; calcification; tumorigenicity and many other physiological or pathological processes (thoroughly studied in vertebrates and some pathogens) [113]. In algae, plants and some bacteria CAs play an important role in photosynthesis and biosynthetic reactions connected to it [2,3,5,15,16]. In diatoms δ- and ζ-CAs play a crucial role in carbon dioxide fixation, whereas in protozoans the role of the η-CAs is poorly understood for the moment but they may be involved in de novo purine/pyrimidine biosynthetic pathways [17]. Many CAs from vertebrates, nematodes, fungi, protozoa and bacteria are drug targets [1,413], since interfering with their activity (by inhibition or activation) leads to a pharmacological response.

Reactions catalysed by CAs

Figure 1
Reactions catalysed by CAs

Only 1 seems to possess physiological functions in plants and animals [14], whereas some bacteria rely on 2 and 3 for survival [18,19]. Reactions 4–11 were less investigated but prove the high catalytic versatility of these enzymes [2028].

Figure 1
Reactions catalysed by CAs

Only 1 seems to possess physiological functions in plants and animals [14], whereas some bacteria rely on 2 and 3 for survival [18,19]. Reactions 4–11 were less investigated but prove the high catalytic versatility of these enzymes [2028].

REACTIONS CATALYSED BY THE CAs

In addition to the CO2 hydration/bicarbonate dehydration processes (reaction 1 in Figure 1), some classes of CAs possess a certain catalytic versatility, with the possibility to hydrate small molecules similar to CO2 such as COS (reaction 2) [18], CS2 (reaction 3) [19] and cyanamide (reaction 4) [20,21] leading to H2S (and CO2) for the first reactions and urea for the last one. Aldehydes were also shown to be hydrated to gem diols (reaction 5) [22], whereas the esterase activity with carboxylic acid esters (reaction 6) [2325], sulfonic acid esters (reaction 7) [26,27], phosphate esters (reaction 8) [24,25] and thioesters (reaction 9) [28,29] were also reported. Some other less investigated hydrolytic processes (reactions 10 and 11 in Figure 1) were also found for at least the α-CAs [30]. It is presently not known whether reactions other than the CO2 hydration/bicarbonate dehydration may have physiological relevance, although the recently reported thioesterase activity [29] may interfere with the generation/hydrolysis of acyl-coenzyme A derivatives, and thus possess an important physiological role.

STRUCTURE OF THE CAs

Metal ion and its coordination

The CAs are metalloenzymes, catalytically effective only with one metal ion bound within the active site cavity, the apoenzymes being devoid of any catalytic action [13,5,7,3133]. The active centre normally comprises M(II) ions in a tetrahedral geometry, with three amino acid residues as ligands, in addition to a water molecule/hydroxide ion coordinating the metal (Figure 2). Zn(II) is the metal ion which may be present in all six CA genetic families, but Cd(II) is interchangeable with Zn(II) in the ζ-CAs [3], Fe(II) seems to be present in γ-CAs, at least in anaerobic conditions [34], whereas Co(II) may substitute the zinc ion in many α-CAs without a significant loss of the catalytic activity [13,5,7,3133]. These metal ions were rarely observed in trigonal bipyramidal or octahedral coordination geometries within the CA active site [13,5,7,3133], but the catalytically effective species are probably the ones shown in Figures 2(A), 2(B), 2(D) and 2(E). Indeed, in the α-, γ- and δ-CAs the Zn(II) is coordinated by three histidine residues and a water molecule/hydroxide ion (Figure 2A). In type I β-CAs (i.e. enzymes with an open active site) the Zn(II) is coordinated by two cysteine and one histidine residue, with water/hydroxide as the fourth ligand (Figure 2B), whereas in the type II β-CAs (i.e. enzymes with a closed active site), an aspartate residues is the fourth zinc ligand, as shown in Figure 2(C). Such enzymes are devoid of catalytic activity due to the lack of a nucleophilic water molecule/hydroxide ion coordinated to the zinc. However, an elegant crystallographic study [10] showed that the closed active site is transformed to an open one at pH values > 8.3, when a conserved arginine residue in all β-CAs (from the so-called catalytic dyad, comprising an Asp-Arg motif) participates in the formation of ionic interactions with the coordinated aspartic acid residue, allowing an incoming water molecule to take its place as the fourth zinc ligand, with the ‘opening’ of the active site. In the ζ-CAs the metal ion coordination is similar to the one present in the type I β-CAs, except that the metal ion may be Cd(II) or Zn(II), as shown in Figure 2(D) [3]. In the η-CAs the pattern of the metal ion coordination [which presumably is Zn(II)] seems to be completely different from those present in the other five classes, with two histidine and one glutamine residue in addition to the water molecule/hydroxide ion binding the Zn(II), as suggested by a computational approach [33]. In all CA classes a metal hydroxide derivative (L3-M2+-OH, where L is the coordinated amino acid residue) of the enzyme is the catalytically active species, acting as a strong nucleophile (at neutral pH) on the CO2 molecule bound in a hydrophobic pocket nearby [35]. This metal hydroxide species is generated from a water coordinated to the metal ion which by itself is not nucleophilic enough to act as a catalyst (see later in the text for discussion of the catalytic mechanism).

The metal ion coordination pattern in the CAs

Figure 2
The metal ion coordination pattern in the CAs

(A) α-, γ- and δ-CAs (in the α- and δ-classes the coordinating residues are from the same monomer, whereas in γ-CAs the third His is from an adjacent monomer, see also later in the text). (B) β-CAs (type I, opened active site). (C) β-CAs (type II, closed active site, an aspartate residues as the fourth zinc ligand). (D) ζ-CAs with Cd(II) bound within the active site. (E). η-CAs [13,3133].

Figure 2
The metal ion coordination pattern in the CAs

(A) α-, γ- and δ-CAs (in the α- and δ-classes the coordinating residues are from the same monomer, whereas in γ-CAs the third His is from an adjacent monomer, see also later in the text). (B) β-CAs (type I, opened active site). (C) β-CAs (type II, closed active site, an aspartate residues as the fourth zinc ligand). (D) ζ-CAs with Cd(II) bound within the active site. (E). η-CAs [13,3133].

Protein fold and active site architecture of CAs

The oligomeric state of the CAs is highly variable. Most α-class enzymes are monomers but homodimers were also reported for some human and bacterial enzymes [1,7,31,32,35]. As shown in Figure 3(A) for the human (h) isoform hCA II, the most common situation for these enzymes is the monomeric protein [36]. hCA II has an egg-like shape (Figure 3A) with the approximate dimensions of 50×40×40 Å3 (1 Å=0.1 nm) and a typical fold characterized by a central ten-stranded antiparallel β-sheet surrounded by several helices and additional β-strands [36]. Based on the high sequence identity of the cytosolic hCAs, their 3D structures are rather similar, with all secondary structure elements strictly conserved [1,7,31,32,35,36]. However, an accurate structural comparison of all hCAs crystallized so far revealed a number of small local structural differences, which were mainly localized around residues 125–131 (hCA II amino acid numbering system), thus occurring both on the surface of the protein (residues 125–130) and in the middle of the active site (residue 131). The active site is located in a large, cone-shaped cavity that reaches the centre of the molecule (Figure 3A). The catalytic Zn(II) ion is located at the bottom of this cavity, being coordinated by the three conserved histidine residues and a water molecule/hydroxide ion. The Zn2+-bound solvent molecule is engaged in hydrogen bond interactions with another water molecule (the so-called the deep water) and with the hydroxyl moiety of the conserved threonine residue (Thr199), which in turn is bridged to the carboxylate moiety of a conserved glutamic acid residue (Glu106) [1,7,31,32,35]. These interactions enhance the nucleophilicity of the Zn2+-bound water molecule, and orient the CO2 substrate in a location favourable for the nucleophilic attack. This is why the residues Thr199-Glu106, an important catalytic dyad for all α-CAs, are called gate-keeping residues [1,7,31,32,36].

Protein fold in CAs for which the X-ray crystal structure has been published

Figure 3
Protein fold in CAs for which the X-ray crystal structure has been published

(A) Monomeric α-CA: human CA II (PDB file 1F2W) [20]; (B) tetrameric β-CA, VchCA from V. cholerae, PDB file 5CKX [39]; (C) homotrimeric γ-CA (upper part) and protein fold of the monomer (lower part) Cam, from the archaeon M. thermophila, PDB file 1QRG [49,50]; (D) monomeric ζ-CA, R3 domain of Cd(II)-bound T. weissflogii enzyme, PDB file U3K8 [52].

Figure 3
Protein fold in CAs for which the X-ray crystal structure has been published

(A) Monomeric α-CA: human CA II (PDB file 1F2W) [20]; (B) tetrameric β-CA, VchCA from V. cholerae, PDB file 5CKX [39]; (C) homotrimeric γ-CA (upper part) and protein fold of the monomer (lower part) Cam, from the archaeon M. thermophila, PDB file 1QRG [49,50]; (D) monomeric ζ-CA, R3 domain of Cd(II)-bound T. weissflogii enzyme, PDB file U3K8 [52].

A striking feature of the hCA II active site (as well as that of all other CAs crystallized so far) [31,32,36] is that half of it is lined with hydrophobic residues whereas the remaining, opposing half is lined with hydrophilic amino acid residues. Residues in position 121, 131, 141, 143, 198 and 207 delimit the hydrophobic region, whereas those in position 62, 64, 67 and 92 identify the hydrophilic one. Furthermore, the bulky Phe131 residue in hCA II, roughly in the middle of the hydrophobic half, subdivides this part of the active site in two sub-sites in which various classes of inhibitors bind in a specific manner [31,32,35,36]. This very particular ‘bipolar’ active site architecture is probably due to the very different chemical nature of the substrate (CO2) and its hydration reaction products: the hydrophobic part is used to entrap the CO2 molecule (a hydrophobic gas). Indeed, the CO2-binding site was found at the bottom of the hydrophobic part of the cavity, as shown in the interesting study by McKenna's group for hCA II [35]. On the other hand, the hydrophilic half of the active site facilitates the binding of the polar components generated from the CO2 hydration reaction (bicarbonate and protons) and their release from the cavity, towards the environment. At least for the protons, it is in fact well demonstrated that a relay of water molecules and several histidines (proton shuttling residues others than His64) are involved in such processes [31,32,35,37]. His64 on the other hand is one of the very few amino acid residues having a high flexibility within the hCA II active site (the scaffold of the protein is very rigid, and no major conformational changes of the protein or its active site are observed when inhibitors or activators belonging to a variety of classes bind to it) [1,7,12,31,32,37]. Indeed, two conformations of the imidazole moiety of this important amino acid residue have been observed by means of X-ray crystallography: an ‘in’ one, pointing towards the Zn(II) ion, and an ‘out’ one, pointing towards the exit of the cavity [37]. The two conformations are part of the proton transfer mechanism by which this residue shuttles protons between the active site and the reaction medium [31,32,37].

The β-CAs are catalytically active mostly as dimers [1,7,11,38], or tetramers (dimers of dimers) (Figure 3B) [39] or as multiples of such homodimers, especially for the higher plant enzymes [16]. This is also true for the COS hydrolase from Thiobacillus thioparus [18], and the CS2 hydrolase from Acidianus A1–3 [19], which are tetramers and octadecamers, respectively, with an occluded active site entrance which induces the discrimination for the binding of COS and CS2 over the more hydrophilic CO2 to these enzymes [18,19]. In the β-CAs from Pisum sativum [40], Methanobacterium thermoautotrophicum [41], Mycobacterium tuberculosis Rv1284 [10], the active site zinc ion is coordinated by one histidine and two cysteine residues, with a fourth coordination site occupied by water or a substrate analogue (the so-called open conformation, as shown in Figure 2B). In contrast, the other subclass of β-CAs, exemplified by structures of the enzymes from Haemophilus influenzae [42], Escherichia coli [43], Porphyridium purpureum [44] and M. tuberculosis Rv3588c [10], has a diverse zinc coordination geometry, where the water molecule has been replaced by an aspartate side chain, forming a non-canonical CA active site (the closed conformation, shown in Figure 2C). However the protein fold of the two types of β-CAs is quite similar. Each monomer of the catalytically active dimer (or tetramer) adopts an α/β fold, being composed of an N-terminal arm, which extends away from the rest of the molecule and makes significant contacts with an adjacent monomer, a conserved zinc binding core and a C-terminal domain dominated by α-helices (see Figure 3B, in which the Vibrio cholerae tetrameric enzyme VchCAβ is shown) [39]. The zinc binding core resembles the classical nucleotide binding Rossmann fold motif [45] with a β-sheet composed of four parallel strands flanked on both sides by α-helix segments. The active site is located at one end of the central β-sheet at the interface of one dimer [39]. In the active site of VchCAβ the zinc ion is coordinated to four amino acid residues, Cys42, Asp44, His98, Cys101, in an approximately tetrahedral geometry. A type I β-CA is Can2 from the pathogenic fungus Cryptococcus neoformans [11]. The 3D structure of this dimeric enzyme is similar to that of other β-CAs, with the N-terminal sub-domain of the Can2 core being formed by four antiparallel α-helices (α1–4), and the C-terminal sub-domain containing a five-stranded β-sheet, with four parallel β-strands (β1–4) and β5, formed by the Can2 C-terminus, attached in an antiparallel orientation [11]. The C-terminal subdomain is packed between the N- and C-terminal domains of a partner monomer, resulting in a tight dimer interaction [11].

A particular case is represented by the so-called ε-CAs, a class initially reported by Cannon's group [46], but subsequently recognized by the same authors [47] to represent a particular case of β-CAs. Indeed, in cyanobacteria and many chemolithoautotrophic bacteria CAs are packed together with ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in order to achieve an efficient photosynthesis, in structures called carboxysomes. CsoS3, such an enzyme from Halothiobacillus neapolitanus, was considered to be the first member of the ε-CA class, but when its X-ray crystal structure was resolved, it was obvious that this was an erroneous assignment. The crystal structure of CsoSCA revealed that it is a representative member of a subclass of β-CAs, distinguished by a lack of active site pairing, typical of all other representatives of this class investigated earlier [11,3844]. In fact, as shown above for VhCAβ and Can2, in the typical β-CAs a pair of monomers form the active sites, organized within a 2-fold symmetric homodimer or pair of fused, homologous domains. However, the two domains present in CsoSCA have diverged to the point that only one domain in the pair retains a viable active site [47]. It has been suggested that this ‘defunct’ and somewhat diminished domain has evolved a new function, specific to its carboxysomal environment [47]. Despite the level of sequence divergence that separates CsoSCA from the other two subclasses of β-CAs, there is a remarkable level of structural similarity among active site regions, which suggests a common catalytic mechanism for the interconversion of carbon dioxide and bicarbonate by this enzyme. Crystal packing analysis suggested that CsoSCA exists within the carboxysome shell either as a homodimer or as extended filaments [47].

The prototype γ-class CAs has been characterized from the methanogenic archaeon Methanosarcina thermophila, by Ferry's group [48–50]. This enzyme, Cam, adopts a left-handed parallel β-helix fold, as shown by X-ray crystallography (Figure 3C). This fold is of particular interest since it contains only left-handed crossover connections between the parallel β-strands, which have been observed so far very infrequently in proteins. The active form of the enzyme is a trimer with three zinc-containing active sites (Figure 3C, upper side), each located at the interface between two monomers, shown in Figure 3(C) (lower part). As shown above (Figure 2A), there are structural similarities in the zinc coordination environment of the γ- and α-CAs, suggestive of convergent evolution dictated by the chemical requirements for catalysis of the same reaction [4850]. A subsequent work from the same group, presenting a structure at higher resolution, showed the side chains of Glu62 and Glu84 to share a proton. Furthermore, Glu84 exhibited multiple conformations, suggesting that this residue may act as a proton shuttle (similar to His64 in α-CAs, as discussed above), which seems to be an important aspect of the reaction mechanism of all CA classes. A hydrophobic pocket on the surface of the enzyme was also detected, which probably participates in the trapping of CO2 at the active site [50]. Few other γ-CA structures except Cam were reported to date, one of which is Ccm, again from carboxysomes [51].

The ζ-CA are presumably monomeric enzymes, as shown in Figure 3(D) for CdCA1-R3 from Thalassiosira weissflogii [3,52]. However the full length enzyme from this diatom is composed of three almost identical repeats (R1, R2 and R3) forming an active enzyme of approximately 69 kDa [3,52]. In all these repeats, CDCA1-R1, CDCA1-R2 and CdCA1-R3, the active site is located in a funnel-shaped pocket, with the metal ion located on the bottom, being coordinated in a highly distorted tetrahedral geometry by three conserved protein ligands, Cys263, His315 and Cys325 (as in the β-CAs with an open active site, see above) and a water molecule (Figure 3D). The structure consists of seven α-helices, three 310-helixes and nine β-strands, which are organized in three β-sheets. The first β-sheet is formed by strands β1, β6, β8 and β9; the second by strands β2 and β7 and the third one by strands β3, β4 and β5 (Figure 3D) [3,52]. The active site is located in a cleft on the protein surface with the catalytic metal ion placed at its bottom where it is coordinated by Cys473, His525 and Cys535, as mentioned above.

The active site cavities of all CA classes are rather large considering the fact that the substrates/reaction products are quite small molecules/ion: the surface areas of these active sites are in fact comparable, being of approximately 280 Å3 for hCA II (α-class), of 160 Å3 for the Cd(II)-containing ζ-CA from T. weissflogii and of 140 Å3 for the β-CA from C. neoformans, Can2 [11,36,52,53]. As no representatives of the δ- and η-CA classes were crystallized to date, the structure of these enzymes will be not discussed here, although partial homology models are available at least for η-CAs [33].

A salient feature of all these enzymes active sites has been recently noticed [53]: half of the cavity is aligned only with hydrophobic amino acid residues whereas the opposite half with hydrophilic ones, leading to a ‘bipolar’ active site architecture unique only to this enzyme superfamily [53]. A probable explanation for this particular active site architecture is that the hydrophobic part has the role of entrapping the CO2 molecule (a hydrophobic gas), whereas the hydrophilic half is involved in the release from the cavity of the ions generated from the CO2 hydration reaction (bicarbonate and protons). This fact is well demonstrated for the proton release pathway, in which a relay of water molecules and several histidines (acting as proton shuttling residues) were shown to be involved [54,55]. More importantly, these particular active sites also have interesting consequences for the binding of CA inhibitors (CAIs) and activators to the enzymes [1,7,8,31,53].

CATALYTIC, INHIBITION AND ACTIVATION MECHANISMS

As discussed above, a divalent metal (Zn, Cd, Fe or Co) hydroxide species generated from the metal-coordinated water molecule (Figure 4A), is the catalytically active species of all CAs. This is due to the enhanced nucleophilicity that the hydrophobic environment within the active site and the metal ion coordination exerts on the coordinated water molecule, which has a pKa between 6.5 and 7.4 (compared with a pKa 14 of water in the bulk) [56]. Furthermore, for many CA classes/isoforms, the rate determining step of the entire catalytic turnover (which for some α- and ζ-CAs achieves kcat/KM values > 108 M−1·s−1) is just the formation of the metal hydroxide species of the enzyme, by the transfer of a proton from the metal-coordinated water molecule to the environment [1,31,54,55].

Catalytic (for the physiological reaction), inhibition (with zinc binders) and activation mechanisms of α-CAs (hCA I amino acid numbering of the zinc ligands [20])

Figure 4
Catalytic (for the physiological reaction), inhibition (with zinc binders) and activation mechanisms of α-CAs (hCA I amino acid numbering of the zinc ligands [20])

A similar catalytic/inhibition/activation mechanism is valid also for CAs from other classes although the metal ion is coordinated by other amino acid residues (shown in Figure 2) or another metal ion is present instead of zinc at the active site. In the D to A step, proton shuttle residues (usually a His placed in the middle of the cavity, such as for example His64 in CA II) play a crucial role in the proton transfer processes.

Figure 4
Catalytic (for the physiological reaction), inhibition (with zinc binders) and activation mechanisms of α-CAs (hCA I amino acid numbering of the zinc ligands [20])

A similar catalytic/inhibition/activation mechanism is valid also for CAs from other classes although the metal ion is coordinated by other amino acid residues (shown in Figure 2) or another metal ion is present instead of zinc at the active site. In the D to A step, proton shuttle residues (usually a His placed in the middle of the cavity, such as for example His64 in CA II) play a crucial role in the proton transfer processes.

The substrate CO2 was observed bound (at least for the α- [35] and β-class enzymes [57]) in a hydrophobic pocket near the Zn(II) ion (Figure 4B), defined among others by residues Val121, Val143 and Leu198 {in the human (h) isoform hCA II [35]}. It is interesting to mention that the orientation of bound CO2 is very similar in the α- [35] and β-class enzymes [57]. Oriented in this favourable position for the nucleophilic attack by the zinc hydroxide species of the enzyme, CO2 is transformed into bicarbonate which is coordinated to the Zn(II) ion (Figure 4C). As the binding of bicarbonate to zinc is rather labile, this intermediate readily reacts with an incoming water molecule, liberating the bicarbonate into solution. The anions probably exit the active site by using the hydrophilic half of the active site [53]. In this way, the acidic species of the enzyme, with water as the fourth zinc ligand is formed (Figure 4D). Generation of the nucleophilically active species of the enzyme (with hydroxide bound to zinc, Figure 4A) is achieved through a proton transfer reaction from the zinc-coordinated water to the buffer, which, as mentioned above, is the rate-determining step of the entire process [5356]. In many CA isoforms and probably in many enzyme classes (not only the α ones which are the most investigated CAs), this process is assisted by an active site residue (the proton shuttle) able to participate in proton transfer processes, e.g. a histidine residue placed in the middle of the active site [5357]. For hCA II and several other human isoforms this residue is His64, which may be further assisted in the proton transfer process by a cluster of histidine residues extending from the middle of the cavity to its entrance and to the surface of the enzyme around the edge of the cavity, including (in hCA II) residues His4, His3, His10 and His15 [55]. The CA activators (CAAs) enhance the catalytic process by favouring this rate-determining step of the catalytic cycle. They form enzyme–activator (E–A) complexes in which the proton transfer reaction becomes intra- not inter-molecular, and thus, more rapid compared with the same reaction without the activator [53,55,58] (Figure 4). Indeed, most classes of CAAs incorporate in their molecules moieties able to participate in proton transfer processes, with pKa values between 6.5 and 8 (e.g. amines, amino acids, oligopeptides) [55,5860].

For a long period, the only CA inhibition mechanisms known were those illustrated in Figure 4(E), with the inhibitors binding to the metal ion in tetrahedral geometry of the metal ion, as shown in Figure 4(E), or by adding to the metal coordination sphere and generating trigonal bipyramidal geometries [1,7,31,53]. However, this situation changed dramatically in the last decade, with a large number of novel CA inhibition mechanisms reported (Figure 5) and new classes of CAIs discovered apart from the zinc binders shown in Figure 4 [61].

CA inhibition mechanisms

Figure 5
CA inhibition mechanisms

(A) Zinc binding. (B) Anchoring to the metal ion coordinated water. (C) Occlusion of the active site entrance. (D) Out of the active site binding [61].

Figure 5
CA inhibition mechanisms

(A) Zinc binding. (B) Anchoring to the metal ion coordinated water. (C) Occlusion of the active site entrance. (D) Out of the active site binding [61].

Many classes of CAIs act as zinc binders (Figure 5A). The classical ones are the sulfonamides and their isosteres (sulfamates, sulfamides) [1,47,31], in which the zinc-binding group (ZBG) is a SO2NH moiety. Such compounds also incorporate a scaffold which may interact with either the hydrophobic, the hydrophilic (or both) halves of the active site, as well as tails which bind towards the exit of the active site cavity which is the most variable region of the various CA isoforms [1,47,31,53,61]. Other classes of inhibitors which bind to the metal ion include carboxylates [6163]; hydroxamates [64,65]; dithiocarbamates and isosteres [66,67], etc. (for a detailed review see [61]).

A second CA inhibition mechanism, anchoring to the metal ion coordinated water molecule/hydroxide ion, is shown in Figure 5(B). These inhibitors incorporate an anchoring group (AG) which is hydrogen bonded to the metal ion coordinated nucleophile, eventually making additional hydrogen bonds with neighbouring residues such as the gate keeping ones (Thr199, Glu106) in the α-class enzymes. Phenol was the first inhibitor for which this mechanism was documented by X-ray crystallography [68], followed thereafter by polyamines [69], esters [70] and sulfocoumarins [71]. Like the zinc binders, the scaffold of this type of inhibitor may interact with one or both halves of the active site, and a tail may also be present for enhancing the isoform selectivity profile of the inhibitors [61]. Situated even further away from the metal ion, at the entrance of the active site cavity are the inhibitors which occlude the active site entrance, shown in Figure 5(C). Such compounds incorporate a sticky group (SG) which may be of the OH, amino, COOH and other types, as well as the scaffold/tail, as the previous classes of inhibitors discussed above. This mechanism was discovered for coumarins [72,73] and it was later shown that many other classes of structurally similar compounds bind in this manner to the enzyme [74,75]. More recently a fourth CA inhibition mechanism (Figure 5D) was also documented by X-ray crystallography [76]. A benzoic acid derivative was observed bound outside the active site cavity, in a hydrophobic pocket adjacent to the entrance within the active site. The inhibition is achieved by blocking the proton shuttle residue (His64) in its out conformation, which leads to the collapse of the entire catalytic cycle [76].

The CAIs have clinical applications and such drugs belonging to the sulfonamide class have been used for decades [1,7,77] as antiglaucoma [78], diuretics [79], antiobesity [80,81], neurologic [82] and more recently antitumour agents [83]. The first and second generation drugs, as mentioned above belonging to the sulfonamide/sulfamate classes, have a range of side effects due to the indiscriminate inhibition of most human CA isoforms (15 CAs are presently known in our species) [1,31]. However the new generation inhibitors, such as those mentioned in this paragraph, act by new inhibition mechanisms which have been unravelled only recently, and may show fewer side effects due to the fact that their inhibition profiles are much more selective compared with the first/second generation inhibitors. In fact a sulfonamide antitumour agents, SLC-0111, recently entered Phase I clinical trials for the management of metastatic, advanced solid tumours [84].

CONCLUSIONS

The CAs represent a unique example of highly investigated enzymes both for basic science studies, which allowed the understanding of intricate structure–function relationships in proteins at the atomic level, but also from the practical viewpoint, as the inhibitors of these enzymes possess clinical applications for a variety of disorders [7784]. Possibly, the CAs themselves might be useful for biotechnological applications in CO2 capture processes or transformation of this gas in useful organic compounds (e.g. oxaloacetate), in order to mitigate the global warming effects due to the accumulation of this gas in the atmosphere [8587]. Six genetically diverse CA families are known to date, the α-, β-, γ-, δ-, ζ- and η-CAs, which evolved in an astonishing manner: the α-, β-, γ-CAs are probably convergently evolved, whereas δ-, ζ- and η-CAs seem to be highly divergent variants of the β- or α-CAs, constituting however diverse genetic families of their own [5]. Detailed kinetic data are available for representatives of all classes, whereas X-ray crystallographic studies are available only for four of them. Such data allowed a deep understanding of the structure–function relationship in this superfamily of proteins. All CA families use a metal hydroxide nucleophilic species of the enzyme, and possess a unique active site architecture, with half of it hydrophilic and the opposing part hydrophobic, allowing these enzymes to act as some of the most effective catalysts known in Nature. The protein fold in the diverse CA classes is highly different, as it is the oligomeric state of these enzymes. The CA activation and inhibition mechanisms are known in detail, with a large number of new inhibitor classes being described in recent years. Apart from the zinc binders, which are the classic CAIs known since the 40s (i.e. the primary sulfonamides), some other classes of inhibitors were shown to possess a similar mechanism of action (e.g. carboxylates, hydroxamates, dithiocarbamates and phosphonates). Other classes of inhibitors were shown to anchor to the metal ion coordinated nucleophile binding in a more distant part of the active site cavity compared with the zinc binders. Other inhibitors occlude the entrance of the active site cavity, whereas more recently, compounds binding outside the active site were also described. As CA inhibition has therapeutic applications for drugs acting as diuretics, antiepileptics, antiglaucoma, antiobesity and antitumour agents, the discovery of the new inhibition mechanisms mentioned here may also lead to isoform-selective inhibitors which should possess a better pharmacological profile compared with the first/second generation inhibitors still in clinical use. Recently, targeting of pathogen CAs has also been proposed as a new strategy for designing anti-infectives with a new mechanism of action [58,11]. Successful structure-based drug design campaigns allowed the discovery of highly isoform selective CAIs, which may lead to new generation of drugs targeting these widespread enzymes. The use of CAs in the CO2 capture processes for mitigating the global temperature rise has also been recently investigated with some success. Overall, this fascinating field led to important discoveries of several widely used drug classes. Although the biotechnological applications of these enzymes started to be investigated only recently, they may resolve important environmental problems such as the global heating due to excessive CO2 production.

CONFLICT OF INTEREST

I report conflict of interest, being author on many patents dealing with CA inhibitors.

I am very much grateful to all my collaborators, in Florence and elsewhere, for their wonderful enthusiasm and help in many projects which led to interesting discoveries in the CA field. I particularly thank Dr Marta Ferraroni for help with some of the figures from the article.

FUNDING

This work was supported by the Euroxy [grant number LSH-CT-2003-502932]; the DeZnIT; the Metoxia [grant number Health-F2-2009-222741]; and the Dynano [grant number PITN-GA-2011-289033].

Abbreviations

     
  • CA

    carbonic anhydrase

  •  
  • CAA

    CA activator

  •  
  • CAI

    CA inhibitor

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