Carbonic anhydrase hydrophobic pocket

The system illustrated by (272) forms the basis of a model for the zinc-containing metalloenzyme, carbonic anhydrase (Tabushi Kuroda, 1984). It contains Zn(n) bound to imidazole groups at the end of a hydrophobic pocket, as well as basic (amine) groups which are favourably placed to interact with a substrate carbon dioxide molecule. These are both features for the natural enzyme whose function is to catalyze the reversible hydration of carbon dioxide. The synthetic system is able to mimic the action of the enzyme (although side reactions also occur). Nevertheless, the formation of bicarbonate is still many orders of magnitude slower than occurs for the enzyme. 

Figure 24 shows a possible CO2 diffusion pathway into the carbonic anhydrase II active site, and Fig. 25 shows the binding of CO2 in the hydrophobic pocket (Liang and Lipscomb, 1990). Also in Fig. 25 is the binding of CO2 in this pocket as independently calculated by Merz (1990,... 

The results of kinetic and X-ray crystallographic experiments on mutant carbonic anhydrases II, in which side-chain alterations have been made at the residue comprising the base of the hydrophobic pocket (Val-143), illuminate the role of this pocket in enzyme-substrate association. Site-specific mutants in which smaller hydrophobic amino acids such as glycine, or slightly larger hydrophobic residues such as leucine or isoleucine, are substituted for Val-143 do not exhibit an appreciable change in CO2 hydrase activity relative to the wild-type enzyme however, a substitution to the bulky aromatic side chain of phenylalanine diminishes activity by a factor of about 10 , and a substitution to tyrosine results in a protein which displays activity diminished by a factor of about 10 (Fierke et o/., 1991). 

Fig. 25. (a) Proposed binding of substrate CO2 (the dark stick in the center) in the hydrophobic pocket of carbonic anhydrase II, as calculated in a molecular dynamics simulation. [Reprinted with permission from Liang, J.-V., Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3675-3679.] (b) Proposed carbonic anhydrase II-CO2 complex, as calculated in an independent investigation (Merz, 1990, 1991, personal communication). 

Fig. 26. Electron density map, calculated with Fourier coefficients 2 Fo - [fj and phases calculated from the final model, of the hydrophobic pocket of the Val-14S- Tyr mutant of human carbonic anhydrase II (Alexander etai, 1991). This mutation nearly obliterates the pocket and results in a 10 -fold loss of activity (Fierke et ai, 1991).

It is interesting that although the Val-143— His mutation leads to a bulky side chain at the base of the hydrophobic pocket, the mutant enzyme exhibits only a 10 -fold loss of CO2 hydrase activity relative to the wild-type enzyme (Fierke et ai, 1991). In this mutant the Val-I43- His side chain packs differently in the pocket relative to the side chains of the Val-143—>Phe and Val-143- Tyr mutants (Alexander et ai, 1991). It is likely that differences in side-chain packing, as well as differences involving active-site solvent structure, are responsible for differences in enzyme-substrate association behavior among the residue-143 mutants of carbonic anhydrase II. 

Subsequent to CO2 association in the hydrophobic pocket, the chemistry of turnover requires the intimate participation of zinc. The role of zinc is to promote a water molecule as a potent nucleophile, and this is a role which the zinc of carbonic anhydrase II shares with the metal ion of the zinc proteases (discussed in the next section). In fact, the zinc of carbonic anhydrase II promotes the ionization of its bound water so that the active enzyme is in the zinc-hydroxide form (Coleman, 1967 Lindskog and Coleman, 1973 Silverman and Lindskog, 1988). Studies of small-molecule complexes yield effective models of the carbonic anhydrase active site which are catalytically active in zinc-hydroxide forms (Woolley, 1975). In addition to its role in promoting a nucleophilic water molecule, the zinc of carbonic anhydrase II is a classical electrophilic catalyst that is, it stabilizes the developing negative charge of the transition state and product bicarbonate anion. This role does not require the inner-sphere interaction of zinc with the substrate C=0 in a precatalytic complex. 

XH NMR data of copper-carbonic anhydrase (CuCA) complexes in the presence of different anions indicated that water is present in the coordination sphere along with the anions (137). The three histidines, the anion, and the coordinated water molecule arrange themselves to maintain essentially a SQPY. His-94 would be in the apical position of the SQPY and two other histidine residues (His-96 and His-119) along with the anion and the coordinated water are positioned in the basal plane. Most likely the anion is present in the hydrophobic pocket or in the site and the coordinated water molecule is present in the C site or the hydrophilic binding site. 

Crystallographic studies indicated that in the active-site of Zn-Cam, Zn2+ has three residues of the protein and two solvent ligands arranged in a TBP geometry (219). The zinc ion is bound by three histidines (protein residues) of two monomers His-81 and His-122 are contributed by one monomer and His-117 is contributed by an adjacent monomer. The active-site of -class carbonic anhydrase in Cam is located at the base of the cleft between two monomers. The proposed hydrophobic pocket consisting of Met-135b (where b denotes that the residue belongs to the adjacent monomer), Phe-138, Phe-140,... 

Carbonic anhydrase II, present in human red blood cells (RBCs), catalyzes the reversible hydration of C02. It is one of the most efficient enzymes and only diffusion-limited in its turnover numbers. The catalytic Zn11 is ligated by three histidine residues and OH this ZnOH+ structure renders the zinc center an efficient nucleophile which is able to attack the C02 molecule and capture it in an adjacent hydrophobic pocket. The catalytic mechanism is shown in Figure 9.5. 

We designed the model complex for the active site of carbonic anhydrase providing (i) imidazole ligand which corresponds to histidine imidazole, (ii) coordinated water molecule and (iii) hydrophobic pocket, as mentioned in Experimental Section. Since the ligand, tris(2-benzimidazolylmethyl)amine L used in this work plays a role of steric hindrance, it will be able to reproduce the tetrahedral geometry which is identical with the active site of carbonic anhydrase. Furthermore, the benzene rings of benzimidazolyl groups can fix a hydrophobic pocket, in which there exists the active site of the native enzymes. 

Alexander RS, Nair SK, Christianson DW (1991) Engineering the hydrophobic pocket of carbonic anhydrase-II. Biochemistry 30 11064-11072... 

Multivalency can be used to design inhibitors of proteins as well. Multivalent inhibitors have been shown to bind (i) an active site and an adjacent hydrophobic patch (e.g., inhibitors of carbonic anhydrase or the Bcl-2 family of proteins ), (ii) two adjacent binding pockets in a protein (e.g., inhibitors of acetylcholinesterase that bind to the active site and a peripheral area at the edge of the active site ), or (iii) more than two binding pockets distributed over the surface of a protein (e.g., pentavalent ligands that bind to SLT ). These studies reveal that even weak monovalent interactions (e.g., = mM ) can form... 

On treatment with potassium iodide, the capped disulfonate j8-cyclo-dextrin discussed above could easily be converted to the corresponding diiodide jS-cyclodextrin. With appropriate nucleophiles (imidazole, histamine) a new route to bis(iV-imidazolyl)-j8-cyclodextrin and bis(iV-histamino)-j8-cyclodextrin was developed by Tabushi s team (182). In the presence of Zn(II) ion, both regiospecifically bifunctionalized cyclodextrins hydrate CO2 and are the first successful carbonic anhydrase models. The Zn(II) ion binds to the imidazole rings located in the edge of the cyclodextrin pocket and the presence of an additional basic group, as with bis(histamino)-cyclodextrin-Zn(II), enhances the activity. Therefore, the present models show that all three factors, Zn(II)-imidazole, hydrophobic environment, and a base seem to help to generate the carbonic anhydrase activity (182). The chemistry of this enzyme is further discussed in Section 6.2, p. 331. 

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