Thermolysin active site zinc

An 80- to 90-residue N-terminal propeptide domain contains a cysteine whose -S group binds to the active site zinc, screening it from potential substrates. The central catalytic domain is followed by a hinge region and a C-terminal domain that resembles the serum iron binding and transporting hemopexin.427/436 The mechanism of action is probably similar to that of thermolysin.430... 

The N-hydroxy amino acid derivatives are likely to be applicable to other metalloproteases. Thermolysin is inhibited irreversibly at pH 7.2 by ClCH2CO-DL-HOLeu-OCH3 where HOLeu is N-hydroxyleucine (47). The inhibition reaction involves coordination of the hydroxamic acid functional group to the active-site zinc atom of the enzyme. This then places the chloroacetyl group adjacent to Glu-143, an essential catalytic residue of thermolysin (see Figure 9). An ester linkage is formed and the enzyme is inactivated irreversibly. This reagent also inactivated two neutral metalloproteases from B. subtilis, but reacted only very slowly with carboxypeptidase A (t1/2 > 3 d). 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

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.

Thus, many metal ions catalyze the hydrolysis of esters [7,8], amides [9], and nitriles [10] via electrophilic activation of the C=0 or C=N group. This type of catalysis is characteristic of coordination complexes and is very common in metalloenzyme-mediated processes. Zinc(II), for example, is a key structural component of more than 300 enzymes, in which its primary function is to act as a Lewis acid (see Chapter 4). The mechanism of action of zinc proteases, e.g., thermolysin, involves electrophilic activation of an amide carbonyl group by coordination to zinc(II) in the active site (Figure 4). 

Zinc proteinases contain a tightly-bound active site Zn2 + ion and a carboxylate group in the two zinc proteinases whose X-ray crystal structures are known, carboxypeptidase A and thermolysin, these are Glu-270 and Glu-143 (Lipscomb, 1983). One thus has at least the following three possibilities for the initial catalytic event ... 

Wasserman and Hodge (290) used molecular dynamics to dock thermolysin inhibitors to an approximate model of the enzyme, with flexibility in the active site (3 8 of 314 residues) and ligand and with the rest of the enzyme represented by a grid approximation. A solvation model was used to compensate for desolvation in complex formation. To get 22 of 25 runs to orient the hydroxamate function correctly, the hydroxamate oxygens of the starting conformation were initialized within 4 A of the zinc. If they were allowed to vary to 8 A, then only 3 of 24 runs placed the ligand correctly. Obviously, there is a serious sampling problem. 

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. 

As was mentioned earlier, by far the largest number of zinc enzymes are involved in hydrolytic reactions, frequently associated with peptide bond cleavage. These include both exopeptidases, like carboxypeptidases A and B, which remove amino acids from the carboxyl-terminus of proteins, albeit with different specificities, and endopeptidases, like thermolysin, which cleave peptide bonds in the interior of the polypeptide chain. They have almost identical active sites (Figure 12.5) with two His and one Glu ligands to the Zn +. It appears that the Glu residue can be bound either in a mono- or bidentate manner. The two classes of enzymes are expected to follow similar reaction mechanisms. 

Gluzincins are proteins with the consensus HexxH( > 20)E, where the third zinc ligand, a glutamate, lies at least 20 residues C-terminal to the zincin motif (Table 2). The archetypical protein of this class, thermolysin consists of two domains, with the active site located between the N-terminal catalytic domain and the all alpha C-terminal domain (Fig. 7). With the limited data available, a comparison of topological matches of the observed structures with their cor-... 

Figure 7. The structure of thermolysin. Ribbon representation of the structure of thermolysin (silver, Brookhaven Databank [53] code 3TLN) shown with a bound inhibitor (green). The catalytic zinc atom (cyan) and structural calcium atoms (magenta) are shown. The active site is located between the N-terminal zinc protease domain and the alpha helical C-terminal domain. Zinc binding residues are in blue and the residue assisting catalysis is shown in red.

Figure 9. Thermolysin-inhibitor complex - an example of inhibitors bound to zinc endoproteases with the long spacer consensus. A stereo view of the active site site of thermolysin showing details of enzyme-inhibitor 2 interactions (Brookhaven Databank Code 5TLN). Enzyme side chains involved in inhibitor recognition are shown in magenta. The color code is as given for Fig. 6.

A structurally similar active site to that of carboxypeptidase A is found for the endopeptidase thermolysin.77 While several crystallographic and biochemical studies favor a zinc hydroxide mechanism for thermolysin (involving Glu-143 as a general base),80 in an alternative proposed mechanism for this enzyme, the zinc center is proposed to activate the substrate for nucleophilic attack by a non-coordinated water molecule (Scheme 14).81,82... 

Leucine aminopeptidase is interesting in that its active site contains two zinc atoms which together bind and activate the water molecule [74]. Despite this enzyme containing a dinuclear metal center at its active site, its mechanism, and specifically its mode of proton transfers reactions, appear to follow the general theme established by thermolysin and carboxypeptidase Adenosine deaminase and other members of the family of nucleoside and nucleotide deaminases utilize zinc-bound water as the catalytic nucleophile to displace ammonia from the 6-position of purines or the 4-position of pyrimidines and in all cases display inverse solvent deuterium isotope effects ranging from 0.3 to 0.8 on fec/Kni [75-80]. These effects are reminiscent of those observed for metallopro-teases and have their origins, like those of the proteases, in fractionation factors for the protons of the bound water that are less than one. 

In the C-terminal domain are five helices in a closed bundle. This characteristic fold is typical of thermolysin-like peptidases. Clan MC contains metallocarbox-ypeptidases which belong to only one family (M14) which is divided into the subfamilies A, B and C. Typical for this clan is that one zinc ion is tetrahedrally coordinated by a water molecule, two histidine and one glutamate residues. Clan MF includes aminopeptidases that require cocatalytic zinc ions for their enzymatic activity. The well-known leucyl aminopeptidase has a two-domain structure bearing the active site in the C-terminal domain. Whereas exopeptidases of clan MG require cocatalytic ions of cobalt or manganese, clan MH contains the third group of metallopeptidases that also require cocatalytic metal ions, but here these are all zinc ions. The third clan in which cocatalytic metal ions are necessary is clan MF with zinc or manganese. Only one catalytic zinc ion is required for peptidases of clans MA, MB, MC, MD and ME. 

---