Thermolysin specificity

Thermolysin, originally derived from Bacillus thermoproteolyticus, can be categorized as a neutral metalloprotease. The optimum conditions for this enzyme are pH 7.0 and 60-80 °C. Thermolysin also requires catalytically active zinc,and calcium for thermostability (Whitaker, 1994). When these conditions are met, heat-stable thermolysin specifically hydrolyzes peptide bonds involving isoleucine, leucine, valine, and phenylalanine (Adler-Nissen, 1986). It has been extensively utilized in the production of antihypertensive peptides which will be discussed later in this review. 

The metalloprotease thermolysin has been widely used for peptide synthesis [66,67]. Thermolysin is produced and secreted by the thermophilic bacterium Bacillus thermo-proteolyticus. Catalysis involves water activation by a zinc ion that is coordinated by side chains of two histidines and a glutamate. Thermolysin specificity is defined by its Sr pocket accepting large hydrophobic, polar, and charged residues. In the SI pocket hydrophobic residues are preferred, and Leu > Ala > Phe > Gly is the preference order for the S2 and S2 pockets. This hydrolytic specificity of thermolysin is reflected in its synthetic specificity. 

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]. 

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). 

T4 lysozyme 33,497 helix stability of 528, 529 hydrophobic core stability of 533, 544 Tanford j8 value 544, 555, 578, 582-Temperature jump 137, 138, 541 protein folding 593 Terminal transferase 408,410 Ternary complex 120 Tertiary structure 22 Theorell-Chance mechanism 120 Thermodynamic cycles 125-131 acid denaturation 516,517 alchemical steps 129 double mutant cycles 129-131, 594 mutant cycles 129 specificity 381, 383 Thermolysin 22, 30,483-486 Thiamine pyrophosphate 62, 83 - 84 Thionesters 478 Thiol proteases 473,482 TNfn3 domain O-value analysis 594 folding kinetics 552 Torsion angle 16-18 Tbs-L-phenylalanine chloromethyl ketone (TPCK) 278, 475 Transaldolase 79 Tyransducin-o 315-317 Transit time 123-125 Transition state 47-49 definition 55... 

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. 

Independently, simple peptide hydroxamic acids (Z-Gly-L-Leu-NHOH and others) were first observed to inhibit the metalloprotease thermolysin in 1977 9,101 The structure was then further improved to the hydroxamidoalkylmalonyl-peptide moiety by considering the substrate specificity of thermolysin and other metalloproteases 10-121 A summary of hydroxamic acids reported to be inhibitors of various metalloenzymes up to 1983 has been published 131 In 1985 hydroxamido-benzylsuccinyl-L-alanine (kelatorphan) was synthesized and found to be one of the best enkephalinase inhibitors 141... 

If the desired hydroxamic acids are sufficiently hydrophobic, workup is easily carried out using extraction and flash chromatography. However, many hydroxamic acids are soluble in polar solvents, which causes problems during isolation and purification. The structure of hydroxamic acid thermolysin inhibitors can be made more potent by the introduction of a malonyl moiety to match the specificity of thermolysin (Scheme 3). For example, in the synthesis of HONHCOCH(Bzl)CO-L-Ala-Gly-NH2 (4), O-benzylhydroxylamine was employed in the synthetic scheme to facilitate the isolation and purification of the intermediate 2.[101 The final precursor is a benzyl-protected hydroxamate 3 that can be deprotected by hydrogenolysis without byproducts contaminating the desired hydroxamic acid 4. 

Limited proteolysis by thermolysin, papain, subtilsin and a-chymotrypsin of the plasma-membrane-bound adenylate cyclase from rat liver had no effect on activity when Mn2+ was the cofactor, but stimulated activity in the presence of Mg2+. However, the addition of Mg2+ over MnATP did not result in stimulation on proteolysis. The simplest interpretation is that proteolysis exerts its main effect on the regulatory components of adenylate cyclase.329 Adenylate cyclase, solubilized from rat liver membranes by Lubrol PX or deoxycholate, loses its regulatory components, and becomes much more specific for MnATP.330... 

All proteolytic enzymes described are fairly non-specific serine endoproteases, cleaving peptide chains preferentially at the carboxyl side of hydrophobic amino acid residues. The enzymes convert their substrates into small, readily soluble fragments which can be removed easily from fabrics. Only serine protease can be used in detergent formulations, as thiol proteases such as papain would be oxidized by the bleaching agents, acidic proteases are not active at common laundry conditions, and metalloproteases such as thermolysin would lose their metal cofactors because of complexation with the water-softening agents or hydroxyl ions. 

Enantioselective acylation of amine and hydrolysis of amide are widely studied. These reactions are catalyzed by acylases, amidases and lipases. Some examples are shown in Figure 21.22 Aspartame, artificial sweetener, is synthesized by a protease, thermolysin (Figure 21(a)).22a In this reaction, the L-enantiomer of racemic phenylalanine methyl ester reacted specifically with the a-carboxyl group of N-protected L-aspartate. Both the separation of the enantiomers of the phenylalanine and the protection of the y-carboxyl group of the L-aspartate were unnecessary, which simplified the synthesis. 

Abz was combined with a broad variety of non-fluorescent acceptors such as p-nitrobenzyl for leucine aminopeptidase (Carmel et al., 1977), pNA for trypsin (Bratanova and Petkov, 1987), 4-ni-trophenylalanine [Phe(NC>2)] for HIV protease (Toth and Marshall, 1990), and V-(2,4-di n itrophenyl) ethylenediamine (EDDnp) for thermolysin and trypsin (Nishino et al., 1992). Lecaille et al. (2003) described a FRET quench assay based on a specific substrate for cathepsin K labeled with Abz and EDDnp. This substrate is not cleaved by the other Cl cysteine cathepsins and serine proteases in contrast to methoxycoumarin (Mca)-based substrates described earlier (Aibe et al., 1996 Xia et al., 1999) and merely covered the non-primed site of the scissile bond. The 5-[(2-aminoethyl)amino] naphthalene-l-sulfonic acid (EDANS) compound is a second example of a fluorescence donor historically used for many FRET quench-based protease assays, e.g., in combination with tryptophan as a quencher in an ECE activity assay (Von Geldren et al., 1991). The FRET-1 example in Table 2.2 shows the typical dynamic range that can be achieved with an EDANS/DABCYL-based assay. 

SMPI M4 metaiioproteases Standard fold-specific thermolysin -0.1 nM (3)... 

Both peptides have been prepared in solution for the last 30 years (136, 137), and many solid-phase syntheses also have been reported. In addition, syntheses of these peptides have employed enzymatic methods, which could be done in low-water content systems on a preparative scale (138). A"-Cbz and A -Boc protected Met-enkephalin and Leu-enkephalin were prepared by means of a-chymotrypsin, papain, thermolysin, and bromelain adsorbed on Celite. a-Chymotrypsin has a primary specificity for bulky and hydrophobic amino acids (Phe, Tyr,... 

Trp, Leu, Met), and it was selected for making the Tyr-Gly, Phe-Leu, and Phe-Met peptide bonds. Papain was selected for Gly-Gly and Gly-Phe peptide bonds. Bromelain is very similar to papain as far as its specificity. All these proteases are serine or cysteine type, and the peptide bond formation can be done under kinetically controlled conditions. Thermolysin is an asparyl protease, and it was selected mainly for Phe-Leu and Gly-Phe bonds. MeCN, EtOAc, and methyl caproate were used as solvents with a controlled amount of buffer or at fixed water amount. 

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

In order to determine the number of histidine residues involved in zinc coordination, the L chains of TeTx and BoNT/A, B and E were modified with diethyl pyrocarbonate (DEPC), a reagent that specifically modifies histidine residues. In each case, two additional histidines were modified in the apo-toxin that were not affected in the holo-neurotoxin (Schiavo etal., 1992 b, c). These results indicate that the zinc atom of CNTs is coordinated via two histidines and a Glu-bound water molecule, as in thermolysin. Mutations at the two histidines of the motif inactivate TeTx and suppress its ability to bind radio-labeled Zn " (Yamasaki etal., 1994 b). In addition, mutations of the conserved Glu-271 and Glu-272 of TeTx, predicted to be in an a-helical segment (Lebeda and Olson, 1994), result in decreased zinc binding and loss of activity. Based on these experimental results, it has... 

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. 

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