Zinc proteases

Before our work [39], only one catalytic mechanism for zinc dependent HDACs has been proposed in the literature, which was originated from the crystallographic study of HDLP [47], a histone-deacetylase-like protein that is widely used as a model for class-I HDACs. In the enzyme active site, the catalytic metal zinc is penta-coordinated by two asp residues, one histidine residues as well as the inhibitor [47], Based on their crystal structures, Finnin et al. [47] postulated a catalytic mechanism for HDACs in which the first reaction step is analogous to the hydroxide mechanism for zinc proteases zinc-bound water is a nucleophile and Zn2+ is five-fold coordinated during the reaction process. However, recent experimental studies by Kapustin et al. suggested that the transition state of HDACs may not be analogous to zinc-proteases [48], which cast some doubts on this mechanism. 

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. 

Schiavo, G., Rossetto, O., Benfenati, F., Poulain, B. and Montecucco, C., Tetanus and botulinum neurotoxins are zinc proteases specific for components of the neuroexcytosis apparatus, Ann. N. Y. Acad. Sci., 710, 65-75, 1994. 

Fig. 20. Schematic diagram of indirect carboxylate-zinc interactions through bridging hydroxyl groups, as observed in the (a) zinc proteases and (b) carbonic anhydrases.

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. 

Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988 Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine or leucine. The exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 A resolution (Rees et ai, 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127, (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). 

Fig. 30. Important active-site residues of carboxypeptidase A (CPA) and thermolysin (TLN) and a general scheme for the active sites of related zinc proteases.

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)]. 

Scheme 4. Zinc proteases nucleophilic adduct is a reaction intermediate carbonic anhydrases nucleophilic adduct is the product.

There are five distinct families of zinc proteases, classified by the nature of the zinc binding site. These families, and their variously proposed mechanisms, have recently been reviewed in depth.143 The most studied member is the digestive enzyme bovine pancreatic carboxypeptidase A, which is a metalloenzyme containing one atom of zinc bound to its single polypeptide chain of 307 amino acids and Mr 34 472. It is an exopeptidase, which catalyzes the hydrolysis of C-terminal amino acids from polypeptide substrates, and is specific for the large hydrophobic amino acids such as phenylalanine. The closely related carboxypeptidase B catalyzes the hydrolysis of C-terminal lysine and arginine residues. The two en-... 

Other well-known zinc proteases are collagenase angiotensin-converting enzyme (important in regulating blood pressure) thermolysin, a bacterial endopep-tidase of Mr 34 600 containing 316 residues in its single polypeptide chain 144 and the Zn2+ G protease from Streptomyces albus, a D-alanyl-D-alanine car-boxypeptidase that catalyzes. carboxypeptidation and transpeptidation in cell wall metabolism. 

Matrixin is implicated in arthritis and invasive cancer. There are also aminopeptidases, specific for the N-termini of proteins, that are zinc proteases. One example, leucine aminopeptidase, has a complex hexameric structure, which is unusual for a protease.145... 

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). 

Formation of the peptide bond is catalyzed by fhermolysin, a neutral zinc protease (E.C. 3.4.24.4). The advantages of this process are ... 

Zinc proteases, such as carboxypeptidases A and B or thermolysin, contain a tightly bound zinc which is bound to two imidazole residues (His), a carboxylate (Glu), and a molecule of H20. Zinc interacts with the substrate carbonyl oxygen and increases its polarization, thereby displacing water. 

Zinc proteases are metalloenzymes containing tightly bound zinc examples are carboxypeptidases A and B, collagenase, and thermolysin. The zinc atom is bound to the imidazole moiety of two histidines and the carboxylate of Glu the fourth ligand is a molecule of H20. 

---