Water anhydrase active 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. 

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... 

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 most ordered surface waters are those around charged side chains or in surface crevices. Occasionally those crevices can be very deep, such as the active site pocket in carbonic anhydrase, which extends about 15 A in from the surface, with a network of water molecules (Lindskog f al., 1971). The well-ordered waters at the protein surface are usually part of an approximately tetrahedral (but sometimes planar trigonal) network of hydrogen bonds to the protein and to other waters. An example from rubredoxin is shown in Fig. 60. 

The importance of maintaining the active site water network in CA II for efficient proton transfer was investigated by substituting different amino acids of varying size at position 65 and measuring the rate constants for proton transfer in the variant carbonic anhydrases... 

Roberts et al. reported a 27 kDa monomeric carbonic anhydrase, TWCAl, from the marine diatom Thalassiosira weissflogii (221). X-ray absorption spectroscopy indicated that the catalytic zinc is coordinated by three histidines and a water molecule, similar to the active sites of the a- and y-CAs (222). Also, the active site geometry is similar to that of a-CAs. Based on these results the catalytic mechanism is expected to be similar to that of the -class carbonic anhydrases. Tripp et al. (223) proposed that this TWCAl is the prototype of a fourth class carbonic anhydrase designated as 8-class CAs. In the... 

One of the simplest biochemical addition reactions is the hydration of carbon dioxide to form carbonic acid, which is released from the zinc-containing carbonic anhydrase (left, Fig. 13-1) as HC03-. Aconitase (center, Fig. 13-4) is shown here removing a water molecule from isocitrate, an intermediate compound in the citric acid cycle. The H20 that is removed will become bonded to an iron atom of the Fe4S4 cluster at the active site as indicated by the black H20. An enolate anion derived from acetyl-CoA adds to the carbonyl group of oxaloacetate to form citrate in the active site of citrate synthase (right, Fig. 13-9) to initiate the citric acid cycle. 

Despite the fact that carbonic anhydrase was the first zinc metalloenzyme identified1233 and a good deal is known of its structure, there is still controversy about the nature of the various active-site species and the detailed mechanisms of their action. In particular, the identity of the group with a pXa of 7 that is involved in the mechanism, and the stereochemistry around the zinc ion during catalysis, are still in dispute. The various mechanisms proposed assume either ionization of a histidine imidazole group (bound or not to the zinc) and nucleophilic attack on C02 by the coordinated imidazolate anion,1273,1274 or ionization of the Znn-coordinated water and nucleophilic attack on C02 by OH. 1271 Many papers on this problem have appeared recently and the extensive literature is the subject of the several review articles referred to above. 

This is of relevance to the mechanism of carbonic anhydrase. This enzyme, which catalyzes the hydration of C02, has at its active site a Zn2+ ion ligated to the imidazole rings of three of its histidines. The classic mechanism for the reaction is that the fourth ligand is a water molecule which ionizes with a pKa of 7.37 The reactive species is considered to be the zinc-bound hydroxyl. Chemical studies show that zinc-bound hydroxyls are no exception to the rule of high reactivity. The H20 in structure 2.31 ionizes with a pKa of 8.7 and catalyzes the hydration of carbon dioxide and acetaldehyde.38... 

The 2.0 A electron density map of carboxypeptidase A shows three zinc-protein contacts (91). The ligands have been identified as histidine-69, glutamic acid-72 and histidine-196 (91, 101), where the numbers indicate the positions of the residues in the sequence counted from the N-terminal end. The geometry of the complex is irregular but resembles a distorted tetrahedron with an open position directed towards the active site pocket, and presumably occupied by water in the resting enzyme (91). The similarity with the tentative structure of the metal-binding site in carbonic anhydrase is striking. 

There is general agreement that carbonic anhydrase activity is linked to the zinc ion and its ligands. At the active site water is bound to the zinc but the state and involvement of the water in the actual catalysis is a matter of controversy. According to a widely accepted opinion the zinc-bound water is ionized and the activity is a function of pH1S6. ... 

By the use of a model system, Kimura et al. [17] tried to mimic the function of the two mechanistically most typical zinc(II) enzymes. Carbonic anhydrase (CA, EC 4.2.1.1) catalyses the reversible hydration of carbon dioxide to bicarbonate ion and its zinc(II) active site is bound to three histidine residues and a water molecule. Carboxypeptidase A (CPA, EC 3.4.17.1) catalyses the hydrolysis of the hydrophobic C-terminal amino acids from polypeptides, and its active-site zinc(II) is bound to two histidine residues, a glutamine residue and a water molecule which is hydrogen bound to a glutamine residue (Scheme 19). 

The active site of bovine carbonic anhydrase consists of a tetrahedrally coordinated zinc ion (1) [27] with a coordinated water molecule whose p(7.5) [28, 29] is con-... 

Ab-initio and density functional theory are used to calculate the probability of proton conduction via a chain of water molecules from Zn+ to its residue in the active site of carbonic anhydrase (Isaev and Scheiner, 2001). They conclude that proton conduction occurs as a concerted process and includes the shortening of each H-bonds as the proton donor and acceptor move towards one other. 

Metal ions are vital to the function of many enzymes that catalyze hydrolytic reactions. Coordination of a water molecule to a metal ion alters its acid-base properties, usually making it easier to deprotonate, which can offer a ready means for catalyzing a hydrolytic reaction. Also, the placement of a metal center in the active site of a hydrolytic enzyme could permit efficient delivery of a catalytic water molecule to the hydrolyzable substrate. In fact, the first enzyme discovered, carbonic an-hydrase, is a metalloenzyme that requires a Zn2+ center for its catalytic activity (32). The function of carbonic anhydrase is to catalyze the hydrolysis of carbon dioxide to bicarbonate ... 

In fact, transient assembly of H-bonded water files is probably common in enzyme function. In carbonic anhydrase, for example, the rate-limiting step is proton transfer from the active-site Zn2+-OH2 complex to the surface, via a transient, H-bonded water network that conducts H+. Analysis of the relationship between rates and free energies (p K differences) by standard Marcus theory shows that the major contribution to the observed activation barrier is in the work term for assembling the water chain (Ren et al., 1995). 

Another way of bringing reactants into close proximity, which is encountered commonly in transition metal chemistry, is through metal ion complexation. The coordination of a reactant to a metal ion complex often activates its reactivity and can bring the reactant into close proximity with a second reactant or with a catalytic group. One example, shown in Fig. 6, is a zinc (11) complex of 1,5,9-triazacyclononane, as a model for the enzyme carbonic anhydrase, which contains a zinc (11) cofactor in its active site (4). In the aqua complex, the bound water molecule has a dramatically reduced pKa value of 7.3, which is similar to the pKa of the active site nucleophihc water. The corresponding cobalt (111) complex catalyzed ester hydrolysis at twice the rate because Co(lll) can coordinate both the hydroxide nucleophile and the ester carbonyl via a... 

Hg. 19.23 Active site of carbonic anhydrase. resting engine a water molecule (O = > coordinates to the zjnc atom. All hydrogen atoms have been omitled for clarity. 

The molecular components of many buffers are too large to reach the active site of carbonic anhydrase. Carbonic anhydrase II has evolved a proton shuttle to allow buffer components to participate in the reaction from solution. The primary component of this shuttle is histidine 64. This residue transfers protons from the zinc-bound water molecule to the protein surface and then to the buffer (Figure 9.30). Thus, catalytic function has been enhanced through the evolution of an apparatus for controlling proton transfer from and to the active site. Because protons participate in many biochemical reactions, the manipulation of the proton inventory within active sites is crucial to the function of many enzymes and explains the prominence of acid-base catalysis. 

Carbonic anhydrases catalyze the reaction of water with carbon dioxide to generate carbonic acid. The catalysis can be extremely fast molecules of some carbonic anhydrases hydrate carbon dioxide at rates as high as 1 million times per second. A tightly bound zinc ion is a crucial component of the active sites of these enzymes. Each zinc ion binds a water molecule and promotes its deprotonation to generate a hydroxide ion at neutral pH. This hydroxide attacks carbon dioxide to form bicarbonate ion, HCO3 ". Because of the physiological roles of carbon dioxide and bicarbonate ions, speed is of the essence for this enzyme. To overcome limitations imposed by the rate of proton transfer from the zinc-bound water molecule, the most active carbonic anhydrases have evolved a proton shuttle to transfer protons to a buffer. 

Step 1 A water molecule is attracted to the zinc ion at the active site of carbonic anhydrase. The positively charged zinc ion displaces a proton from the water molecule. The displaced proton finds a new place of residence — the histidine residue. This histidine residue prt>bably aids in the removal of the proton from the water molecule, in concert wdth the action of the zinc ion. Combination of the zinc ion (Zn ) vedth the hydroxyl group does not form a complex with the structure Zn OH. The zinc atom does not change its valence (Its number of charges). Instead, the complex has the structure Zn (OH ). [Calcium ions behave similarly to zinc ions. In contrast, iron and copper ions readily change their valences when they participate in biochemical reactions.)... 

---