Thermolysin active-site residues

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

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.

Fig. 2. Thermolysin active site. The structure shown is of a substrate bound to the active site 2n2+ and amino acid residues. From Kessler and Matthews (JO). Reprinted with permission of The American Chemical Society.

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). 

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). 

Standard mechanism inhibitors are classified strictly as inhibitors of serine proteases. There have been reports of inhibitors of other classes of proteases that have similar mechanisms to those of standard mechanism inhibitors, though. Initial studies on the streptomyces metalloprotease inhibitor (SMPl) suggest that it inhibits the metalloprotease thermolysin through a substrate-like binding mechanism (2). Similarly, staphostatin B, a cysteine protease inhibitor from Staphylococcus aureus, binds in a substrate-like manner in the active site of staphopain cysteine proteases. However, staphostatin B has a glycine PI residue, which adopts a backbone conformation that seems to prevent nucleophilic attack of the scissiie bond (3). 

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. 

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.

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. 

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