Carbonic anhydrases zinc site

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. 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

Carbonic anhydrase an insight into the zinc binding site and into the active cavity through metal substitution. I. Bertini, C. Luchinat and A. Scozzafava, Struct. Bonding (Berlin), 1982, 48, 46-92 (296). 

Bertini I, Luchinat C, Scozzafava A (1982) Carbonic Anhydrase An Insight into the Zinc Binding Site and into the Active Cavity Through Metal Substitution. 48 45-91 Bertrand P (1991) Application of Electron Transfer Theories to Biological Systems. 75 1-48 Bill E, see Trautwein AX (1991) 78 1-96 Bino A, see Ardon M (1987) 65 1-28 Blanchard M, see Linares C (1977) 33 179-207 Blasse G, see Powell RC (1980) 42 43-96... 

Bertini, /., Luchinat, C., Scozzafava, A. Carbonic Anhydrase An Insight into the Zinc Binding Site and into the Active Cavity Through Metal Substitution. Vol. 48, pp. 45-91. 

X-ray diffraction studies on several forms of the enzyme have demonstrated that the active site is composed of a pseudo-tetrahedral zinc center coordinated to three histidine imidazole groups and either a water molecule [(His)3Zn-OH2]2+ (His = histidine), or a hydroxide anion [(His)3Zn-OH] +, depending upon pH (156,157). On the basis of mechanistic studies, a number of details of the catalytic cycle for carbonic anhydrase have been elucidated, as summarized in Scheme 22... 

The value of the tris(pyrazolyl)hydroborato complexes [TpRR ]ZnOH is that they are rare examples of monomeric four-coordinate zinc complexes with a terminal hydroxide funtionality. Indeed, [TpBut]ZnOH is the first structurally characterized monomeric terminal hydroxide complex of zinc (149). As such, the monomeric zinc hydroxide complexes [TpRR ]ZnOH may be expected to play valuable roles as both structural and functional models for the active site of carbonic anhydrase. Although a limitation of the [TpRR ]ZnOH system resides with their poor solubility in water, studies on these complexes in organic solvents... 

Fig. 42. Comparison of the coordination environment about zinc in the active site of carbonic anhydrase in its deprotonated form with that of [TpRR ]ZnOH. Reprinted with permission from Ref. (151). Copyright 1993 American Chemical Society.

As an illustration, we briefly discuss the SCC-DFTB/MM simulations of carbonic anhydrase II (CAII), which is a zinc-enzyme that catalyzes the interconversion of CO2 and HCO [86], The rate-limiting step of the catalytic cycle is a proton transfer between a zinc-bound water/hydroxide and the neutral/protonated His64 residue close to the protein/solvent interface. Since this proton transfer spans at least 8-10 A depending on the orientation of the His 64 sidechain ( in vs. out , both observed in the X-ray study [87]), the transfer is believed to be mediated by the water molecules in the active site (see Figure 7-1). To carry out meaningful simulations for the proton transfer in CAII, therefore, it is crucial to be able to describe the water structure in the active site and the sidechain flexibility of His 64 in a satisfactory manner. 

The zinc acetate complex of tris(3-/-butyl-5-methylpyrazol-l-yl)borate was prepared as a structural model for carbonic anhydrase and comparison with the enzyme active site structures confirmed that the complexes are excellent structural models.239 A mononuclear zinc hydroxide complex can also be formed with the tris(pyrazolyl) borate ligand system as a structural model for carbonic anhydrase.240... 

The ligand tris[2-(l-methylbenzimidazol-2-yl)ethyl] nitromethane (25) has been used in the formation of zinc complexes as models of the active site of carbonic anhydrase, and the formed complexes reveal affinity for the sulfonamide-containing enzyme inhibitor acetazolamide.248... 

Theoretical calculations have been carried out on a number of zinc-containing enzymatic systems. For example, calculations on the mechanism of the Cu/Zn enzyme show the importance of the full protein environment to get an accurate description of the copper redox process, i.e., including the electronic effects of the zinc ion.989 Transition structures at the active site of carbonic anhydrase have been the subject of ab initio calculations, in particular [ZnOHC02]+, [ZnHC03H20]+, and [Zn(NH3)3HC03]+.990... 

The first zinc enzyme to be discovered was carbonic anhydrase in 1940, followed by car-boxypeptidase A some 14 years later. They both represent the archetype of mono-zinc enzymes, with a central catalytically active Zn2+ atom bound to three protein ligands, and the fourth site occupied by a water molecule. Yet, despite the overall similarity of catalytic zinc sites with regard to their common tetrahedral [(XYZ)Zn2+-OH2] structure, these mononuclear zinc enzymes catalyse a wide variety of reactions, as pointed out above. The mechanism of action of the majority of zinc enzymes centres around the zinc-bound water molecule,... 

Carbonic anhydrase is a metalloprotein with a co-ordinate bonded zinc atom immobilized at three histidine residues (His 94, His 96 and Hisl 19) close to the active site of the enzyme. The catalytic activity of the different isoenzymes varies but cytosolic CA II is notable for its very high turnover number (Kcat) of approximately 1.5 million reactions per second. 

Carbonate anhydrase (carbonic anhydrase, EC 4.2.1.1) catalyzes the reversible interconversion of C02 and HCO3 (see Sect. 3.7.3). The enzyme is found in erythrocytes, and in kidney and gastric juices where it contributes to the control of the acid-base balance. The esterase activity of carbonic anhydrase is probably due to the similarity between its active site and that of the zinc proteases. A possible physiological role of the esterase activity of this enzyme remains to be established. 

The carbonic anhydrase (Cam) in M. thermophila cells is elevated several fold when the energy source is shifted to acetate, suggesting a role for this enzyme in the acetate-fermentation pathway. It is proposed that Cam functions outside the cell membrane to convert CO2 to a charged species (reaction A4) thereby facilitating removal of product from the cytoplasm. Cam is the prototype of a new class (y) of carbonic anhydrases, independently evolved from the other two classes (a and P). The crystal structure of Cam reveals a novel left-handed parallel P-helix fold (Kisker et al. 1996). Apart from the histidines ligating zinc, the activesite residues of Cam have no recognizable analogs in the active sites of the a- and P-classes. Kinetic analyses establish that the enzyme has a zinc-hydroxide mechanism similar to that of Cab (Alber et al. 1999). 

X-Ray crystallographic studies of native human carbonic anhydrase II have been reported at 2.0 A resolution (Eriksson et al, 1986, 1988a). Important active-site residues include Thr-199, Thr-200, Glu-106, His-64, Trp-209, Val-121, Val-143, the zinc ion (liganded by His-94, His-96, and His-119), and the zinc-bound hydroxide ion. A schematic view of the active site is found in Fig. 22. The globular protein has a molecular... 

There may be two proton transfers in the carbonic anhydrase II-catalyzed mechanism of CO2 hydration that are important in catalysis, and both of these transfers are affected by the active-site zinc ion. The first (intramolecular) proton transfer may actually be a tautomerization between the intermediate and product forms of the bicarbonate anion (Fig. 28). This is believed to be a necessary step in the carbonic anhydrase II mechanism, due to a consideration of the reverse reaction. The cou-lombic attraction between bicarbonate and zinc is optimal when both oxygens of the delocalized anion face zinc, that is, when the bicarbonate anion is oriented with syn stereochemistry toward zinc (this is analogous to a syn-oriented carboxylate-zinc interaction see Fig. 28a). This energetically favorable interaction probably dominates the initial recognition of bicarbonate, but the tautomerization of zinc-bound bicarbonate is subsequently required for turnover in the reverse reaction (Fig. 28b). 

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