The zinc proteases

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. 

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. 

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 chemical mechanism of the zinc proteases is controversial because there is no direct evidence for intermediates, and the Zn2+ ion can act as an electrophile to polarize the > C=0 group of a substrate and/or as a source of metal-bound nucleophilic OH ions (see Chapter 2, section B7). 

The metalloproteases constitute the final major class of peptide-cleaving enzymes. The active site of such a protein contains a bound metal ion, almost always zinc, that activates a water molecule to act as a nucleophile to attack the peptide carbonyl group. The bacterial enzyme thermolysin and the digestive enzyme carboxypeptidase A are classic examples of the zinc proteases. Thermolysin, but not carboxypeptidase A, is a member of a large and diverse family of homologous zinc proteases that includes the matrix metalloproteases, enzymes that catalyze the reactions in tissue remodeling and degradation. 

Proteins which have the consensus HexxHxxgxxH, where the separation between the first and third (histidine) zinc ligands is nine residues. When the primary sequence in regions other than the zinc binding motif are considered, these proteases to be grouped into four [4, 5,33] classes (Fig. 2). Since most of the zinc proteases are multimodular proteins, with indi-... 

Table 1. Pairwise comparison of the topology and primary sequence of members of the short spacer family. The alpha carbon atoms defining the zinc protease fold (orange segment. Fig. 3) have been used in the topological superposition [56]. The distances refer to the root mean square deviations of this fold between pairs of structures. The corresponding pairwise primary sequence homology is also shown.

Unknown elements of primary sequence and zinc consensus determine the tertiary fold, indicating that the parallel structural and sequence categories classifing the zinc proteases is a result of the fortuitious selection of the proteases used for structural analysis. 

The reversal of the peptidic functional groups is often used in peptide chemistry. The obtained retropeptides are generally more resistant to enzymatic attacks (Figure For thiorphan and rctro-thiorphan an identical binding mode to the zinc protease thermolysin was demonstrated. Similar inhibition values for thermolysin and neutral endopeptidase were observed, whereas, for another zinc protease, angiotensin-converting enzyme (ACE), noticeable differences for inhibition were found (Figure 15.47). 

Fig. 2 (a) Schematic representation of the zinc-protease active site with subsite (S) nomenclature. X features a zinc-chelating group such as thiolate, carboxylate or hydroxamate. (b) Generic structure of a phosphinic peptide covering the S2 to S2 subsites of the active site... 

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