Carbonic anhydrase zinc coordination

Zinc is found only in the + 2 state in biological systems so we need consider only this oxidation level as we examine the mechanism of carbonic anhydrase. A zinc atom is essentially always bound to four or more ligands in carbonic anhydrase, three coordination sites are occupied by the imidazole rings of three histidine residues and an additional coordination site is occupied by a water molecule (or hydroxide ion, depending on pH). Because all of the molecules occupying the coordination sites are neutral, the overall charge on the Zn(His)3 unit remains +2. 

In carbonic anhydrase, zinc is coordinated to a water molecule and to three imidazole groups belonging to hystidine residues (Figure 8.11.1). The classi-... 

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.

Mn(II)-substituted carbonic anhydrase, which is less active than the zinc enzyme, has also been postulated to exhibit bidentate bicarbonate coordination. 

The X-ray structure of the unsubstituted tris(pyrazolyl)borato zinc nitrate has been solved showing a unidentate coordination mode for nitrate, in contrast with the t-butyl substituted ligand, which shows anisobidentate nitrate coordination due to the steric effects.232 A partial explanation of the reduced activity of cadmium-substituted carbonic anhydrase is offered by Parkin on the basis of the comparison of nitrate coordination to cadmium and zinc trispyrazo-lylborate moieties. A contributing factor may be the bidentate coordination supported by the cadmium that does not allow the facile access to a unidentate bicarbonate intermediate, which could be highly important to carbonic anhydrase activity.233... 

There has been particular recent interest in zinc nitrate complexes as coordination models for bicarbonate binding in carbonic anhydrase. The mono- or bidentate coordination modes have been studied with tris-pyrazolyl borate complexes and can be rationalized in the context of the enzyme activity.433 Caution in this comparison is introduced by ab initio calculations on these model systems demonstrating both monodentate and bidentate coordination energy minima for nitrate binding to zinc 434... 

Synthesis of functional models of carbonic anhydrase has been attempted with the isolation of an initial mononuclear zinc hydroxide complex with the ligand hydrotris(3-t-butyl-5-methyl-pyrazolyl)borate. Vahrenkamp and co-workers demonstrate the functional as well as the structural analogy to the enzyme carbonic anhydrase. A reversible uptake of carbon dioxide was observed, although the unstable bicarbonate complex rapidly forms a dinuclear bridged complex. In addition, coordinated carbonate esters have been formed and hydrolyzed, and inhibition by small ions noted.462 A number of related complexes are discussed in the earlier Section 6.8.4. 

Zinc in carbonic anhydrase Open-sided 5-coordinate... 

Four examples of catalytic or regulatory zinc proteins are reviewed here, and the discussion of metalloprotein function is set within the context of the metal ion and its coordination polyhedron. In the zinc enzymes carbonic anhydrase (carbonate dehydratase) II and carboxypeptidase A, the coordination polyhedron of the metal ion changes as the... 

The catalysis of CO2 hydration by carbonic anhydrase II occurs via the two chemically independent steps outlined in Scheme 2 a general mechanistic profile is found in Fig. 23. The first step involves the association of substrate with enzyme and the chemical conversion of substrate into product. The second step is product dissociation and the regeneration of the catalytically active nucleophile zinc hydroxide (Coleman, 1967). Below, we address the structural aspects of zinc coordination in each of these steps. 

Another contrast between the zinc proteases and the carbonic an-hydrases concerns the zinc coordination polyhedron. The carbonic an-hydrases ligate zinc via three histidine residues, whereas the zinc proteases ligate the metal ion through two histidine residues and a glutamate (bidentate in carboxypeptidase A, unidentate in thermolysin). Hence, the fourth ligand on each catalytic zinc ion, a solvent molecule, experiences enhanced electrostatic polarization in carbonic anhydrase II relative to carboxypeptidase A. Indeed, the zinc-bound solvent of carbonic anhydrase II is actually the hydroxide anion [via a proton transfer step mediated by His-64 (for a review see Silverman and Lindskog, 1988)]. 

The engineering of zinc-binding sites in a-helical peptides, where metal binding stabilizes protein tertiary structure, has been reported by Handel and DeGrado (1990). In these experiments zinc-binding sites are incorporated into a dimeric helix-loop—helix peptide (H3 2) and a protein composed of four helices connected by three short loop sequences (H3 4). a model of one subunit of the H3 2 dimer is found in Fig. 47. In addition to metal complexation by two histidine residues at positions n and n+4 of one a helix, the metal is coordinated by a third histidine residue of an adjacent a helix. The composition of the zinc coordination polyhedron is like that of carbonic anhydrase (i.e., Hiss), and spectroscopic results suggest that all three histidine residues are involved in zinc complexation. This work sets an important foundation... 

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