Thermolysin-like

M. anisopliae thermolysin thermolysin-like metallo 43 7.3 1,10-phenanthioline, phosphoramidon St. Leger et al. (1994a)... 

The mechaiusm by which metalloproteinases execute catalysis has been of interest for many years. Most studies focused on carboxypeptidase A and thermolysin-like proteases for which extensive stmctural, chemical, and biochemical data are available. The first peptide hydrolysis mechanisms to be proposed... 

In spite of the overall similarity of tertiary structure, a detailed analysis of the binding of inhibitors (Fig. 10), shows important differences in inhibitor-binding properties between the classes. In the short spacer family, the inhibitor is bound in an extended conformation while in the thermolysin family inhibitors adopt a twisted conformation. The contrasting requirements of the two classes is illustrated by the selectivity of the classical non-specific zinc-pro-tease inhibitor phosphoramidon which is active in nanomolar concentrations against the thermolysin-like enzymes [28] but has little or no inhibitory activity against the enzymes of the short spacer family [42]. 

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. 

Fig. 2. Catalytic motifs of thermolysin-like metalloproteases (a) a H2O chelates Zn + as part of the catalytic motif, (b) no H2O participate in the catalytic motif (modified from Auld, 1997, and Pelmenschikov Siegbahn, 2002, respecively).

Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, van Den BB, Venema G, Ulbrich-Hofmann R, Eijsink VG (1997) Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond. J Biol Chem 272 11152-11156 Matsuda A, Matsuyama K, Yamamoto K, Ichikawa S, Komatsu K (1987) Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. J Bacteriol 169 5815-5820 Matsumura I, Wallingford JB, Surana NK, Vize PD, Ellington AD (1999) Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase. Nat Biotechnol 17 696-701... 

Many related so-called thermolysin-like proteinases (TLPs) from various Grampositive strains have been described [47], including neutral proteases from Bacillus subtilis, and some of these variants are applied in peptide synthesis. Several metal-loenzymes acting as carboxy- or aminopeptidase have also been characterized, but these variants have not been extensively used in peptide synthesis. A bovine carboxy-peptidase A [39] and orange carboxypeptidase C [68] have been applied for dipeptide synthesis in water-organic solvent mixtures, both under thermodynamic and xmder kinetic control. 

The improvement of enzyme like MIP is currently another area of intense research. Beside the use of the MIP themselves as catalysts, they may also be applied as enhancer of product yield in bio-transformation processes. In an exemplary condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to Z-aspartame, the enzyme thermolysin was used as catalyst. In order to shift the equilibrium towards product formation, a product imprinted MIP was added. By adsorbing specifically the freshly generated product from the reaction mixture, the MIP helped to increase product formation by 40% [130]. MIP can also be used to support a physical process. Copolymers of 6-methacrylamidohexanoic acid and DVB generated in the presence of calcite were investigated with respect to promotion of the nucleation of calcite. Figure 19 (left) shows the polymer surface with imprints from the calcite crystals. When employing these polymers in an aqueous solution of Ca2+ and CO2 the enhanced formation of rhombohedral calcite crystals was observed see Fig. 19 (right) [131]. 

Thermolysin belongs to a class of proteases (called neutral proteases) which are distinct from the serine proteases, sulfhydryl proteases, metal-loexopeptidases, and acid proteases. Neutral proteases A and B from Bacillus subtilis resemble thermolysin in molecular weight, substrate specificity, amino acid content, and metal ion dependence. Since physiological substrates are most likely proteins, it is difficult to design simple experiments that can be interpreted in terms of substrate specificity and relative velocities. Therefore, studies of substrate specificity and other kinetic parameters must be carried out on di- and tripeptides so that details of the mechanism of catalysis can be obtained and interpreted simply. 

Alkaline phosphatases form a well-known class of proteins that perform quite interesting and complicated reactions. As previously reported, Zn enzymes, like carboxypeptidases, thermolysin, and carbonic anhydrases, consist of only one Zn atom per active center. Most of the alkaline phosphatases consist of two 96-kDa subunits, each containing two Zn and one Mg ion. The alkaline phosphatase from E. coli has been crystallized and described in full detail [4], and a mechanism has been proposed. Several enzymes in this category have been mentioned in recent years, some of them also containing different metal ions, such as iron and zinc, as in the purple acid phosphatase [5], It is likely that the detailed structure and mechanism of many more examples of enzymes that remove or add phosphate groups to proteins will become available in the next decade. 

Peptide synthesis is an extremely important area of chemistry for the pharmaceutical industry, and like any specialized area of chemistry, has its own set of unique problems associated with it. Racemization and purification of final products are two of the most difficult problems in this area. The use of enzymes has been explored as a possible answer to these problems since 1938 [29]. However, proteases needed to catalyze peptide synthesis are subject to rapid autolysis under the conditions needed to affect peptide coupling, so this has generally not been a practical approach until cross-linked enzyme crystals of proteases became available. The synthetic utility of protease-CLCs was demonstrated by the thermolysin CLC (PeptiCLEC -TR)-catalyzed preparation of the aspartame precursor Z-... 

Protein structure determinations have identified several examples of one domain inserted within another. One example is the E. coli DsbA protein, which catalyzes the formation of disulfide bonds in the periplasm. The enzyme consists of two domains a thioredoxin-like domain that contains the active site, and an inserted helical domain similar to the C-terminal domain of thermolysins (Martin et al., 1993). The inserted domain forms a cap over the active site, suggesting that it plays a role in binding to partially folded polypeptide chains before oxidation of... 

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). 

Oral administration of hydrolysates (doses of 500 and 2000mg/kg of body weight) from upstream chum salmon muscle prepared with thermolysin resulted in significant reductions in the blood pressure of SHRs in comparison with control rats. Blood pressure in SHRs remained significantly lower than control rats for up to 8h, with the maximum reduction occurring 4h after administration, and levels returning to normal after 24h (Ono et al., 2003). This trend implies that the key constituents of the hypotensive effect were likely short-chain peptides from protein hydrolysates. In contrast, if the main... 

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). 

For these thermodynamically controlled syntheses a variety of enzymes may be used, e.g. serine proteases like chymotrypsin and trypsin, cysteine proteases (thiol proteases) like papain, aspartate proteases like pepsin and metalloproteases like thermolysin. ... 

Endothelin converting enzyme (ECE) has yet to be fully characterized. It is a membrane-located enzyme found in the vascular endothelium. It is essential in the production of endothelin in the body since it converts the inactive precursor big ET-1 to endothelin-1. It is an unusual enzyme because it cleaves at a Tyr-Val link. Like endopeptidase-24.11, it is inhibited by phosphoramidon (so is a metalloproteinase), and these two enzymes are often colocated. Furthermore, monoclonal antibody co-precipitation studies indicate they share a common epitope, and a homology to the extent of about 39%, ECE is also similar to the bacterial metalloprotease thermolysin, but is more specific. If a specific inhibitor is discovered that can act in vivo, then clearly such a drug could modulate endothelin production throughout the body, which would have important consequences (e.g. in antihypertensive therapy). 

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. 

An important feature of long spacer zinc-endoproteases like thermolysin, as revealed by comparisons of the free enzyme and that complexed with inhibitors [40,41] is that conformational change is an essential component for catalytic activity. A similar conformational change is also probable [15,18,26] for the short spacer family, although a definite confirmation must await more structural information on equivalent free and inhibited proteins of this class. 

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