Ординатура / Офтальмология / Английские материалы / Carbonic Anhydrase Its Inhibitors and Activators_Supuran, Scozzafava, Conway_2004

Taylor et al. 1970) and also the hydrated trichloroacetaldehyde (Bertini et al. 1979a) and α-aminoacids in their zwitterionic form (Bertini et al. 1977b).

Class C Inhibitors have an intermediate pH dependence of Kapp (between Class A and Class B; Figure 7.1C). They can bind to the active site over a wide range of pH values, owing to their particular structural and ionizing properties (Scheme 7.2.). This class includes imidazole, 1,2,4-triazole and 1,2,3-triazole (Alberti et al. 1981). The behavior of these ligands clearly reveals the direct influence of the binding mode on the inhibitory properties of the compound.

SCHEME 7.2

In general, both anions and sulfonamides bind to CA (as well as to Co-CA) in the stoichiometric ratio of 1:1 (Bertini and Luchinat 1983; Bertini et al. 1982). On binding, the electronic and magnetic properties of both the metal ion and the inhibitor are altered. Consequently, coordination behavior can be revealed by electronic, NMR-, EPRand UV-VIS spectra (Bertini and Luchinat 1983). Ultimate details are provided by x-ray diffraction experiments on enzyme–inhibitor adducts, but because of the inherent difficulties associated with this technique, data are available for only a limited number of such inhibitors.

A basic fact revealed by 1H-NMR studies on the cobalt protein is that the histidines remain coordinated on binding of inhibitors (Bertini et al. 1981). As a direct consequence, inhibitors can give rise to tetrahedral species, to five-coordinated species or to equilibrium between the two (Bertini and Luchinat 1983):

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The position of the equilibrium can be evaluated by electronic spectroscopy, by monitoring the intensity of the overall absorption spectrum, or the presence or absence of a weak band, in the range of 13 to 15 ∞ 103 cm–1 for Co-CA, characteristic of the five-coordinated complexes (Bertini et al. 1980a, 1978a). Similar results were obtained from EPR spectra (Bencini et al. 1981) performed on Cu(II)-CA (Taylor and Coleman 1971), V(IV)-CA (Bertini et al. 1979b; Fitzgerald and Chasteen 1974) and Mn(II)-CA (Bertini et al. 1978b; Lanir et al. 1973; Meirovitch and Lanir 1978). Also, a very powerful technique for studying the inhibition mechanism of CA is nuclear magnetic resonance spectroscopy. The large majority of the studies were done on Co(II)-CA (Alberti et al. 1981; Banci et al. 1990, 1989; Bertini et al. 1981; Luchinat et al. 1990; Moratal et al. 1992a), Ni(II)-CA (Moratal et al. 1992b), Cd(II)- CA (Armitage et al. 1978; Jarvet et al. 1989), as well as on the native enzyme (Jarvet et al. 1989).

The correlated results of all these studies allow a good overview (Bertini and Luchinat 1983) of the coordination behavior of different anionic and neutral inhibitors to the Zn(II) ion of the active site. In light of these experiments, anions such as CN–, NCO–, SH–, as well as ligands such as aniline, anthranilate, trichloroacetaldehyde, thiadiazole, imidazole (at high pH), 1,2,4-triazole, tetrazole and all the sulfonamides generate pseudotetrahedral species. The anions SCN–, HSO3–, NO3–, I–, Au(CN)2–, Ag(CN)2–, formate, acetate, bromoacetate, oxalate, malonate, succinate, glutarate and ligands such as glycine, L(+)-alanine, D(–)-alanine, 2,4-pen- tanedione and 1,2,3-triazole yield five-coordinated species.

Between these two extremes are HCO3–, F–, Cl–, Br–, N3–, phosphate and benzoate anions and imidazole at low pH, all generating fourto five-coordinated species, as shown by the electronic spectra and NMR experiments on the Co-CA adducts (Banci et al. 1989; Bertini et al. 1981, 1978a, 1979b, 1977a, 1982; Bertini and Luchinat 1983, 1984), which can be extrapolated to the native enzyme (Bertini and Luchinat 1983; Supuran and Manole 1999).

These results are supported, in general terms, by x-ray crystallographic data. Pseudotetrahedral coordination was confirmed for HS– (Mangani and Haakansson 1992), 1,2,4-triazole (Mangani and Liljas 1993) and sulfonamides (Eriksson et al. 1988b; Haakansson and Liljas 1994; Vidgren et al. 1990, 1993). Thus, for HS–, Mangani and Haakansson (1992) showed that the strong inhibitor hydrosulfide coordinates the zinc atom of hCA II at a distance of 2.2 Å, replacing the metalbound water/hydroxide (Wat-263) of the native enzyme (Figure 7.2). The binding mode is further stabilized by a hydrogen bond donated to the Oγ of Thr-199 (similar to the case of sulfonamide binding). The zinc ion maintains its tetrahedral coordination geometry, and differences from the native structure are minimal. The structured water network within the active site is also little affected by the inhibitor binding (Mangani and Haakansson 1992).

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FIGURE 7.2 Coordination detail for the complex between human CA II and hydrogen sulfide (From Mangani, S. and Haakansson, K. 1992. European Journal of Biochemistry 210, 867–871.); the structure was generated from the pdb file (1CAO) by using the program RasWin for Windows, version 2.7.1.1.

FIGURE 7.3 Coordination detail for the complex between human CA II and 1,2,4-triazole (From Mangani, S. and Liljas, A. 1993. Journal of Molecular Biology 232, 9–14.); the structure was generated from the pdb file (1CRA) by using the program RasWin for Windows, version 2.7.1.1.

A related pattern is valid for the adduct of 1,2,4-triazole with hCA II (Mangani and Liljas 1993). The five-member ring of triazole coordinates to zinc through the N4 atom at a distance of 2.05 Å, displacing the water/hydroxyl ion, and generates a distorted tetrahedral geometry for the metal ion (Figure 7.3). It also forms two bent hydrogen bonds with the Oγ of Thr-200 (by N1) and the amide nitrogen atom

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FIGURE 7.4 Coordination detail for the complex between human CA II and thiocyanate anion (From Eriksson, A.E. et al. 1988b. Proteins: Structure, Function, and Genetics 4,

283–293.); the structure was generated from the pdb file (2CA2) by using the program RasWin for Windows, version 2.7.1.1.

of Thr-199 (with N2). Despite the smaller bond energy, the presence of the N2Thr199 hydrogen bond accounts for the different coordination pattern observed for the enzyme with 1,2,4-triazole (clearly tetrahedral) as compared with the case of imidazole (tetrato pentacoordinated; Alberti et al. 1981; Kannan et al. 1977; Luchinat et al. 1990; Mangani and Liljas 1993). In contrast to the hydrosulfide ion, the larger size of the 1,2,4-triazole molecule causes substantial changes in the network of water molecules present in the active-site cavity. Besides replacing the zinc-bound water, the inhibitor displaces the deep water molecule (Wat-338) and also another two water molecules from the active-site network. The positive entropic contribution arising from the release of these molecules also accounts in the total free energy of binding for the 1,2,4-triazole molecule (Mangani and Liljas 1993). Mention should be made that the location of this inhibitor overlaps the binding site for bicarbonate (Haakansson and Wehnert 1992; Xue et al. 1993) as well as the proposed binding site for carbon dioxide (Lindahl et al. 1993). This fact can explain the observed competitive binding of 1,2,4-triazole with respect to CO2/HCO3– under equilibrium conditions (Tibell et al. 1985).

Alternatively, the binding of SCN– ion to hCA II was shown to be a pure pentacoordinated one (Eriksson et al. 1988b). The three histidines, a water molecule and the SCN– ion coordinate the zinc ion. The nitrogen atom of the thiocyanate is 1.9 Å away from the metal and shifted 1.3 Å with respect to the hydroxyl ion in the native structure, being at van der Waals distance from the Oγ of Thr-199 (Figure 7.4; Eriksson et al. 1988b).

The authors assigned this conformation to the inability of the Oγ1 atom of Thr199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen.

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FIGURE 7.5 Coordination detail for the complex between human CA II and nitrate (From Mangani, S. and Haakansson, K. 1992. European Journal of Biochemistry 210, 867–871.); the structure was generated from the pdb file (1CAN) by using the program RasWin for Windows, version 2.7.1.1.

The zinc-bound water was found to be located 2.2 Å away from the metal and 2.4 Å from the nitrogen atom of the thiocyanate ion. This coordinated water is hydrogen bonded to the Oγ1 atom of Thr-199 and to another water molecule (Wat-318). It was also shown that the sulfur atom of the SCN– displaces the deep water molecule of the native enzyme and makes van der Waals interactions with Val-143, Leu-198 and Trp-209 side chains. Besides these modifications, the hydrogen network in the active site is almost identical to the native structure (Eriksson et al. 1988a, 1988b).

Mangani and Haakansson (1992) reported the structure of the hCA II–NO3– complex and showed that this anion is also bound in a pentacoordinated geometry at the zinc ion. It shares a similar position with SCN– within the active site of the enzyme, being located in the deep-water pocket of the protein (delimited by residues Val-121, Leu-141, Val-143, Leu-198, Thr-199, Val-207 and Trp-209). However, the interaction of the nitrate anion [in a monodentate fashion, which was calculated (Kumar and Marynick 1993) to be more stable than the bidentate form] is weaker than in the case of thiocyanate ion, the distance between the zinc and the ligand being 2.8 Å (Figure 7.5). The coordinated water (Wat-263) is placed 1.9 Å away from the zinc ion, slightly moved from its original position.

The same major characteristics can be found in the complexes of formate and acetate with hCA II (Haakansson et al. 1992, 1994). The formate anion is bound to zinc in a pentacoordinated manner, the other ligand besides the three histidines being a water molecule (Figure 7.6). The HCOO– ion is linked in a monodentate fashion to the metal, with its O2 atom 2.5 Å away from the zinc. The other oxygen of the formate ion forms a hydrogen bond with the Thr-199 amide nitrogen. The coordinated water is 2.2 Å from the zinc ion, only 0.7 Å away from its native position in the active protein.

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FIGURE 7.6 Coordination detail for the complex between human CA II and formate (From Haakansson, K. et al. 1992. Journal of Molecular Biology 227, 1192–1204.); the structure was generated from the pdb file (2CBC) by using the program RasWin for Windows, version 2.7.1.1.

FIGURE 7.7 Coordination detail for the complex between human CA II and acetate (From Haakansson, K. et al. 1994. Acta Crystallographica, Section D: Biological Crystallography

D50, 101–104.); the structure was generated from the pdb file (1CAY) by using the program RasWin for Windows, version 2.7.1.1.

In the case of acetate (Haakansson et al. 1994), one carboxylate oxygen is coordinated to the zinc at a distance of 2.4 Å, 1.7 Å away from the position of the zinc-bound water in the native protein. The zinc water itself is displaced 0.8 Å from its native position (Figure 7.7). The other carboxylate oxygen atom is hydrogen bonded to the Thr-199 backbone NH (3.0 Å away). The similarity with the formate

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FIGURE 7.8 Coordination detail for the complex between human CA II and bisulfite (From Haakansson, K. et al. 1992. Journal of Molecular Biology 227, 1192–1204.); the structure was generated from the pdb file (2CBD) by using the program RasWin for Windows, version 2.7.1.1.

and nitrate ions bonding is obvious. The methyl group of the inhibitor makes van der Waals interactions with the hydrophobic pocket of the protein, being in contact with Val-143 (3.3 Å), Leu-198 (3.9 Å) and Trp-209 (3.6 Å; Haakansson et al. 1994). Haakansson et al. (1992) also analyzed, in conjunction with the formate adduct, the complex of CA II with the bisulfite ion. Although bisulfite differs geometrically from bicarbonate, being pyramidal instead of planar as is the latter, it was assumed that its binding is analogous to the binding of the bicarbonate substrate (Haakansson et al. 1992). The results of the study showed that, in contrast to formate, the bisulfite anion is coordinated to the zinc active site in a tetrahedral geometry (Figure 7.8).

The explanation resides in the ability of bisulfite to act as a hydrogen-bond donor to the Oγ of Thr-199 and also to establish another hydrogen bond to the amide nitrogen atom of the same residue (2.9 Å away), similar to the cases of HS– and the sulfonamides (Eriksson et al. 1988b; Haakansson et al. 1992; Haakansson and Liljas 1994; Mangani and Haakansson 1992; Vidgren et al. 1990, 1993). These additional H bonds account for sufficient energy to fully stabilize a tetrahedral geometry at the zinc ion. From this point of view, the Thr-199, along with H-bonded Glu-106, was considered to act as a door-keeper to the zinc ion, selecting protonated atoms for the water position on the zinc ion and excluding nonprotonated atoms from this coordination site (Eriksson et al. 1988b; Liljas et al. 1994; Lindskog and Liljas 1993; Lindskog and Silverman 2000; Merz 1990). Other structural characteristics of the bisulfite–hCA II complex are very similar to those of the formate ion. The HSO3– also displaces the deep water molecule and Wat-338 (similar to the cases of 1,2,4- triazoleand NO3– binding), thus interfering with the residues Val-121, Val-143, Leu198, Thr-199 and Trp-209. The ligand, obviously bound in the monodentate mode, is 3.1 Å away from the Zn ion. Again, as in the case of formate, no changes in the

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peptide chain conformation are observed (Haakansson et al. 1992). The results are consistent with the Laue crystallographic data generated by Lindahl et al. (1992) for the same ligand; they are pH independent, as determined previously (Yachandra et al. 1983).

The very important role of Thr-199 for the binding mode of inhibitors into the active site of CA II (e.g., bisulfite vs. nitrate or formate) is also revealed in the case of adducts of strong inhibitors cyanide and cyanate with CA II. A common conclusion of several electronic absorption studies (Bertini et al. 1980b; Lindskog 1963, 1966) and ligand NMR studies (Banci et al. 1990; Bertini and Luchinat 1983) on Co-CA, carried out in solution, was that the strong inhibitors NCO– and CN– will substitute the zinc-bound water and coordinate to the metal in a tetrahedral mode (Banci et al. 1990; Bertini et al. 1978a), whereas the SCN– ion, a weaker inhibitor, will coordinate as a fifth ligand, thus forming a distorted trigonal bipyramid. Eventually, this was proved to be true in the case of SCN– (Eriksson et al. 1988b).

However, Lindahl et al. (1993) determined the structure of the hCA II inhibited by cyanide and cyanate. Their puzzling conclusion was that the two inhibitors bind in the close vicinity of the zinc ion, displacing the deep water molecule and forming a hydrogen bond with NH of Thr-199 backbone; the coordination of the metal ion was left unaltered, with a molecule of water coordinated in a tetrahedral fashion. The shortest distance between the zinc and the cyanate ion was found to be 3.2 Å, whereas the shortest Zn–CN-distance was a little longer –3.3 Å (Lindahl et al. 1993). It was hypothesized that this coordination behavior of the two ions is because of their inability to form a donor H bond with the Oγ1 of Thr-199, which can stabilize a tetrahedral coordination as in the case of hydrosulfide-, 1,2,4-triazole- and sulfonamide binding (Lindahl et al. 1993). In response to these findings, Bertini’s group (Bertini et al. 1992) reinvestigated the binding of NCO–, CN– and SCN– by spectroscopic techniques on Co-CA and 67Zn-CA (which has a large quadrupolar moment). They arrived at the same conclusion that they considered previously, that the CN– and CNO– bind, in solution, the metal ion in bCA II. The discrepancies between their findings and the results of the crystallographic study were attributed to the existence of two different free-energy minima accessible to the system in solution and in the solid state.

These contradictory reports prompted the hypothesis (Supuran 1992; Supuran et al. 1994, 1995) that cyanate, which is isoelectronic and isosteric with the physiological substrate of the enzyme, CO2, might behave as a poor substrate for CA (Supuran et al. 1997). Theoretical calculations by Merz’s group (Peng et al. 1993) for the binding of cyanate, cyanide and thiocyanate to CA suggested that cyanide and cyanate might act as substrates; they tried to solve the controversy between the two approaches — the crystallographic methods and the spectroscopic ones — by suggesting that crystallographic methods observe the hydrated complexes of HCN and HNCO whereas the spectroscopic methods do not (Peng et al. 1993). Supuran et al. (1997) investigated whether different CA isozymes were able to catalyze the hydrolysis of cyanide or cyanate and found that a clear-cut answer was difficult to provide. They affirmed that either the process is unfavored kinetically or the two anions are actually hydrolyzed by CA but the corresponding products (carbamic acid for cyanate, formamide for cyanide) are very tightly bound to the enzyme and

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FIGURE 7.9 Coordination detail for the complex between human CA II and urea (From Briganti, F. et al. 1999. Journal of Biological Inorganic Chemistry 4, 528–536.); the structure was generated from the pdb file (1BV3) by using the program RasWin for Windows, version 2.7.1.1.

do not dissociate from the active site (Supuran et al. 1997). The latter hypothesis is sustained by the recent finding (Briganti et al. 1999; Guerri et al. 2000) that cyanamide, which is also isoelectronic with CO2, acts as a potent suicide substrate for CA. The urea formed after the hydrolytic reaction of the cyanate ion remains blocked within the enzyme active site, where it is directly coordinated to the zinc ion through a protonated nitrogen atom (Figure 7.9; Briganti et al. 1999). The hCA II–urea adduct was found to be quite a regular tetrahedron, with the distances and angles around the zinc atom similar to those of the native metalloprotein. The position of the ureate in the active site is further stabilized by hydrogen bonding with Thr-199, Thr-200 and several water molecules (Figure 7.9). These strong interactions at the active site explain why the zinc-bound ureate cannot be displaced even by high concentrations of very potent sulfonamide inhibitors (Briganti et al. 1999). The authors also provided a reaction mechanism to explain the cyanamide hydration, based on electronic spectroscopy and kinetic and x-ray crystallographic studies on the system.

The precise binding mode of cyanamide to the enzyme was clarified in a subsequent investigation by the same group (Guerri et al. 2000), using cryocrystallographic techniques. The crystal structure, obtained at 100 K and after soaking the protein crystal for a limited period in cyanamide solutions, shows that two different adducts are formed under the experimental conditions, with different occupancy in the crystal. The high-occupancy form consists of a binary hCA II–cyanamide complex, wherein the substrate has replaced the zinc-bound hydroxide/water, maintaining the tetrahedral geometry around the metal ion (Figure 7.10). The second, low-occupancy form consists of a hCA II–cyanamide–water ternary complex, wherein the catalytic zinc ion is still coordinated by a cyanamide molecule, but is also approached by a molecule of water. In the high-occupancy form, the zinc ion, besides the three

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FIGURE 7.10 Coordination detail for the complex between human CA II and cyanamide (Wat-51 right) (From Guerri, A. et al. 2000. Biochemistry 39, 12391–12397.); the structure was generated from the pdb file (1F2W) by using the program RasWin for Windows, version 2.7.1.1.

histidines, binds one cyanamide molecule at 2.09 Å; the water molecule (Wat-51) approaches zinc at 2.91 Å and can be considered as a zinc-outer sphere fifth ligand, generating a 4 + 1 coordination type for the metal ion. The deep water molecule (Wat-338) usually occupying the hydrophobic pocket of the enzyme is absent in this structure. The Wat-51 is closer to cyanamide (~2.5 Å) than to zinc (2.91 Å). The interaction of cyanamide with the CA II active site is completed by two strong hydrogen bonds. The first one involves the NH attached to zinc and the Oγ1 of Thr199 proved to stabilize a tetrahedral geometry in the case of HS–, 1,2,4-triazole, HSO3– and sulfonamides. Another hydrogen bond is formed with the side chain of Thr-200. Except for seven water molecules displaced on binding of the ligand, the remaining water molecules and the overall structure of CA II remain unaltered on interaction with cyanamide. The low-occupancy form of the hCA II–cyanamide adduct is a ternary hCA II–cyanamide–water (Wat-51) complex, which can be interpreted as a frozen intermediate state of the hCA II-catalyzed conversion of cyanamide to urea. Wat-51 is considered to be added to the Zn coordination sphere, generating a five-coordination geometry. The coincidence of this water molecule position with that of the zinc-bound oxygen and nitrogen atoms of the five-coordi- nated adducts with nitrate and thiocyanate favors this hypothesis (Guerri et al. 2000).

Thus, the possibility of the existence of equilibria between fourand fivecoordinate species in CA (Bertini and Luchinat 1983; Bertini et al. 1982) is once again confirmed. Moreover, formation of a pentacoordinated adduct in the presence of cyanamide was anticipated on the basis of spectroscopic investigations on Co-CA (Briganti et al. 1999).

If the ternary complex represents a snapshot along the reaction coordinate of the hCA II-catalyzed cyanamide hydration, this reaction will follow a different

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