Zinc enzymes structural aspects

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. 

A number of important structural aspects of zinc complexes as found in enzymes are introduced in this section to serve as background information for the subsequent sections. Aquated Zn(II) ions exist as octahedral [Zn(H20)6] + complexes in aqueous solution. The coordinated water molecules are loosely bound to the Zn + metal center and exchange rapidly with water molecules in the second coordination sphere (see Figure 1) with a rate constant of ca 10 s at 25 °C extrapolated from complex-formation rate constants of Zn + ions with a series of nucleophiles. The mechanism of the water exchange reaction on Zn(II) was studied theoretically, from which it was concluded that the reaction follows a dissociative mechanism as outlined in Figure 2. ... 

Another important structural aspect in terms of the reaction mechanisms of zinc enzymes concerns the coordination mode of water and carboxylates, which can bind in a monoden-tate or bidentate fashion to the Zn(II) center, as shown in Figure 4. 

A number of general reviews on the biochemistry of zinc are available.461 472 Some of these cover the important question of analysis,461,471 while others deal specifically with structural aspects.467,468 Reviews on specific enzymes will be given as appropriate. 

Fig. 8.1 Classification of metallopeptidases by zinc-binding motifs. The major amino acid motif in zincins has two histidine residues that coordinate with the metal ion and a glutamate residue (E) for catalysis. A third residue that coordinates with the metal may be glutamate, aspartate (D), or histidine. In metzincins, the third coordinating residue is histidine or aspartate (H/D), but the name is taken from the presence of a downstream invariant methionine residue (see Fig. 8.2 and text). The red type indicates enzymes or enzyme subfamilies encoded in the human genome (Slightly modified from Fig. 1A of F.X. Gomis-Ruth, Structural aspects of the metzincin clan of metal-loendopeptidases. Mol. Biotechnol. 24(2) 157-202, 2003)...

All of these recent CA models qualitatively or quantitatively illustrate the importance of nucleophilicity of zinc(II)-bound OH species in aqueous or nonaqueous solutions. This aspect is important in almost all of the zinc enzymes and will be presented again for models of other hydrolytic enzymes. However, in CA and many other zinc enzymes, the basicity of the zinc(II)-bound hydroxide is similarly important. Although I offers an excellent CA model, its catalytic activity is moderate in comparison to CK. Model 1 simply shows the essence of the intrinsic properties of zinc (II) at the active center of CA. Other important features such as the hydrophobic pocket for CO2, proton relay (network), and other structures in... 

Fig. 34. Glu-72- Zn interactions in native carboxypeptidase A and in carboxypep-tidase A-inhibitor complexes (inhibitors have been reviewed by Christianson and Lipscomb, 1989). When substrates or inhibitors bind to the enzyme active site and interact with the zinc ion, the interaction of the metal with Glu-72 tends from bidentate toward uniden-tate coordination. The flexibility of protein-zinc coordination may be an important aspect of catalysis in this system, and the Glu-72->Zn - coordination stereochemistry observed here is consistent with the stereochemical analysis of carboxylate-zinc interactions from the Cambridge Structural Database (Carrell et al., 1988 see Fig. 4).

These experimental studies leave major aspects of the mechanism unresolved. The most important one is whether the catalysis proceeds via a general base mechanism or via a nucleophilic attack by an enzyme residue on the scissile bond. Although the experimental studies of carboxypeptidase A are providing essential information, they cannot show what species are actually involved in the reaction. For example, the suggestion that the zinc bound water acts as the nucleophile was based on the results of x-ray structures of unproductive and static complexes of carboxypeptidase A formed with pseudosubstrates and inhibitors (Christianson and Lipscomb 1985). 

One of the most unusual aspects of the structure of this enzyme is the occurrence of the bridging imidazolate ligand, which holds the copper and zinc ions 6 A apart. Such a configuration is not unusual for imidazole complexes of... 

There are two distinct forms of MMOs one is soluble MMO (sMMO), which contains a nonheme dinuclear iron center, and the other is particulate MMO (pMMO), which is a copper-containing enzyme. Although sMMO has been well characterized and studied (78,79), many aspects of pMMO chemistry and biochemistry remain unclear. Because of the difficulty in the isolation and purification of pMMO, four different research groups have reported various ranges of copper stoichiometries for purified pMMO 2 (80), 2-3 (81) 8-10 (82), and 15-20 (83) copper ions per 100 kDa. The first crystal structure of pMMO firom M. capsulatus Bath (28) shows a heterotrimer of three subunits, and each subunit contains three polypeptides pmoB (a, 47 kDa), pmoA (, 24 kDa), and pmoC (y, 22 kDa). Three types of metal centers were found in pMMO a mononuclear copper, a dinuclear copper center in pmoB, and a zinc center in pmoC. 

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